Synthesis and structure - activity correlation studies of secondary- and tertiary-amine-based glutathione peroxidase mimics

Article abstract of DOI:10.1002/chem.200900818

In this study, a series of secondary- and tertiary-amino-substituted diaryl diselenides were synthesized and studied for their glutathione peroxidase (GPx) like antioxidant activities with H₂O₂, cumene hydroperoxide, or tBuOOH as sub

Full text of DOI:10.1002/chem.200900818

DOI: 10.1002/chem.200900818
Synthesis and Structure–Activity Correlation Studies of Secondary- and
Tertiary-Amine-Based Glutathione Peroxidase Mimics
Krishna P. Bhabak and Govindasamy Mugesh*[a]
Abstract: In this study, a series of sec-
ondary- and tertiary-amino-substituted
diaryl diselenides were synthesized and
studied for their glutathione peroxidase
(GPx) like antioxidant activities with
H2O2, cumene hydroperoxide, or
tBuOOH as substrates and with PhSH
or glutathione (GSH) as thiol cosub-
strates. This study reveals that replace-
ment of the tert-amino groups in ben-
zylamine-based diselenides by sec-
amino moieties drastically enhances
the catalytic activities in both the aro-
matic thiol (PhSH) and GSH assay sys-
tems. Particularly, the N-propyl- and
N-isopropylamino-substituted
nides are 8–18 times more active than
the corresponding N,N-dipropyl- and
N,N-diisopropylamine-based
pounds in all three peroxide systems
when GSH is used as the thiol cosub-
strate. Although the catalytic mecha-
nism of sec-amino-substituted disele-
nides is similar to that of the tert-
amine-based compounds, differences in
disele-
the stability and reactivity of some of
the key intermediates account for the
differences in the GPx-like activities. It
is observed that the sec-amino groups
are better than the tert-amino moieties
for generating the catalytically active
selenols. This is due to the absence of
any significant thiol-exchange reactions
in the selenenyl sulfides derived from
sec-amine-based diselenides. Further-
more, the seleninic acids (RSeO2H) de-
rived from the sec-amine-based com-
pounds are more stable toward further
reactions with peroxides than their tert-
amine-based analogues.
com-
Keywords: amines
activity enzyme mimics
oxidases · selenium · thiol exchange
·
antioxidant
·
·
per-
AHCTUNGTRENNUNG
Introduction
GPx. Therefore, rapid reactions of the selenenic acid (E-
SeOH) with GSH and of the resulting selenenyl sulfide (E-
Se-SG) with a second GSH molecule to produce the selenol
are crucial for good catalytic activity.
As selenium redox chemistry at the active site of GPx
plays an important role in oxidative damage,[2] several
groups have worked toward the design and synthesis of low-
molecular-weight organoselenium compounds that function-
ally mimic GPx activity in the presence of thiols.[3] These
mimics include the well-known anti-inflammatory com-
pound ebselen (1)[4] and its analogues,[5,6] camphor-based se-
lenenyl amide 2,[7] tert-amino-substituted diselenides 3–7,[8]
Glutathione peroxidase (GPx) is a mammalian selenoen-
zyme, which catalyzes the reduction of harmful peroxides by
using glutathione (GSH) as a cofactor.[1] The catalytic cycle
involves the reaction of peroxides with the selenol form of
the enzyme (E-SeH) to produce the corresponding selenenic
acid (E-SeOH), which upon reaction with GSH generates
the enzyme-bound selenenyl sulfide (E-Se-SG). The nucleo-
À
philic attack of a second GSH molecule at the Se S bond
regenerates the active-site selenol, with release of the cofac-
tor in its oxidized form (GSSG; Figure 1).[1e–g] At high con-
centrations of peroxide, the selenenic acid generated in the
reaction may undergo further oxidation to produce the cor-
responding seleninic acid (E-SeO2H) or selenonic acid (E-
SeO3H); these reactions reduce the catalytic efficiency of
[a] K. P. Bhabak, Prof. G. Mugesh
Department of Inorganic and Physical Chemistry
Indian Institute of Science, Bangalore 560 012 (India)
Fax : (+91)80-2360-1552/0683
E-mail: mugesh@ipc.iisc.ernet.in
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200900818.
