molecules
Article
Glutathione Peroxidase-Like Activity of
Amino-Substituted Water-Soluble Cyclic Selenides:
A Shift of the Major Catalytic Cycle in Methanol
Kenta Arai *, Ayako Tashiro, Yuui Osaka and Michio Iwaoka *
Department of Chemistry, School of Science, Tokai University, Kitakaname, Hiratsuka-shi, Kanagawa 259-1292,
Japan; [email protected] (A.T.); [email protected] (Y.O.)
* Correspondence: [email protected] (K.A.); [email protected] (M.I.)
Academic Editors: Claudio Santi and Luana Bagnoli
Received: 31 January 2017; Accepted: 22 February 2017; Published: 25 February 2017
Abstract: We previously reported that water-soluble cyclic selenides can mimic the antioxidative
function of glutathione peroxidase (GPx) in water through a simple catalytic cycle, in which the
selenide (>Se) is oxidized by H2 O2 to the selenoxide (>Se=O) and the selenoxide is reduced by a thiol
back to the selenide. In methanol, however, the GPx-like activity could not be explained by this simple
scenario. To look into the reasons for the unusual behaviors in methanol, monoamino-substituted
cyclic selenides with a variable ring size were synthesized, and the intermediates of the catalytic cycle
were characterized by means of 77 Se-NMR and LC–MS spectroscopies. In water, it was confirmed
that the selenide and the selenoxide mainly contribute to the antioxidative function, though a slight
contribution from the dihydroxy selenane (>Se(OH)2 ) was also suggested. In methanol, on the other
hand, other active species, such as hydroxyselenonium (>Se+ –OH) and hydroxy perhydroxy selenane
(>Se(OH)(OOH)), could be generated to build another catalytic cycle. This over-oxidation would be
more feasible for amino-substituted cyclic selenides, probably because the ammonium (NH3 + ) group
would transfer a proton to the selenoxide moiety to produce a hydroxyselenonium species in the
absence of an additional proton source. Thus, a shift of the major catalytic cycle in methanol would
make the GPx-like antioxidative function of selenides perplexing.
Keywords: antioxidant; enzyme model; glutathione peroxidase; hydroxy perhydroxy selenane;
selenide; selenoxide
1. Introduction
Reactive oxygen species (ROS)—such as hydrogen peroxide (H2 O2 ), superoxide anion (·O2− ),
hydroxyl radical (HO·), and singlet oxygen—which are steadily generated during the consumption
processes of triplet oxygen, have high oxidizability to damage DNAs, cellular lipids, and proteins,
leading various life-threatening disorders, such as protein-misfolding disease in central nervous
systems, myocardial infarction in heart, diabetes in liver, and cataract in eyes [1]. To protect
themselves from such oxidative stress, eukaryotic organisms have evolved systems involving
various antioxidative enzymes. Glutathione peroxidase, a representative antioxidative enzyme and a
well-known selenoenzyme, has selenocysteine (Sec), a selenium analog of natural cysteine, at the active
site. Glutathione peroxidase (GPx) catalyzes the reduction of H2 O2 to harmless water (H2 O) using
glutathione (GSH) as a reducing cofactor. From a view point of drug design for antioxidant therapy,
much effort has been directed for decades toward development of organoselenium compounds [2–7],
which can act as GPx-like catalysts, and also toward elucidation of their catalytic mechanisms [8–11].
Various GPx model compounds, most of which are aromatic diselenides (ArSeSeAr), have been
developed and their catalytic activities have been evaluated [12–25]. In the meantime, aliphatic
Molecules 2017, 22, 354; doi:10.3390/molecules22030354
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meantime, aliphatic and/or aromatic monoselenides (RSeR′) are also known to act as GPx-like
catalystsaromatic
throughmonoselenides
a unique selenide/selenoxide
cycle
(Scheme
1, cyclecatalysts
A), in which
selenide
1 is
and/or
(RSeR0 ) are alsoredox
known
to act
as GPx-like
through
a unique
oxidized by H2O2 to the
corresponding
selenoxide
which
is reduced
back
to oxidized
1 by a thiol
selenide/selenoxide
redox
cycle (Scheme
1, cycle 2,A),
in which
selenide
1 is
by substrate
H2 O2 to
(RSH)
[26–29].
In
addition,
Back
et
al.
reported
that
bis(3-hydroxypropyl)
selenide
can
be
oxidized
to a
the corresponding selenoxide 2, which is reduced back to 1 by a thiol substrate (RSH) [26–29].
reactive
spiroBack
dioxoselenurane
species,
instead of a selenoxide,
whichcan
is also
reducedtoback
to the
In
addition,
et al. reported
that bis(3-hydroxypropyl)
selenide
be oxidized
a reactive
corresponding
selenidespecies,
by thiols
[30,31].
Braga
andiscoworkers
demonstrated
presence of
spiro
dioxoselenurane
instead
of Recently,
a selenoxide,
which
also reduced
back to thethe
corresponding
another by
catalytic
cycle (cycle
B) [32].
According
to their
report, selenoxide
2 is inofequilibrium
with
selenide
thiols [30,31].
Recently,
Braga
and coworkers
demonstrated
the presence
another catalytic
dihydroxy
3a or hydroxyselenonium
3 in methanol,
3 reacts with
with dihydroxy
H2O2 to generate
cycle
(cycleselenane
B) [32]. According
to their report, selenoxide
2 is in and
equilibrium
selenanea
highly
reactive
species
(i.e.,
hydroxy
perhydroxy
selenane
4),
which
is
a
much
greater
oxidant
than 2
3a or hydroxyselenonium 3 in methanol, and 3 reacts with H2 O2 to generate a highly reactive species
for
thiol
substrates.
(i.e., hydroxy perhydroxy selenane 4), which is a much greater oxidant than 2 for thiol substrates.
Cycle A
Cycle B
Proposed
glutathione
peroxidase
catalytic
cycle (A)
ofselenide/selenoxide
a selenide. (A)
Scheme 1.1.Proposed
glutathione
peroxidase
(GPx)-like(GPx)-like
catalytic cycle
of a selenide.
selenide/selenoxide
redox observed
pathways in
mainly
water
[26‒29];
(B) another
cycle
redox
pathways mainly
waterobserved
[26–29];in(B)
another
catalytic
cycle catalytic
mediated
by
mediated
by
hydroxyselenonium
3
and
hydroxy
perhydroxy
selenane
4
as
a
highly
active
oxidant
[32].
hydroxyselenonium 3 and hydroxy perhydroxy selenane 4 as a highly active oxidant [32].