Figure 1. Proposed catalytic mechanism for the reduction of hydroperox-
ides by GPx.
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Chem. Eur. J. 2009, 15, 9846 – 9854

FULL PAPER
diselenides with Se···O interactions (such as 8 and 9),[9] aza-
selenonium chloride 10,[10] allyl and alkyl selenides 11 and
12,[11] and cyclic selenelate ester 13 (Scheme 1).[12] Particular-
4 and 5.[8g] On the other hand, replacement of the N,N-dial-
kylamino side chain in compounds 3–5 by secondary- or ter-
tiary-amide moieties significantly reduces the catalytic acti-
vity.[6e] It has been shown that compounds 17–19 with sec-
amido substituents and compounds 20–22 with tert-amido
groups are 20–30 and 3–4 times, respectively, less active than
the corresponding tert-amine-based compounds 3–5.[6e,8g]
However, the tert-amido-substituted diselenides 20–22 were
almost 6–7 times more active than the corresponding sec-
amide-based compounds 17–19.[6e] The lower antioxidant ac-
tivity of the amide-based compounds (17–22) as compared
to that of the tert-amino-substituted diselenides (3–5) can be
ascribed to the different catalytic mechanisms for these two
classes of compounds.[6e,8g] Although a structure–activity cor-
relation has been clearly established for the sec- and tert-
amide-based compounds, there is no such comparison avail-
able for the amine-based compounds. In this paper, we
report the synthesis, characterization, and GPx-like activity
of a series of sec-amino-substituted diselenides. We also
show, for the first time, that the replacement of tert-amino
groups in N,N-dialkylbenzylamine-based diselenides by sec-
amino moieties significantly enhances the GPx-like activity.
Results and Discussion
Synthesis of diselenides 23–27: The tert-amine-based disele-
nides 3–5 and 23 employed in this study were synthesized by
following the ortho-lithiation methodology[13] by starting
from N,N-dimethylbenzylamine (for compound 3) or 2-
bromo-N,N-dialkylbenzylamines (for compounds 4, 5, and
23). Lithiation of the substituted benzylamines with nBuLi
Scheme 1. Some representative examples of small-molecule GPx mimics.
ly, diselenides such as 3–7 with basic tert-amino groups have
attracted considerable attention because the amino groups
play some key roles in the catalytic cycle. Iwaoka and
Tomoda have shown that tert-amino substituents in close
proximity to selenium atoms abstract the protons from sele-
nols to produce more reactive selenolates during the catalyt-
ic cycle.[8b] These amino groups may also help in reductive
À
cleavage of Se Se bond by thiols to produce the selenenyl
sulfide intermediates. Although most of the diselenides
exert their peroxidase activity via selenol, selenenic acid,
and selenenyl sulfide intermediates, as shown for the natural
enzyme (Figure 1), the differences in the catalytic activities
of the diselenides are due to the varying degrees of thiol-ex-
change reactions that take place in the selenenyl sulfide in-
termediates. In selenenyl sulfides with tert-amino substitu-
ents, the nucleophilic attack of the thiol takes place prefer-
entially at the selenium rather than the sulfur atom and this
reduces the formation of selenol by terminating the forward
reaction.[8]
Recently, we have shown that simple replacement of hy-
drogen atoms by methoxy substituents at the 6-position in
N,N-dialkylbenzylamine-based diselenides 3–5 can lead to
an increase in the catalytic activity.[8g] For example, the cata-
lytic activity of the methoxy-substituted compound 14 has
been shown to be almost one order of magnitude higher
than that of 3. Similarly, compounds 15 and 16 have also
been shown to be much better catalysts than the diselenides
in diethyl ether afforded the corresponding ortho-lithiated
compounds. Subsequent treatment with selenium powder
and oxidative work up afforded the diselenides in moderate
yields. The sec-amine-based diselenides 24–27, on the other
hand, could not be prepared by this conventional ortho-lith-
iation route, and our attempts to synthesize compounds 24–
27 from N-alkylbenzylamines or 2-bromo-N-alkylbenzyl-
AHCTUNGTREGaNNUN mines by treatment with nBuLi were unsuccessful. Al-
though there are numerous examples of diaryl diselenides
with tert-amino groups,[8] diselenides containing sec-amino
moieties are extremely rare. Wirth and co-workers have
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9847

G. Mugesh and K. P. Bhabak
shown that diselenide 28 with a monomethylated amino
moiety can be synthesized through the ortho-lithiation route
by using tBuLi as the lithiating agent.[14] Singh and co-work-
ers reported the synthesis of diselenide 29 by reduction of
the corresponding azomethine diselenide, which was pre-
pared from bis(o-formylphenyl)diselenide.[15]
It is known that the reactions of 2-(chloroseleno)benzoyl
chloride (30) with various amines produce ebselen ana-
logues in good yield,[6b] and this methodology can be used to
synthesize diselenides having sec-amido moieties. For exam-
ple, the diaryl diselenides 17–19 can be synthesized from 30
via the corresponding selenenyl amides 31–33, as shown in
Scheme 2.[6e] Based on this methodology, we have attempted
A comparison of the 77Se NMR spectra of 3–5 and 23 with
those of 24–27 indicates that the chemical shifts of dialkylat-
ed compounds 3–5 are shifted slightly downfield relative to
those of the N-monosubstituted derivatives 24–26. In con-
trast, an upfield shift was observed for compound 23 (d=
406 ppm) relative to the chemical shift for compound 27
(d=414 ppm), which indicates that the introduction of the
sterically bulky N,N-diisopropyl substituent significantly re-
duces the Se···N interactions.[17] This is further confirmed
from the single-crystal X-ray structure of 23, which shows
that the average Se···N distance (2.946 ꢁ) in this compound
is considerably higher than that of compounds 3 (2.859 ꢁ)[18]
and 5 (2.885 ꢁ; Figure 2).
Scheme 2. Synthesis of the sec-amide-based diselenides 17–19 from the
corresponding amide-based cyclic compounds 31–33 and the attempted
synthesis of compounds 35–38.
the synthesis of compounds 24–27 by treating 2-(bromosele-
no)benzyl bromide (34) with the appropriate primary
amines. In contrast to the cyclization of 30, the reaction of
À
34 with alkyl amines did not yield the expected cyclic Se N
derivatives 35–38. Although 77Se NMR experiments indicat-
ed a reaction between the SeBr moiety and the amine, the
reactions generated several selenium compounds that could
not be isolated. However, the sec-amino-substituted disele-
nides 24–27 could be conveniently synthesized by addition
of 2,2-diseleno-bis(bromomethylbenzene) (40),[16a] obtained
from the hydroxymethyl analogue 39,[16b] to the correspond-
ing alkyl amines (Scheme 3). In this reaction, the use of
Figure 2. X-ray crystal structures of compounds 5 and 23. ORTEP dia-
grams with the ellipsoids representing 50% probability. The hydrogen
atoms are omitted for clarity. Significant bond lengths (r in ꢁ) and angles
(q in 8): 5: rSe1···N1 =2.782, rSe2···N2 =2.988, rSe1ÀSe2 =2.348; qN1···Se1ÀSe2 =174.2,
qN2···Se2ÀSe1 =173.3; 23:
r
Se1···N1 =2.943,
r
Se2···N2 =2.950,
rSe1ÀSe2 =2.346;
qN1···Se1ÀSe2 =174.7, qN2···Se2ÀSe1 =171.5.