In our laboratory, a series of water-soluble cyclic selenides, such as 7‒9, have been synthesized,
In our laboratory, a series of water-soluble cyclic selenides, such as 7–9, have been synthesized,
and their potential GPx-like activities were evaluated in water as well as in methanol [26,27,29]. Our
and their potential GPx-like activities were evaluated in water as well as in methanol [26,27,29].
previous analysis revealed that they exhibit the activity through a simple redox interconversion
Our previous analysis revealed that they exhibit the activity through a simple redox interconversion
between a selenide and a selenoxide (i.e., cycle A in Scheme 1) in an aqueous medium, and also that
between a selenide and a selenoxide (i.e., cycle A in Scheme 1) in an aqueous medium, and also that
the magnitude of the activity can be controlled by changing the ring size and the polar functional
the magnitude of the activity can be controlled by changing the ring size and the polar functional
groups attached to the ring. The optimal ring size was five, and the GPx-like catalytic activity
groups attached to the ring. The optimal ring size was five, and the GPx-like catalytic activity generally
generally became lower when the substituent on the ring structure was changed from carboxy
became lower when the substituent on the ring structure was changed from carboxy (CO2 H) to
(CO2H) to hydroxy (OH), and then amino (NH2) groups. Since CO2H should exist as an ionized form
hydroxy (OH), and then amino (NH2 ) groups. Since CO2 H should exist as an ionized form (CO2 − ), this
(CO2−), this substituent would facilitate the conversion of selenide to selenoxide (reaction i in Scheme
substituent would facilitate the conversion of selenide to selenoxide (reaction i in Scheme 1)—which is a
1)—which is a rate-limiting step in the catalytic cycle A—by elevating the HOMO (highest occupied
rate-limiting step in the catalytic cycle A—by elevating the HOMO (highest occupied molecular orbital)
molecular orbital) energy level of the selenide by inductive electron releasing. On the other hand, a
energy level of the selenide by inductive electron releasing. On the other hand, a protonated amino
protonated amino group (NH3+) should decelerate the reaction due to the inductive electron
group (NH3 + ) should decelerate the reaction due to the inductive electron withdrawal. Although a
withdrawal. Although a magnitude of GPx activity of the cyclic selenides correlates well with both
magnitude of GPx activity of the cyclic selenides correlates well with both the HOMO energy level
the HOMO energy level and the second-order rate constant for the oxidation of selenide to
and the second-order rate constant for the oxidation of selenide to selenoxide (reaction i) in water,
selenoxide (reaction i) in water, cyclic selenide 7, with a five-membered ring having one amino
cyclic selenide 7, with a five-membered ring having one amino substituent, showed unexpectedly high
substituent, showed unexpectedly high activity [29].
activity [29].
In methanol, on the other hand, cyclic selenides having amino substituents exhibited higher
In methanol, on the other hand, cyclic selenides having amino substituents exhibited higher
GPx-like activity than those having OH or CO2H substituents. Thus, the activity rank of cyclic
GPx-like activity than those having OH or CO2 H substituents. Thus, the activity rank of cyclic selenides
selenides could not be explained by the simple scenarios, which were applicable in water [29],
could not be explained by the simple scenarios, which were applicable in water [29], suggesting that
suggesting that the presence of other catalytic cycles, such as cycle B. In this context, it would be
the presence of other catalytic cycles, such as cycle B. In this context, it would be important to assess
important to assess an effective GPx-like catalytic cycle in methanol for cyclic selenides, especially
an effective GPx-like catalytic cycle in methanol for cyclic selenides, especially for those having amino
for those having amino substituents, in order to explain the unusual behaviors.
substituents, in order to explain the unusual behaviors.
In this study, monoamino-substituted cyclic selenides with a four- (5) or six-membered ring (6)
In this study, monoamino-substituted cyclic selenides with a four- (5) or six-membered ring
were additionally synthesized, and their GPx-like catalytic activity and the catalytic cycle were
(6) were additionally synthesized, and their GPx-like catalytic activity and the catalytic cycle were
investigated in water and in methanol by using monoamino cyclic selenide 7 (MASred), diamino
Molecules 2017, 22, 354
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354
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2017, 22,
22,
354
investigated
in water
3 of 13
3 of 13
and in methanol by using monoamino cyclic selenide 7 (MASred ), diamino cyclic
red
red
selenide
8 (DAS ), and
cyclic selenideselenide
9 (DHS ) as reference
selenides. The selenide
reddihydroxy
red
cyclic
cyclic selenide
selenide 88 (DAS
(DASred),
), and
and dihydroxy
dihydroxy cyclic
cyclic selenide 99 (DHS
(DHSred)) as
as reference
reference selenides.
selenides. The
The
compounds
employed
in
this
study
are
lined
up
in
Figure
1.
selenide
compounds
employed
in
this
study
are
lined
up
in
Figure
1.
selenide compounds employed in this study are lined up in Figure 1.
Figure
(7‒9).
Figure 1.
1. Target
Target compounds
compounds in
in this
this study
study (5
(5 and
and 6)
6) and
and previous
previous selenides
selenides (7–9).
(7‒9). Compounds
Compounds 88 and
and 99
are
racemic
compounds.
are racemic compounds.
2.
2. Results
Results and
and Discussion
Discussion
2.1.
2.1. Synthesis
Synthesis
Compounds
Compounds
Compounds 55 and
and 66 were
were synthesized
synthesized by
by applying
applying aa similar
similar protocol
protocol reported
reported previously
previously [33]
[33]
2).
Boc-protected
mesylates
12a
and
12b
were
diethyl
(Scheme
Boc-protected
mesylates
12a
and
first
prepared
from
diethyl
aminomalonate
(Scheme 2). Boc-protected mesylates 12a and 12b were first prepared from diethyl aminomalonate
hydrochloride
hydrochloride
-glutamic acid
acid (11),
hydrochloride (10)
(10) or
or LL-glutamic
-glutamic
acid
(11), respectively,
respectively, by
by following
following previous
previous methods
methods with
with slight
slight
modifications
[34,35].
The
mesylates
were
then
converted
into
cyclic
selenides
13a
and
13b
mesylates
converted
into
cyclic
selenides
13a
and
modifications [34,35]. The mesylates were then converted into cyclic selenides 13a and 13b by
by
selenation
with
NaHSe.
Finally,
obtained
13a
and
13b
were
treated
HCl
to
give
Finally,
obtained
13a and
HClwith
to give
target
selenation with
withNaHSe.
NaHSe.
Finally,
obtained
13a 13b
andwere
13btreated
were with
treated
with
HCl
to compounds
give target
target
1
13C, and 77Se-NMR as well as
compounds
55 were
and
were
5compounds
and 6, which
fully
characterized
by 1characterized
H, 13 C, and 77 by
Se-NMR
mass
and 6,
6, which
which
were fully
fully
characterized
by 1H,
H, 13as
C,well
andas77high-resolution
Se-NMR as well
as
high-resolution
mass
spectroscopy
(HRMS).
spectroscopy
(HRMS).
high-resolution mass spectroscopy (HRMS).
N,
Scheme
Synthesis
selenides
and
6.
Reagents
and
conditions:
Boc
Scheme 2.
2. Synthesis
Synthesis of
of monoamino
monoamino selenides
selenides 55 and
and 6.
6. Reagents
Reagents and
and conditions:
conditions: (i)
(i) (1)
(1) Boc
Boc222O,
O, Et
Et333N,
N,
◦ C, 18 h, (2) NaBH , EtOH, reflux, 1
1,4-dioxane/H
O
(5:2),
50
Et
Cl
20
room
1,4-dioxane/H
2O
(5:2),
50
°C,
18
h,
(2)
NaBH
44, EtOH, reflux, 1 h, (3) MsCl,
MsCl,
Et
3N, CH2
2Cl
22, , 20
h,
2
3
1,4-dioxane/H2O (5:2), 50 °C, 18 h, (2) NaBH4, EtOH, reflux, 1 h, (3) MsCl, Et3N, CH2Cl2, 20 h, room
temperature
(rt);(ii)
(ii)(1)
(1)EtOH,
EtOH,AcCl,
AcCl,reflux,
reflux,444h,
(2)Boc
Boc
1,4-dioxane:H
= 5:2,
50 ◦18
C,h,
18(3)
h,
temperature
2O,
Et
3N,
1,4-dioxane:H
2O 2=O5:2,
50 °C,
2 O,
3 N,
temperature (rt);
(rt);
(ii)
(1)
EtOH,
AcCl,
reflux,
h,h,(2)
(2)
Boc
2O,
EtEt
3N,
1,4-dioxane:H
2O = 5:2, 50 °C, 18 h, (3)
(3)
NaBH
,
EtOH,
reflux,
1
h,
(4)
MsCl,
Et
N,
CH
Cl
,
20
h,
rt;
(iii)
NaHSe,
iPrOH/1,4-dioxane,
reflux,
NaBH
4, EtOH,
reflux,
1
h,
(4)
MsCl,
Et
3
N,
CH
2
Cl
2
,
20
h,
rt;
(iii)
NaHSe,
iPrOH/1,4-dioxane,
reflux,
2.5
4
NaBH4, EtOH,
reflux, 1 h, (4) MsCl, Et3N, 3CH2Cl2,220 2h, rt; (iii) NaHSe, iPrOH/1,4-dioxane, reflux, 2.5 h
h
2.5
h
for
synthesis
13a12a;
from
12a; NaHSe,
EtOH/THF,
3 h for synthesis
of 13b
12b;
for
from
NaHSe,
EtOH/THF,
reflux,
h
of
12b;
(iv)
for synthesis
synthesis of
of 13a
13a of
from
12a;
NaHSe,
EtOH/THF,
reflux, 33reflux,
h for
for synthesis
synthesis
of 13b
13b from
from
12b;from
(iv) HCl,
HCl,
a Details of the synthesis are given in Supporting Information.