GPx-like activity: The GPx-like catalytic activity of com-
pounds 24–27 was studied by using PhSH and GSH as thiol
cofactors and H2O2, cumene hydroperoxide (Cum-OOH),
and tert-butyl hydroperoxide (tBuOOH) as substrates, and
the activities were compared to those of the corresponding
N,N-dialkyl derivatives 3–5 and 23. In the PhSH assay, the
formation of PhSSPh was followed by a reversed-phase
HPLC method and the time required for 50% conversion of
PhSH into PhSSPh (t1/2) was calculated by determining the
peak areas at different time intervals.[6a,b,7,8g] A calibration
plot, obtained from known concentrations of PhSSPh, was
used to calculate the amount of PhSSPh formed during the
course of the reactions. To understand the catalytic behavior
of the selenium compounds, t1/2 values obtained by the re-
duction of peroxides by PhSH in the absence of selenium
compounds were used as control values (Table 1).
Scheme 3. Synthetic route to compounds 24–27. a) HBr/AcOH, reflux,
3 h; b) RNH2, tetrahydrofuran (THF), RT, 12 h.
large excesses of the primary amines (ꢀ10 equiv) signifi-
cantly increased the yields of the products. This indicates
that the use of an excess amount of alkyl amine may prevent
the possible N,N-disubstitution reactions that lead to the for-
mation of oligomeric products.
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Glutathione Peroxidase Mimics
FULL PAPER
Table 1. Reduction of peroxides by PhSH in the absence of and the pres-
ence of compounds 3–5 and 23–27.
tivities of both types of compound depend on the nature of
the peroxide. In this regard, the effect of different peroxide
substrates on the catalytic activities of the amine-based com-
pounds is similar to that of tert-amide-based compounds 20–
22, but it is quite different from that of sec-amide-based di-
Compound
t1/2 values [min][a]
Relative activity[b]
H2O2 tBu-
Cum-
OOH
H2O2 tBu-
Cum-
OOH
OOH
OOH
Control
3
4
5
23
24
25
26
27
2905 1130
798
49
63
116
81
17
24
27
40
1.0
43.3 15.7
31.9 11.3
9.9
10.7
78.5 31.4
44.0 19.5
39.2 14.1
24.6 11.3
1.0
1.0
16.3
12.7
6.9
AHCTUNGTREGsNNNU elenides 17–19 and the corresponding cyclic selenenyl
67
91
292
272
37
72
100
206
197
36
amides 31–33.[6b,e]
The catalytic reduction of peroxides by the amino-substi-
tuted diselenides was also studied by using GSH as the co-
substrate. The initial rate (v0) for the reaction in the pres-
ence and absence of selenium compounds were determined
by following the oxidation of reduced nicotinamide adenine
dinucleotide (NADPH) at 340 nm by using a GSH–GSSG
coupled assay in phosphate buffer.[3a,6a,b,8a] In this assay, the
reactions of diselenides with GSH are expected to produce
the corresponding catalytically active selenols. Interestingly,
the sec-amine-based diselenides 24–27 exhibited much
higher activities than their tert-amine-based analogues 3–5
and 23 (Table 2). Particularly, the nPr and iPr derivatives 26
5.5
5.7
9.8
46.9
33.2
29.5
19.9
66
58
74
80
118
100
[a] Assay conditions: The reactions were carried out in MeOH at 228C.
Catalyst: 5.0 mm; PhSH: 1.0 mm; peroxide: 2.0 mm. The control reactions
were performed under identical conditions in the absence of selenium
compounds. [b] Relative activities are given with respect to the corre-
sponding control values.
From Table 1 and Figure 3, it is evident that the sec-
amine-based compounds 24–27 are much better catalysts
than the tert-amino-substituted diselenides 3–5 and 23. For
Table 2. Initial rates for the reduction of peroxides by GSH in the pres-
ence of compounds 3–5 and 23–27.
[a]
Initial rate (v0) [mmminÀ1
tBu-OOH
]
Compound
H2O2
Cum-OOH
3
4
5
23
24
25
26
27
581.1Æ2.4
289.9Æ5.0
39.5Æ2.3
56.2Æ2.2
691.3Æ3.8
488.1Æ1.0
469.1Æ1.4
468.4Æ0.8
361.7Æ5.2
134.3Æ2.7
20.2Æ2.9
40.7Æ2.3
463.6Æ5.6
337.2Æ8.6
356.0Æ4.2
318.1Æ1.2
708.5Æ7.5
456.2Æ9.2
45.3Æ2.0
68.2Æ0.7
763.3Æ4.5
639.8Æ2.3
664.1Æ6.4
631.9Æ2.9
[a] Assay conditions: The reactions were carried out in phosphate buffer
(100 mm, pH 7.5) at 228C. Catalyst: 80.0 mm; GSH: 2.0 mm; NADPH:
0.4 mm; ethylenediaminetetraacetate (EDTA): 1 mm; glutathione disul-
fide reductase: 1.7 unitsmLÀ1; peroxide: 1.6 mm. The control reactions
were performed under identical conditions in the absence of selenium
compounds. For the peroxidase activity, the rates were corrected for the
background reaction between thiol and peroxide.
Figure 3. Catalytic reduction of H2O2 by PhSH in the presence of various
selenium compounds. The formation of PhSSPh was followed by re-
versed-phase HPLC, and the percentage conversion was calculated from
a calibration plot: a) Control; b) 23; c) 27; d) 3; e) 24. Assay conditions:
catalyst: 5.0 mm; PhSH: 1.0 mm; H2O2: 2.0 mm; in MeOH at 228C.
and 27 were found to be almost 8–18 times more active than
their respective N,N-dipropyl and N,N-diisopropyl deriva-
tives 5 and 23. This is probably due to the presence of the
relatively more polar sec-amino group in compounds 26 and
27, which increases the solubility of these compounds in the
assay buffer. Similarly to the effect of peroxides on PhSH-
mediated reactions, the GPx-like activities of both sec- and
tert-amine-substituted diselenides with GSH depend on the
nature of the peroxide used in the reaction (Table 2).
To further understand the catalytic behavior of com-
pounds 3–5 and 23–27, we have carried out detailed kinetic
experiments. The catalytic parameters, such as maximum ve-
locity (Vmax), Michaelis constant (KM), catalytic constant
(kcat), and catalytic efficiency (h), for the reduction of Cum-
OOH by PhSH in the presence of compounds 3–5 and 23–
27 were obtained by plotting the reciprocal of the initial
rates (1/no) against the reciprocal of the substrate concentra-
tion (1/[S]).[6b,e,8d,g] It is clear from Table 3 that the catalytic
example, the t1/2 value for the reduction of H2O2 by com-
pound 24 (37 min) is much lower than that with 3 (67 min),
which indicates that the N-methyl derivative 24 is almost
two times more active than the N,N-dimethyl derivative 3.
Similarly, the other N-alkyl-based compounds 25–27 are sig-
nificantly more active than the corresponding N,N-dialkyl
derivatives 4, 5, and 23 in all three peroxide assays. Al-
though the replacement of the Me group by Et, nPr, or iPr
groups reduces the catalytic activity in both series of com-
pounds, the sec-amine-based compounds 25–27 exhibit much
better activity than the tert-amine-based diselenides 4, 5,
and 23. A comparison of the relative activities of the sec-
and tert-amine-based compounds reveal that the catalytic ac-
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9849

G. Mugesh and K. P. Bhabak
Table 3. Catalytic parameters (maximum velocities (Vmax), Michaelis con-
stants (KM), catalytic constants (kcat) and catalytic efficiencies (h)) for the
reduction of Cum-OOH by PhSH in the presence of diselenides 3–5 and
23–27.[a]
amine-based diselenides 14–16 has been shown to increase
the catalytic activity.[8g] This is due to prevention of the
thiol-exchange reaction by the methoxy substituents, which
allows efficient generation of the catalytically active sele-
nols. In contrast to diselenides 3–5, which produce a mixture
of selenols 41–43 and selenenyl sulfides 45–47 upon treat-
ment with PhSH (1 equiv), compound 23 with a sterically
bulky N,N-diisopropyl substituent generates selenol 44 as
the major product. In this particular case, only a trace
amount of selenenyl sulfide 48 was detected by 77Se NMR
spectroscopy. Selenenyl sulfide 48 could be completely con-
verted into the corresponding selenol 44 by addition of
excess PhSH (ꢀ5 equiv). The 77Se NMR signals for selenols
41–44 disappeared completely upon addition of iodoacetic
acid to produce the monoselenides 49–52.