a ◦ C, 20 h.
(iv)
HCl, H235
O/EtOH,
H
°C,
H22O/EtOH,
O/EtOH,
35
°C, 20
20 h.
h.35a Details
Details of
of the
the synthesis
synthesis are
are given
given in
in Supporting
Supporting Information.
Information.
2.2.
2.2. Redox
Redox Properties
Properties of
of Selenides
Selenides and
and Selenoxides
Selenoxides in
in Water
Water
77
77
The
were
monitored
by
spectroscopy in
O.
The redox
redox reactions
reactions of
of synthesized
synthesized 55 and
and 66 were
were monitored
monitored by
by 77Se-NMR
Se-NMR spectroscopy
spectroscopy
in D
D222O.
O.
The
spectral
changes
observed
for
6
are
shown
in
Figure
S1
as
a
typical
example.
When
6
was
6 are
shown
in Figure
S1 asS1
a typical
example.
When 6When
was reacted
The spectral
spectralchanges
changesobserved
observedforfor
6 are
shown
in Figure
as a typical
example.
6 was
reacted
with
1
equivalent
of
H
2broad
O
2, the
broad
peak
of
6
(136
ppm)
completely
disappeared
after
22 h,
with
1
equivalent
of
H
O
,
the
peak
of
6
(136
ppm)
completely
disappeared
after
2
h,
and
2
2
reacted with 1 equivalent of H2O2, the broad peak of 6 (136 ppm) completely disappeared after two
h,
and
two
signals
(829
ppm
(major)
and
834
ppm
(minor)),
which
should
correspond
to
two
signals
(829
ppm
(major)
and
834
ppm
(minor)),
which
should
correspond
to
two
stereoisomers
of
and two signals (829 ppm (major) and 834 ppm (minor)), which should correspond to two
stereoisomers
of
Quantum
chemical
at
the
selenoxide, appeared
(Figure S1b).appeared
Quantum(Figure
chemicalS1b).
calculation
at B3LYP/6-31+G(d,p)
showed
stereoisomers
of the
the selenoxide,
selenoxide,
appeared
(Figure
S1b).
Quantum
chemical calculation
calculation
at
B3LYP/6-31+G(d,p)
showed
that
the
cis-isomer,
which
has
an
axial
amino
group
on
the
that
the
cis-isomer,
which
has
an
axial
amino
group
on
the
six-membered
ring,
is
6.02
kcal/mol
more
B3LYP/6-31+G(d,p) showed that the cis-isomer, which has an axial amino group on the
six-membered
six-membered ring,
ring, is
is 6.02
6.02 kcal/mol
kcal/mol more
more stable
stable than
than the
the trans-isomer,
trans-isomer, which
which has
has an
an equatorial
equatorial
amino
amino group,
group, in
in implicit
implicit water.
water. Thus,
Thus, the
the major
major peak
peak (829
(829 ppm)
ppm) in
in the
the NMR
NMR spectrum
spectrum was
was assigned
assigned
to
the
cis-isomer,
in
which
a
hydrogen
atom
of
the
protonated
ammonium
(NH
3++) group would form
to the cis-isomer, in which a hydrogen atom of the protonated ammonium (NH3 ) group would form
Molecules 2017, 22, 354
4 of 13
stable than the trans-isomer, which has an equatorial amino group, in implicit water. Thus, the major
peak (829 ppm) in the NMR spectrum was assigned to the cis-isomer, in which a hydrogen atom of
the protonated ammonium (NH3 + ) group would form an intramolecular hydrogen bond with the
oxygen atom of the selenoxide moiety. The selenoxide was reduced back to selenide 6 within 15 min
after addition of 1 equivalent of dithiothreitol (DTTred ). For five-membered cyclic selenide 7, similar
spectral changes were observed in the 77 Se-NMR analysis (Figure S3). Quantum chemical calculation
again suggested that among the two stereoisomers of the selenoxide the cis-isomer is more stable.
According to Scheme 1, selenoxide 2 can be further oxidized by an excess amount of H2 O2
to hydroxy perhydroxy selenane 4 through hydroxyselenonium 3 and/or dihydroxy selenane 3a
(cycle B). To confirm whether such over-oxidation is possible or not, the selenoxide obtained from 6
(Figure S1b) was reacted with 4 equivalents of H2 O2 . However, obvious spectral changes were not
observed (Figure S1c). Nevertheless, when 4 equivalents HCl were further added as a proton source,
the major selenoxide signal disappeared on the NMR chart, leaving the minor trans-isomer with an
equatorial ammonium (NH3 + ) group unreacted (Figure S1d). At this stage, any new signals were
not detected over a range of 0–1500 ppm in the 77 Se-NMR spectrum, probably because of significant
line broadening of the absorption peak for the generated species, which would be the corresponding
hydroxyselenonium 3 or dihydroxy selenane 3a. Preferential conversion of the cis-isomer suggests
that the protonated axial NH3 + group can assist conversion of the selenoxide by interacting with the
selenoxide oxygen, whereas the equatorial NH3 + group cannot assist it. It should be noted that the
minor isomer of the selenoxide was not reacted, even when 6 equivalents in total of HCl was added.
When the resulting solution (Figure S1d) was added with 5 equivalents of DTTred , the selenide 6 was
regenerated, suggesting that the tetrahydroselenopyran skeleton was not decomposed during the
conversion to active species. In addition, when the solution of the selenoxide (Figure S1b) was added
with 4 equivalents HCl in the absence of H2 O2 , only the major peak of the selenoxide disappeared,
probably due to its conversion into hydroxyselenonium 3 or dihydroxy selenane 3a (Figure S1e).
The selenoxide was recovered by neutralization with 4 equivalents of NaOH (Figure S1f), suggesting
that selenoxide 2 and hydroxyselenonium 3 are in equilibrium with each other (reaction iii in Scheme 1).
Similar redox behaviors were observed for other amino selenides 5, 7, and 9 (Figures S2–S5), although
the selenoxide of 5 was decomposed by addition of an excess amount of H2 O2 and HCl (Figure S2).
For the selenoxide of DHSred (9), a new signal (959 ppm), which would correspond to 3 or 3a, appeared
with an accompanying disappearance of the selenoxide peak (926 ppm) when 4 equivalents of HCl
was added to the solution of the selenoxide (Figures S5 and S6). The signal at 959 ppm disappeared
and the selenoxide signal appeared after neutralization with 4 equivalents of NaOH (Figure S6).
In order to identify the products of the above reactions, the sample solution during the redox
reaction of selenides with H2 O2 was analyzed by LC–MS (atmospheric-pressure chemical ionization
(APCI) and electrospray ionization (ESI)) in a positive ion mode under a continuous flow condition
(0.3 mL/min). Typical MS spectra obtained for 6 are shown in Figure 2. After treatment of the selenide
with 1 equivalent of H2 O2 in water for 2 h, the selenoxide (C5 H12 NO80 Se+ ) was observed as a major
product, which was detected at m/z 182.0 (Figure 2a), while the small peak of dihydroxy selenane 3a
(C5 H14 NO2 80 Se+ ), was detected at m/z 200.0. Further addition of H2 O2 (4 equivalents) did not cause
dramatic changes in the MS spectrum (Figure 2b). However, when 4 equivalents of HCl were added,
the peaks of the selenoxide converted into 3a (Figure 2c), which would be generated through reaction
iii to v or reaction iv in Scheme 1. Furthermore, under an ESI+ condition, hydroxy perhydroxy selenane
4 (C5 H14 NO3 Se+ ) was slightly but obviously detected (Figure S7), supporting that the selenoxide can
be over-oxidized to 4 under an acidic condition via hydroxyselenonium 3. Very recently, based on
theoretical calculation, Orian et al. reported that the conversions of a selenoxide into the corresponding
dihydroxy selenane and hydroxy perhydroxy selenane are significantly endothermic (+13.1 and
+16.4 kcal/mol, respectively) [36]. This is consistent with our observation that the conversion of
selenoxide 2 into 3a and 4 was not easy unless HCl and/or excess H2 O2 were added. When the same
experiments were performed for monoamino selenide 7, similar results were obtained (Figure S8).
Molecules 2017, 22, 354
Molecules 2017, 22, 354
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5 of 13
The results of LC–MS analysis strongly indicate that the simple redox reaction between selenide
and selenoxide (i.e., reaction i and ii in Scheme 1) is predominant during the GPx-like catalytic
For selenide 5, on the other hand, the reaction with H2 O2 produced significant amounts of decomposed
reaction under a physiological pH condition (i.e., pH
7.4), while a small portion of the selenoxide,
compounds, for which the structures could not be characterized.
when it has an amino substitution, could be over-oxidized.
182.0
Intensity
a
164.0
200.0
m/z
100
120
b
140
160
180
Intensity
4 eq H2O2, 60 min
200
220
240
182.0
200.0
164.0
m/z
100
120
c
140
160
180
200
Intensity
240
200.0
4 eq HCl, 10 min
100
220
164.0
182.01
120
140
160
180
200
220
240
m/z
Figure 2.
chemical ionization,
ionization,APCI+)
APCI+)spectral
spectral changes
changes during
during the
Figure
2. LC–MS
LC–MS (atmospheric-pressure
(atmospheric-pressure chemical
oxidation of
of the
the selenoxide
selenoxide form
form of
of selenide
selenide 66 in
O at
at 25
25 ◦°C.
C. For
For a–c,
O (100%)
(100%) was
was used
used as
as an
an
oxidation
in H
H2O
a‒c, H
H2O
eluent
for
the
LC
under
a
continuous
flow
at
0.3
mL/min,
and
3
µL
of
the
sample
solution
was
injected
eluent for the LC under a continuous flow at 0.3 mL/min, and 3 μL of the sample solution was
into the into
LC and
analyzed
by the APCI+
mode. mode.
Reaction
conditions:
(a) Selenide
6 (0.038
mmol)
and
injected
the LC
and analyzed
by the APCI+
Reaction
conditions:
(a) Selenide
6 (0.038
mmol)
H2 OH
mmol)
werewere
mixed
in Hin
(800
µL);μL);
(b) to(b)
a was
H2 O2H(0.15
mmol);
(c): to
b to
was
2 (0.038
2 OH
and
2O2 (0.038
mmol)
mixed
2O (800
to a added
was added
2O2 (0.15
mmol);
(c):
b
added
HCl HCl
(0.15(0.15
mmol).
was
added
mmol).
2.3. GPx-Like
Activity
of Selenides
5‒9strongly
at pH 7.4indicate that the simple redox reaction between selenide
The results
of LC–MS
analysis
and selenoxide (i.e., reaction i and ii in Scheme 1) is predominant during the GPx-like catalytic reaction
The GPx-like catalytic activities of the selenides were evaluated in a buffer solution at pH 7.4 in
under a physiological pH condition (i.e., pH 7.4), while a small portion of the selenoxide, when it has
the nicotinamide adenine dinucleotide phosphate (NADPH)-coupled assay [27,37]. In this assay, the
an amino substitution, could be over-oxidized.
decomposition of H2O2 was monitored by NADPH consumption at 340 nm. The results are
graphically
3A.
initial
2.3. GPx-Likeshown
ActivityinofFigure
Selenides
5–9The
at pH
7.4 velocities (ν0) of H2O2 decomposition catalyzed by
various selenides are summarized in Table 1.
The GPx-like
catalytic activities
selenides
were
evaluated
a buffer i),
solution
at pH
7.4 in
Since
the rate-determining
step of
of the
cycle
A is the
oxidation
stepin(reaction
GPx-like
catalytic
the
nicotinamide
adenine
dinucleotide
phosphate
(NADPH)-coupled
assay
[27,37].
In
this
assay,
the
activity of selenides in water at pH 7.4 should depend on the second-order rate constants (kox) for the
decomposition
of
H
O
was
monitored
by
NADPH
consumption
at
340
nm.
The
results
are
graphically
2 2selenide and H2O2. Following the previous protocol [29], the kox value was
reaction between the
shown
in
Figure
3A.
The1).
initial
velocities
of H2for
O2 selenide
decomposition
by variousdue
selenides
determined for 6 (Table
However,
the (ν
kox0 )value
5 couldcatalyzed
not be determined
to the
are
summarized
in
Table
1.
decomposition as mentioned above. The order of kox and ν0 values obtained for amino selenides were 7
stepwhich
of cycle
the oxidation
step (reaction
GPx-like
catalytic
> 6 > Since
8 and the
5 > 7rate-determining
> 6 > 8, respectively,
wereAiniscomplete
agreement
with eachi),
other.
The order
was
activity
of
selenides
in
water
at
pH
7.4
should
depend
on
the
second-order
rate
constants
(k
)
for
ox
also consonant with the order of the HOMO energy levels calculated in water (Table 1), although
the
reaction
between
the
selenide
and
H
O
.
Following
the
previous
protocol
[29],
the
k
value
was
ox two NH3+
2
diamino selenide 8 had a much higher2 HOMO
level, probably due to the presence of
determined for 6 (Table 1). However, the kox value for selenide 5 could not be determined due to
substituents.
the decomposition as mentioned above. The order of kox and ν0 values obtained for amino selenides
were 7 > 6 > 8 and 5 > 7 > 6 > 8, respectively, which were in complete agreement with each other.
The order was also consonant with the order of the HOMO energy levels calculated in water (Table 1),
Molecules 2017, 22, 354
6 of 13
Molecules 2017,
22, 354 selenide 8 had a much higher HOMO level, probably due to the presence of6 two
of 13
although
diamino
NH3 + substituents.