Compound
Vmax [mmminÀ1
]
KM [mm]
kcat [minÀ1
]
h [mÀ1 minÀ1
]
3
4
5
23
24
25
26
27
38.95
14.26
5.36
2.89
1.10
0.80
0.84
0.42
0.45
0.49
0.60
15.58
5.70
2.14
3.09
6.03
4.08
3.99
3.15
5.39ꢂ103
5.18ꢂ103
2.66ꢂ103
3.66ꢂ103
14.20ꢂ103
8.97ꢂ103
8.20ꢂ103
5.28ꢂ103
7.74
15.08
10.21
9.97
7.88
[a] Assay conditions: PhSH: 0.0–4.0 mm; Cum-OOH: 1.0 mm; test com-
pounds: 2.5 mm; in MeOH at 228C.
efficiencies (h) of the sec-amine-based compounds are two
to three times higher than those of the corresponding tert-
amine-based analogues. The higher catalytic efficiencies of
compounds 24–27 relative to those of compounds 3–5 and
23 are mainly due to the differences in their KM values.
Whereas the thiol-exchange reactions in compounds 3–5 and
23 lead to higher KM values, the relatively low KM values for
compounds 24–27 indicate that the selenenyl sulfides de-
rived from 24–27 react readily with PhSH to produce the
corresponding selenols. When the initial rates were mea-
sured with increasing concentrations of PhSH and fixed con-
centrations of catalysts and Cum-OOH, a rapid increase in
the rates was observed at the beginning, then the rate
became constant after a certain concentration of PhSH was
reached. This indicates that compounds 24–27 follow typical
saturation kinetics, similar to those of the native GPx.[8g,19]
To understand the effect of the sec-amino group on the
reactivity of diselenides toward thiols, we carried out the re-
actions of compounds 24–27 with PhSH (Scheme 4). Al-
Scheme 4. Reaction of sec-amine-based diselenides 24–27 with thiophenol
(PhSH) to produce selenols 53–56 and selenenyl sulfides 57–60. Selenols
53–56 were trapped by iodoacetic acid to produce corresponding monose-
lenides 61–64.
Reactivity of the diselenides towards thiol: It has been
shown that ebselen (1) and related cyclic selenenyl amides
readily react with PhSH (1 equiv) to produce the corre-
sponding selenenyl sulfides. In most cases, these selenenyl
sulfides have been shown to be dead-end products due to
the presence of strong Se···O nonbonded interactions, which
lead to thiol-exchange reactions.[6] If there are strong Se···O
interactions in the selenenyl sulfide intermediates, nucleo-
philic attack of thiol takes place at the selenium atom in-
stead of the desired attack at the sulfur atom. This leads to
a thiol-exchange reaction, which hampers the regeneration
though compounds 24–27 reacted with one equivalent of
PhSH to produce the corresponding selenols 53–56 and sele-
nenyl sulfides 57–60 in a similar manner to the tert-amine-
based analogues (3–5 and 23), addition of a second equiva-
lent of PhSH to the reaction mixture completely converted
the selenenyl sulfides into selenols 53–56. These selenols
could be trapped by iodoacetic acid to produce the corre-
sponding selenides 61–64 in nearly quantitative yields. How-
ever, in contrast to selenols 41–44, which exhibited sharp
signals in their 77Se NMR spectra, compounds 53–56 showed
very broad signals, probably due to a rapid proton exchange
between the sec-amino and selenol moieties (Table 4). This
is further confirmed by treating compound 27 with two
equivalents of PhSH and recording the 77Se NMR spectrum
at À408C. At this temperature, a sharp signal at 58 ppm was
observed for the expected selenol 56. The reactivity of sec-
amine-based compounds 24–27 toward PhSH is completely
different from that of sec-amide-based diselenides 17–19,
which do not react with PhSH to produce the corresponding
selenols and selenenyl sulfides.[6e] These observations indi-
cate that the sec-amino group may deprotonate the thiol
À
of the selenol. Reductive cleavage of the Se Se bond in
compounds 3–5 with PhSH (1 equiv) has been shown to pro-
duce the corresponding selenols, 41–43, and selenenyl sul-
fides, 45–47.[8g] Furthermore, the simple replacement of hy-
drogen atoms by methoxy substituents in N,N-dialkylbenzyl-
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Glutathione Peroxidase Mimics
FULL PAPER
Table 4. Experimental 77Se NMR chemical shifts for selenols 41–44 and
53–56, selenenyl sulfides 45–48 and 57–60, and the iodoacetic acid trap-
ped monoselenides 49–52 and 61–64 in CDCl3.
Compound d [ppm][a] Compound d [ppm] Compound d [ppm]
45
46
47
48
57
58
59
60
564
558
555
535
539
536
536
530
41
42
43
44
53
54
55
56
35
54
56
61
49
50
51
52
61
62
63
64
246
239
241
244
252
243
242
237
[a]
–
–
[a]
65[a]
62[a]
[a] Sharp signals were not observed due to NMR line broadening at
room temperature.
Figure 4. X-ray crystal structure of compound 76. ORTEP diagram with
the ellipsoids representing 50% probability. Two water molecules, crystal-
lized with this molecule, are omitted for clarity.
(PhSH) to produce a more reactive thiolate, which would in-
À
crease the possibility of nucleophilic attack at the Se Se
linkage. It should be noted that 3-carboxy-4-nitrobenzene-
thiol, which exists predominantly in its thiolate form, has
been shown to enhance the thiol peroxidase activity of cy-
clodextrin-based ditellurides.[20]
and Tomoda.[8b] In contrast to the selenenic and seleninic
acids, which are expected to show Se···N nonbonded interac-
tions, no such interactions are found in compound 76; this
indicates that the overoxidation of the selenium atom pre-
vents any interaction between the selenium and nitrogen
atoms. This is also reflected in the solution behavior of com-
pound 76. The 77Se NMR signal at d=1019 ppm for com-
pound 76 is significantly shifted upfield relative to the signal
of seleninic acid 72 (d=1193 ppm). This is in agreement
with the report of Iwaoka and Tomoda that the selenonic
acid derived from compound 6 exhibits an upfield shift in
the 77Se NMR spectrum relative to the signals of the corre-
sponding selenenic and seleninic acids.[8b]
Reactions of compounds 24–27 with peroxides: It has been
shown that the sec- and tert-amide-based diselenides 17–22
react readily with H2O2 to produce the corresponding sele-
nenic and seleninic acids.[6e] The tert-amine-based diselenides
3–5 also react with H2O2 to produce the selenenic acids 65–
67 and seleninic acids 69–71. When the concentration of
H2O2 is very high (ꢀ30 equiv), the seleninic acids 69–71 un-
dergo further oxidation to produce the selenonic acids 73–
75 (Scheme 5).[8b,g] An increase in the steric hindrance does
Although the reactivity of sec-amine-based diselenides
24–27 towards H2O2 was similar to that of the tert-amine-
based compounds 3–5 and 23, the reactions produced the se-
leninic acids 81–84 as the only products, as observed by
77Se NMR spectroscopy (Scheme 6). It has been shown that
Scheme 5. Reaction of diselenides 3–5 and 23 with peroxide to produce
selenenic acids 65–68, seleninic acids 69–72, and selenonic acids 73–76.