B
UV absorbance at 340 nm
1.4
1.2
No catalyst
1.0
8 (DASred)
0.8
6
0.6
9 (DHSred)
7 (MASred)
0.4
0.2
5
0.0
0
100
200
300
Conversion of DTTred (%)
A
100
No catalyst
80
9 (DHSred)
5
60
6
40
7 (MASred)
20
6 + 4 eq HCl
8 (DASred)
0
0 10 20 30 40 50 60 70 80 90
400
Reaction time (s)
Reaction time (min)
6 + 10 eq HCl
Figure 3. GPx-like activity assay in buffer solution and in methanol. (A) Nicotinamide adenine
Figure 3. GPx-like activity assay in buffer solution and in methanol. (A) Nicotinamide adenine
dinucleotide phosphate (NADPH)-coupled GPx assay for selenides 5–9. Reaction conditions were
dinucleotide phosphate (NADPH)-coupled GPx assay for selenides 5–9. Reaction conditions were
[GSH]0 = 1.0 mM, [H2 O2 ]0 = 2.5 mM, [NADPH]0 = 0.3 mM, [glutathione reductase] = 4 units/mL, and
[GSH]0 = 1.0 mM, [H2O2]0 = 2.5 mM, [NADPH]0 = 0.3 mM, [glutathione reductase] = 4 units/mL, and
[selenide] = 0.2 mM in pH 7.4 phosphate buffer at 25 ◦ C. (B) Percentages of residual dithiothreitol
[selenide] = 0.2 mM in pH 7.4 phosphate buffer at 25 °C. (B) Percentages of residual dithiothreitol
(DTTred ) as a function of reaction time in the oxidation of DTTred with H2 O2 in the presence of a selenide
(DTTred) as a function of reaction time in the oxidation of DTTred with H2O2 in the presence of a
catalyst (5–9) in CD3 OD. Reaction conditions were [DTTred ]0 = [H2 O2 ]0 = 0.14 M and [selenide] =
selenide catalyst (5–9) in CD3OD. Reaction conditions were [DTTred]0 = [H2O2]0 = 0.14 M and
0.014 M at 25 ◦ C. Data for 7-9 were quoted from Reference [29].
[selenide] = 0.014 M at 25 °C. Data for 7‒9 were quoted from Reference [29].
Table 1. Summary of GPx-like catalytic activities of selenides in water and in MeOH along with
Table 1. Summary of GPx-like catalytic activities of selenides in water and in MeOH along with the
the second-order rate constants for oxidation and the HOMO (highest occupied molecular orbital)
second-order rate constants for oxidation and the HOMO (highest occupied molecular orbital)
energy levels.
energy levels.
Selenides
Selenides
No catalyst
No catalyst
5
5
−1 a
·min−1) a)
0 (µMmin
νν
0 (μM·
32.2 (±3.2)
32.2 (±3.2)
61.3 (±3.4)
61.3 (±3.4)
6
6
52.6 (±2.1)
52.6 (±2.1)
MASred (7) e
59.8 (±1.1)
MASred
(7) e
DASred (8) e
red
DAS (8) e
red (9)e e
DHSred
DHS
(9)
59.8 (±1.1)
47.2 (±4.3)
47.2 (±4.3)
54.2(±4.0)
(±4.0)
54.2
aa
b
t50 (min)
b
t50 (min)
>300
>300
>300(4~10
(4~10equiv.
equiv. HCl)
>300
HCl)24
24 17
5 (+ 17
4 equiv. HCl)
3 (+10 equiv. HCl)
5 (+ 4 equiv. HCl)
14HCl)
3 (+10 equiv.
14
7
7
40 40
−1
(M
ox −1
kox k(M
·s−1) ·cs
‒
−1 ) c
f
f
0.23 (± 0.02)
0.23 (± 0.02)
HOMO
in Water
(eV)d
HOMO
in Water
(eV)
-
‒
–6.39
‒6.39
–6.56 (ax)
‒6.56 (ax)
0.47 (± 0.05)
–6.44
0.47 (± 0.05)
0.14 (±0.02)
0.14 (±0.02)
0.57
(± 0.03)
0.57 (±
0.03)
‒6.44
–5.32
‒5.32
–6.16
‒6.16
d
Substituents
Substituents
-
‒
NH3 + ax
NH3+ax
NH3 + ax
NH3+ax
NH3 + ax
NH
NH33++ax
ax ,
+
NH
ax 3+ax
NH3+ax,3NH
OHax
OHax
OH
ax,, OH
ax
◦ C; b
b Reaction
Initial velocities
buffer
at pH
7.4 7.4
andand
25 25 °C;
Reaction
times
for 50%
Initial
velocities(ν(ν0 )0)ofofHH2 O
2O
reductionininphosphate
phosphate
buffer
at pH
times
for
2 2reduction
red to DTTox in CD OD estimated from Figure 3B; c The second-order rate constants for the
conversion
of DTTof
c The second-order rate
50% conversion
DTTred to DTT3ox in CD3OD estimated
from
Figure
3B;
reaction of selenide + H2 O2 → selenoxide + H2 O in water; d Calculated at B3LYP/6-31+G(d,p)
in water using
constants for the reaction of selenide + H2O2 → selenoxide + H2O in water; d Calculated at
the polarizable continuum model (PCM); e Data were quoted from Reference [29]; f Not determined. MASred :
e Data were quoted from
red : diamino
B3LYP/6-31+G(d,p)
in water
the polarizable
continuum
(PCM);
monoamino
cyclic selenide;
DASusing
cyclic selenide;
DHSred : model
dihydroxy
cyclic selenide
Reference [29]; f Not determined. MASred: monoamino cyclic selenide; DASred: diamino cyclic selenide;
DHSred: dihydroxy
cyclic in
selenide
2.4. GPx-Like
Activity Assay
Methanol
The GPx-like
activities
evaluated by the NMR method as described previously [26,27].
2.4. GPx-Like
Activity
Assay inwere
Methanol
Oxidation of dithiothreitol (DTTred ) was initiated by addition of H2 O2 to a mixture of DTTred and a
The amount
GPx-like(10
activities
were
evaluated
by3 OD.
the NMR
methodwas
as described
[26,27].
catalytic
mol %) of
a selenide
in CD
The reaction
carried outpreviously
in a NMR sample
red) was initiated by addition of H2O2 to a mixture of DTTred and a
1
Oxidation
of
dithiothreitol
(DTT
tube at 298 K. The H-NMR spectrum of the solution was measured after a certain period of time.
ox (δ
catalytic
amount
mol %)signal
of a selenide
in CD
3OD.red
The
wasand
carried
in=a 3.03,
NMR2H)
sample
The
change
of the(10
relative
intensities
of DTT
(δ reaction
= 3.67, 2H)
DTTout
was
1
red to
tube at 298to
K.calculate
The H-NMR
spectrum
of the solution
measured
after a 3B).
certain
of time.
integrated
the percent
conversion
of DTTwas
DTTox (Figure
Theperiod
times for
50%
red (δ = 3.67, 2H) and DTTox (δ = 3.03, 2H) was
The
change
of
the
relative
signal
intensities
of
DTT
conversion (t50 ) obtained are summarized in Table 1. All selenides catalyzed the oxidation of DTTred
integrated to calculate the percent conversion of DTTred to DTTox (Figure 3B). The times for 50%
conversion (t50) obtained are summarized in Table 1. All selenides catalyzed the oxidation of DTTred
with H2O2. The order of the catalytic activity (i.e., the inverse of t50) was 8 > 7 > 6 > 5 > 9, which did not
match with the order in water (i.e., the magnitude of ν0), 5 > 7 > 9 > 6 > 8. In addition, it is obvious
Molecules 2017, 22, 354
7 of 13
with H2 O2 . The order of the catalytic activity (i.e., the inverse of t50 ) was 8 > 7 > 6 > 5 > 9, which did
not match with the order in water (i.e., the magnitude of ν0 ), 5 > 7 > 9 > 6 > 8. In addition, it is obvious
that the amino groups on the rings had a proclivity to enhance the activity in methanol. Similar results
had been observed in our previous study [29]. These observations suggest that the reaction should be
catalyzed through not only cycle A, but also through another cycle, which would involve hydroxy
perhydroxy selenane 4 (cycle B). This assumption was indeed supported by the following analyses.
2.5. Components of the Reaction Solution in Methanol
To obtain information about a shift of the catalytic cycle in methanol, the components of the
reaction mixture were analyzed by means of 77 Se-NMR and LC–MS (APCI+ or APCI−) spectroscopies.
The results of NMR analysis obtained for selenide 7 are shown in Figure S9. 77 Se-NMR spectra in
CD3 OD showed that 7 (δ = 148 ppm, Figure S9a) was oxidized by 1 equivalent of H2 O2 to the selenoxide
(δ = 967 ppm (major) and 949 ppm (minor), Figure S9b). The two peaks can be assigned to cis- and
trans-stereoisomers, respectively, because ab initio calculation showed that the cis-isomer, which
forms an intramolecular hydrogen bond, is 6.24 kcal/mol more stable than the trans-isomer. When 1
equivalent of DTTred was added, the selenoxide was rapidly reduced to selenide 7. On the other hand,
when 4 equivalents H2 O2 were added to the selenoxide, the major isomer (δ = 968 ppm) preferentially
converted into other species (Figure S9c). This is in significant contrast to the reaction in water, where
the selenoxide was unreacted in the absence of HCl. The selenoxide was completely converted into
other species, for which no signal was detected over the range of 0–1500 ppm, by further addition of
4 equivalents HCl (Figure S9d). When 5 equivalents of DTTred were added to the resulting sample
solution, the selenide was regenerated, suggesting that the selenoxide did not decompose but was
converted into active species. In addition, when the solution of the selenoxide (Figure S9b) was added
with 4 equivalent of HCl, the selenoxide completely reacted, probably to produce hydroxyselenonium
3 or dihydroxy selenane 3a, as observed for 6 in aqueous solution (Figure S9e). By neutralization
with NaOH, the selenoxide was recovered (Figure S9f). On the other hand, the LC–MS (APCI+)
analysis showed that by reacting selenide 7 (Figure 4a) with 1 equivalent of H2 O2 , generation of
a considerable amount of dihydroxy selenane 3a (m/z 186.0; C4 H12 O2 80 Se+ ) was observed along
with the corresponding selenoxide (m/z 168.0; C4 H10 NO80 Se+ ) (Figure 4b), suggesting that 3a was in
equilibrium with selenoxide 2 in methanol although HCl was absent. When four equivalents of H2 O2
were further added to the resulting solution, small signals were observed at around m/z 202.0, which
corresponded to species 4 (C4 H12 NO3 80 Se+ ) (Figure S10). Similar redox behaviors were also observed
for selenide 6, although the generation of the corresponding dihydroxy selenane 3a was less than
that of selenide 7 when 6 was reacted with excess H2 O2 (Figure S11). On the other hand, oxidation
of selenide 5 by one equivalent of H2 O2 in methanol provided complicated MS spectra, showing the
formation of not only selenoxide and species 3a but also several decomposed species. For 9, NMR
analysis showed that conversion of the selenoxide to an active species was progressed when excess
H2 O2 and HCl were both added (Figure S12).
The NMR and LC–MS analyses suggested that in methanol amino-substituted selenides could
be over-oxidized to 4 by excess amounts of H2 O2 even in the absence of HCl. It was, therefore,
assumed that in addition to cycle A of Scheme 1, cycle B would work in methanol for monoamino
selenides, except for 5. It is known that dihydroxy selenane 3a acts as a good oxidant for nucleophilic
substrates through the exchange of the hydroxy ligands, as shown in Scheme 3 [38–41]. Dihydroxy
tellurane, which is a tellurium analog of 3a, can also oxidize thiols to disulfides through similar ligand
exchange [42]. Thus, the 3a could be involved in the catalytic reaction as an active intermediate via the
cycle other than cycles A and B.
the generation of the corresponding dihydroxy selenane 3a was less than that of selenide 7 when 6
was reacted with excess H2O2 (Figure S11). On the other hand, oxidation of selenide 5 by one
equivalent of H2O2 in methanol provided complicated MS spectra, showing the formation of not
only selenoxide and species 3a but also several decomposed species. For 9, NMR analysis showed
that conversion of the selenoxide to an active species was progressed when excess H2O2 and HCl
Molecules 2017, 22, 354
8 of 13
were both added (Figure S12).
135.0
Molecules 2017, 22, 354
Molecules 2017, 22, 354
152.0
Intensity
a
7
8 of 13
8 of 13
Figure 4. LC–MS (APCI+) spectral changes during the redox reactions of 7 in MeOH at 25 °C. MeOH
Figure 4. LC–MS (APCI+) spectral changes during the redox reactions of 7 in MeOH
at 25 °C. MeOH
m/z
(100%) was used as an eluent
conditions:
(a)
160
100 for the
140
220 mmol) in MeOH (800
180 selenide
200 7 (0.038
120LC. Reaction
(100%) was used as an eluent
for the
LC. Reaction
conditions:
(a) selenide
7 (0.038
mmol) in MeOH (800
μL); (b) to a was added H2O2 (0.038 mmol).
eq H2O2, 40 min 168.0
μL); (b) to a was added H2O2 (0.038 1
mmol).
b
Intensity
The NMR and LC–MS analyses suggested that in methanol amino-substituted selenides could
The NMR and LC–MS analyses suggested
that in methanol amino-substituted selenides could
135.0
be over-oxidized to 4 by excess amounts
of H2152.02
O2 even in the absence of HCl. It was, therefore,
be over-oxidized to 4 by excess amounts of H2O2 even in the 186.0
absence of HCl. It was, therefore,
assumed that in addition to cycle A of Scheme 1, cycle B would work in methanol for monoamino
assumed that in addition to cycle A of Scheme 1, cycle B would work in methanol for monoamino
selenides, except for 5. It is known that dihydroxy selenane 3a acts as a good oxidant for nucleophilic
selenides, except for 5. It is known that dihydroxy selenane 3a acts as a goodm/z
oxidant for nucleophilic
160
100
140
180shown
220
200in Scheme
substrates through the exchange
of 120
the hydroxy
ligands,
as
3 [38–41]. Dihydroxy
substrates through the exchange of the hydroxy ligands, as shown in Scheme 3 [38–41]. Dihydroxy
tellurane, which is a tellurium analog of 3a, can also oxidize thiols to disulfides through
similar
◦ C. MeOH
tellurane,
is a(APCI+)
tellurium
analog
of 3a,
can the
alsoredox
oxidize
thiols
similar
Figurewhich
4. LC–MS
spectral
changes
during
reactions
of 7toindisulfides
MeOH at 25through
ligand exchange [42]. Thus, the 3a could be involved in the catalytic reaction as an active
ligand
exchange
[42].
Thus,
be involved
catalytic
reaction
asMeOH
an active
(100%)
was used
as an
eluentthe
for 3a
the could
LC. Reaction
conditions:in
(a)the
selenide
7 (0.038
mmol) in
intermediate via the cycle other than cycles A and B.
intermediate
viatothe
cycle
other
cycles
A and B.
(800 µL); (b)
a was
added
H2than
O2 (0.038
mmol).
Scheme
of a nucleophile (NuH) by dihydroxyselenane 3a.
Scheme 3.
3. Oxidation
Oxidation of a nucleophile (NuH) by dihydroxyselenane 3a.
3a.
2.6. Determinant Factors of the GPx-Like Activity of Selenides
2.6. Determinant Factors of the GPx-Like Activity of Selenides
According to component analysis during the redox reaction in methanol, it appeared that in
According to component analysis
According
analysis during
during the redox reaction in methanol, it appeared that in
methanol convertibility of selenoxides to 3, 3a, and 4 would correlate to the GPx-like activity. Indeed,
methanol convertibility of selenoxides
selenoxides to
to 3, 3a, and 4 would correlate to the GPx-like activity. Indeed,
the catalytic reaction of selenide 6 in methanol was dramatically accelerated when 4 or 10 equivalents
the catalytic reaction of selenide 6 in methanol was dramatically accelerated when 4 or 10 equivalents
of HCl were added to the reaction solution, probably due to the enhancement of convertibility of 6 into
of HCl
thethe
reaction
solution,
probably
due todue
the to
enhancement
of convertibility
of 6 into
HClwere
wereadded
addedtoto
reaction
solution,
probably
the enhancement
of convertibility
such active species (Figure 3B). It should be noted that the reaction was not accelerated when HCl was
such
active
species
(Figure
3B).(Figure
It should
beItnoted
thatbethe
reaction
accelerated
when
HCl was
of
6 into
such
active
species
3B).
should
noted
thatwas
the not
reaction
was not
accelerated
added to the blank solution in the absence of a selenide catalyst. For the series of
added HCl
to was
the added
blank tosolution
in solution
the absence
of a selenide
catalyst.
For the
series
of
when
the blank
in the absence
of a selenide
catalyst.
For the
series
monoamino-substituted selenides, it was suggested that over-oxidation of the selenoxide to 4 can be
monoamino-substituted
selenides,
it was
suggested
that over-oxidation
of the of
selenoxide
to 4 cantobe
of
monoamino-substituted
selenides,
it was
suggested
that over-oxidation
the selenoxide
4
progressed by an excess amount of H2O2 even in the absence of a proton source (Figures 4, S9 and S11).
progressed
by an excess
H2O2 even
the
absence
of
a
proton
source
(Figures
4,
S9
and
S11).
can
be progressed
by anamount
excess of
amount
of Hin
O
even
in
the
absence
of
a
proton
source
(Figure
4,
2 2
This is probably because the cis-isomer of the selenoxide having a protonated ammonium (-NH3++)
This is probably
because
cis-isomer
of the the
selenoxide
having
protonatedhaving
ammonium
(-NH3 )
Figures
S9 and S11).
This the
is probably
because
cis-isomer
of thea selenoxide
a protonated
group in an axial direction
is in equilibrium with hydroxyselenonium 3 as shown in Scheme 4. Such
+
group in an (-NH
axial3direction
is in
with
hydroxyselenonium
3 as shown in Scheme
Such
ammonium
) group in
an equilibrium
axial direction
is in
equilibrium with hydroxyselenonium
3 as 4.
shown
equilibrium, however, cannot occur for the selenoxide of hydroxy-substituted selenide 9. A
equilibrium,
cannot occur
for cannot
the selenoxide
selenide 9. A
in
Scheme 4. however,
Such equilibrium,
however,
occur for of
thehydroxy-substituted
selenoxide of hydroxy-substituted
preferential conversion of the cis-isomer as observed in Figures S1 and S9 would support the
preferential
the cis-isomer
as observed
in Figures
S1 andS1S9and
would
support
the
selenide
9. A conversion
preferential of
conversion
of the cis-isomer
as observed
in Figures
S9 would
support
presence of the equilibrium of Scheme 4.
presence
of the
equilibrium
of Scheme
4. 4.
the
presence
of the
equilibrium
of Scheme
Scheme 4. A proposed equilibrium between an amino-substituted cyclic selenoxide and the corresponding
Scheme 4. A proposed equilibrium between an amino-substituted cyclic selenoxide and the
Scheme 4. A proposed
equilibrium between an amino-substituted cyclic selenoxide and the
hydroxyselenonium
3.
corresponding hydroxyselenonium 3.
corresponding hydroxyselenonium 3.
In aqueous medium, on the other hand, generation of species 3 was not obviously observed for
In aqueous medium, on the other hand, generation of species 3 was not obviously observed for
any selenides in the absence of HCl. This is probably because hydration around the selenoxides
any selenides in the absence of HCl. This is probably because hydration around the selenoxides
inhibits the proton transfer from NH3++ to oxygen atom to form 3, which might further react with
inhibits the proton transfer from NH3 to oxygen atom to form 3, which might further react with
H2O2 to form 4. Indeed, distances of the intramolecular hydrogen bonds between NH3++ and the
H2O2 to form 4. Indeed, distances of the intramolecular hydrogen bonds between NH3 and the
selenoxide moiety calculated for selenides 6 and 7 were ca. 0.01 Å longer in water than those in
selenoxide moiety calculated for selenides 6 and 7 were ca. 0.01 Å longer in water than those in
methanol, supporting more difficult conversion of the selenoxide to 3 in water than in methanol.
Molecules 2017, 22, 354
9 of 13
In aqueous medium, on the other hand, generation of species 3 was not obviously observed for
any selenides in the absence of HCl. This is probably because hydration around the selenoxides inhibits
the proton transfer from NH3 + to oxygen atom to form 3, which might further react with H2 O2 to form
4. Indeed, distances of the intramolecular hydrogen bonds between NH3 + and the selenoxide moiety
calculated for selenides 6 and 7 were ca. 0.01 Å longer in water than those in methanol, supporting
more difficult conversion of the selenoxide to 3 in water than in methanol. Thus, the strength of the
hydrogen bonding to the selenoxide moiety may be one of the factors to determine the convertibility
of cyclic selenoxides into other active species, such as 3, 3a and 4, and hence enhance the GPx-like
activity in methanol. It should be noted though that the calculated hydrogen bond distance of the
selenoxide of 7 was slightly longer than that of the selenoxide of 6, the catalytic activity of 7 was larger
than that of 6.
3. Material and Methods
3.1. General
1H
(500 MHz), 13 C (125.8 MHz), and 77 Se (95.4 MHz) NMR spectra were recorded at 298 K, and
coupling constants (J) are reported in hertz. High-resolution mass spectra (HRMS) and low-resolution
mass spectra (MS) were recorded under atmospheric-pressure chemical ionization (APCI+ or APCI−)
or electrospray ionization (ESI+) conditions. All reactions for the synthesis of selenides were monitored
by thin-layer chromatography (TLC). Gel permeation chromatography (GPC) was performed with a
general isocratic HPLC system using CHCl3 as the eluent. Ultraviolet (UV) spectra were measured
at 25.0 ◦ C using a circulating water-bath system. Selenides 7 [33], 8 [29], and 9 [43] were prepared as
described. All other chemicals were used as purchased without further purification. NADPH-coupled
GPx activity assay in buffer solution at pH 7.4, GPx-like activity assay by NMR in CD3 OD, and kinetic
analysis for selenide oxidation with hydrogen peroxide in water were performed by following our
previous literatures [26,27,29].
3-(tert-Butoxycarbonylamino)selenetane (13a). Selenium powder (0.289 g, 3.66 mmol) and sodium
borohydride (0.383 g, 9.11 mmol) were placed in a two-necked round-bottomed flask. After replacement
of air with argon gas in the flask, anhydrous isopropyl alcohol (25 mL) was added. The mixture was
stirred and heated under reflux conditions for 1 h under argon atmosphere to generate sodium
hydrogen selenide (NaHSe) in situ. A solution of 12a (0.482 g, 1.28 mmol) in 1,4-dioxane (20 mL) was
added to the resulting colorless solution. After further stirring under reflux conditions for 3 h, the
reaction solution was cooled to room temperature and evaporated. To the residual material were
added water (20 mL) and CH2 Cl2 (20 mL), and extraction was conducted with CH2 Cl2 (20 mL × 3).
The combined organic layers were washed with brine (40 mL), dried over MgSO4 , and concentrated
under vacuum. The residual yellow solid was purified by silica gel column chromatography
(EtOAc/n-hexane 1:4) and then by GPC to give a white solid of 13a. Yield: 0.149 g, 49%; M.p.
120–122 ◦ C; 1 H-NMR (500 MHz, CDCl3 ): δ = 1.37 (s, 9H), 1.56 (s, 1H), 3.02–3.17 (m, 2H), 3.18–3.32 (m,
2H), 4.93 ppm (br s, 1H); 13 C-NMR (125.8 MHz, CDCl3 ): δ= 24.8, 28.3, 49.5, 79.9, 153.8 ppm; 77 Se-NMR
(CDCl3 ): δ = 94.3 ppm; MS (APCI+): m/z calcd for C8 H16 NO2 Se+ : 238.03 [M + H]+ ; found: 238.04.
(S)-3-(tert-Butoxycarbonylamino)tetrahydroselenopyran (13b). A similar protocol to the synthesis of 13a
was applied, though EtOH was used as a solvent for generation of NaHSe and THF was used as a
solvent to dissolve the starting material. Starting with 12b (0.489 g, 1.30 mmol), 13b was obtained
as a white solid. Yield: 0. 235 g, 68%; M.p. 93–94 ◦ C; 1 H-NMR (500 MHz, CDCl3 ): δ = 1.38 (s, 9H),
1.42–1.49 (m, 1H), 1.66–1.70 (m, 1H), 1.83–1.90 (m, 1H), 2.36–2.58 (m, 3 H), 2.79–2.89 (m, 1H), 3.80
(m, 1H), 4.97 ppm (br s, 1H); 13 C-NMR (125.8 MHz, CDCl3 ): δ = 19.1, 24.7, 26.0, 28.4, 32.7, 46.4, 49.4,
154.8 ppm; 77 Se-NMR (CDCl3 ): δ = 115.8 ppm; MS (APCI+): m/z calcd for C10 H20 NO2 Se+ : 266.07
[M + H]+ ; found: 266.08.
Molecules 2017, 22, 354
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3-Aminoselenetane Hydrochloride (5). HCl (4 mL, 5 M) was added to a solution of 13a (0.14 g, 0.59 mmol)
in Et2 O (4 mL). The mixture was vigorously stirred for 18 h at 35 ◦ C. After removal of the ether by
evaporation, the resulting aqueous layer was diluted with water (50 mL) and lyophilized to give a
white solid of 2 (99 mg, 97%). M.p. 172 ◦ C (decomp); 1 H-NMR (D2 O): δ = 2.96–3.07 (m, 2H), 3.34–3.45
(m, 2H), 4.56–4.68 ppm (m, 1H); 13 C-NMR (D2 O) δ = 17.5, 48.6 ppm; 77 Se-NMR (D2 O): δ = 144.5 ppm;
HRMS (APCI+): m/z calcd for C3 H8 NSe+ : 137.9816 [M − Cl]+ ; found:137.9802.
(S)-3-Aminotetrahydroselenopyran Hydrochloride (6). A similar protocol to the synthesis of 5 was applied.
Using 13b (0.23 g, 0.89 mmol) as a starting material, 6 was obtained as a white solid. Yield: 0.16 g,
90%; M.p. 192 ◦ C (decomp); 1 H-NMR (D2 O): δ = 1.41–1.51 (m, 1H), 1.79–1.89 (m, 1H), 1.91–1.95 (m,
1H), 2.20–2.26 (m, 1H), 2.46–2.61 (m, 2H), 2.63–2.76 (m, 2H), 3.42–3.54 ppm (m, 1H); 13 C-NMR (D2 O):
δ = 18.7, 19.9, 26.5, 30.5 49.4 ppm; 77 Se-NMR (D2 O): δ = 136.2 ppm; HRMS (APCI+): m/z calcd for
C5 H12 NSe+ : 166.0129 [M − Cl]+ ; found: 166.0130.
3.2. Quantum Chemical Calculation
The Gaussian 09 software package (revision B.01) [44] was employed. The structures were
optimized in water and in methanol at the B3LYP/6-31+G(d,p) level, using the polarizable
continuum model (PCM). For the cyclic selenides 6 and 7, all possible configurations were
tested, and the global-energy-minimum structure was thus determined. Frequency calculations
were performed for all the obtained structures to confirm that the structures had no imaginary
frequencies. The energy-minimum structures were used for analyzing the HOMO energy levels and
the intramolecular hydrogen-bond distances.
4. Conclusions
Here, we analyzed in-depth the GPx-like catalytic cycle of water-soluble amino-substituted cyclic
selenides in water and in methanol on the basis of the component analysis of the reaction solution by
means of 77 Se-NMR and LC–MS spectroscopies. Under an aqueous condition at a physiological pH, it
was revealed that two components (i.e., the selenide and the corresponding selenoxide), can mainly
contribute to the antioxidative function though a slight contribution from the dihydroxy selenane
3a was also suggested. Thus, cycle A in Scheme 1 should be a major cycle in water as reported
in the literature [26–29]. In methanol, however, other active species, such as hydroxyselenonium 3
and hydroxy perhydroxy selenane 4 would be generated by over-reaction or over-oxidation of the
corresponding selenoxide by an excess amount of H2 O2 . These species (i.e., 3 and 4), should work as
active oxidants against thiol substrates in consort with selenoxide 2 and 3a if the velocities of reactions
iii and/or vi in Scheme 1 are comparable or faster than that of reaction ii. This situation would be
more feasible for amino-substituted cyclic selenides probably because the NH3 + group would transfer
a proton to the selenoxide moiety to give hydroxyselenonium 3 in the absence of an additional proton
source. Thus, a shift of the major catalytic cycle in methanol would make the GPx-like antioxidative
function of selenides perplexing. The information obtained here will provide a valuable clue for design
of novel selenide-based antioxidative enzyme mimics.
Supplementary Materials: Supplementary materials (Synthesis of mesylates 12a and 12b; 1 H, 13 C, and 77 Se-NMR
spectra of 5, 6, 13a and 13b; Figures S1-S12; Quantum chemical calculations of the selenoxides corresponding 6
and 7) are available online.
Acknowledgments: This work was supported by KAKENHI Grant Number 26888016 for Research Activity
Start-up (to K.A.), in part by Research and Study Project of Tokai University, Educational System General Research
Organization (to K.A. and M.I.). A part of analysis of NMR and LC-MS was supported by Technology Joint
Management Office of Tokai University. We also gratefully acknowledge Haruhito Ueno (Tokai University) for
technical support.
Author Contributions: K.A. conceived, designed, and performed the overall of experiments and wrote the main
manuscript text; A.T. synthesized amino-substituted selenides and performed the GPx-like activity assay of
selenides and NMR and LC-MS analyses. Y.O. performed GPx-like activity assay. M.I. performed the quantum
chemical calculations and supervised this work.
Molecules 2017, 22, 354
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Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the
decision to publish the results.
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© 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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