Scheme 6. Various intermediates involved in the reaction of diselenides
24–27 with H2O2.
not appear to affect these oxidation reactions because the
N,N-diisopropylamine-based diselenide 23 undergoes similar
reactions with H2O2. However, the selenenic acid 68 could
not be detected by 77Se NMR spectroscopy due to its con-
version into the corresponding seleninic acid 72 and further
conversion into the overoxidized selenonic acid 76 at higher
peroxide concentrations. The structure of selenonic acid 76
was unambiguously confirmed from the single-crystal X-ray
structure (Figure 4). This structure is very similar to that of
the selenonic acid obtained from compound 6 by Iwaoka
the selenenic acids derived from the sec-amide-based disele-
nides 17–19 undergo a cyclization to produce the corre-
sponding selenenyl amides 31–33.[6e] However, no such cycli-
zation was observed in the reaction of diselenides 24–27
with H2O2. This is probably due to the rapid oxidation of se-
lenenic acids 77–80 into the corresponding seleninic acids
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G. Mugesh and K. P. Bhabak
(Scheme 6). Furthermore, seleninic acids 81–84 did not un-
dergo any further oxidation, even in the presence of a large
excess of H2O2 (ꢀ50 equiv). In this regard, the reactivity of
seleninic acids 81–84 towards H2O2 is different from that of
the tert-amine-based seleninic acids 69–72. This may also ac-
count for the higher GPx-like activity of 24–27 relative to
that of the tert-amine-based diselenides 3–5 and 23 because
overoxidation generally reduces the GPx-like activity of or-
ganoselenium compounds.[8b,d,g]
Reduction of seleninic acids 81–84 by PhSH: As seleninic
acids 81–84 were the major products in the reactions of dise-
lenides 24–27 with H2O2, it is important to study the reactiv-
ity of 81–84 toward thiols. As expected, the addition of
PhSH to the reaction mixture containing seleninic acids 81–
84 produced the corresponding selenenyl sulfides 57–60.
When an excess of PhSH (ꢀ5 equiv) was added, the sele-
nenyl sulfides were completely converted into the corre-
sponding selenols 53–56. The reactivity of selenenyl sulfides
57–60 towards thiol is significantly different from that of the
tert-amine-based selenenyl sulfides 45–48, which undergo ex-
tensive thiol-exchange reactions. These thiol-exchange reac-
tions reduce the possibility of nucleophilic attack at the
sulfur atom and prevent the formation of the corresponding
selenols. For example, the reaction of selenenyl sulfide 45
with p-thiocresol (4-Me-C6H4-SH) produces the selenenyl
sulfide 85 as the major product due to the thiol-exchange re-
action (Scheme 7). This type of reactivity has been previous-
ly observed for selenenyl sulfides derived from 3 and other
related diselenides with tert-amine-based substituents.[8c–g]
The selenenyl sulfide derived from ebselen has also been
shown to undergo extensive thiol-exchange reactions due to
the presence of strong Se···O interactions.[6] In contrast to
the result with compound 45, the reaction of sec-amine-
based selenenyl sulfide 57 with p-thiocresol produces the
corresponding selenol 53 as the only product (Scheme 7).
This indicates that the sec-amino groups are better than the
tert-amino substituents in generating the catalytically active
selenols. The reactivity of selenenyl sulfides 57–60 toward
PhSH is very similar to that of the selenenyl sulfides derived
from compounds 14–16, for which the thiol-exchange reac-
tions are suppressed by the methoxy substituent.[8g] The ab-
Scheme 8. The proposed catalytic mechanism for the reduction of perox-
ides with the sec-amine-based diselenides 24–27 in the presence of thiol.
sence of any significant thiol-exchange reactions in selenenyl
sulfides 57–60 is responsible for the higher GPx-like activity
of compounds 24–27 relative to that of their tert-amine-
based analogues.
Catalytic mechanism with sec-amine-based diselenides 24–
27: From the experimental observations, we propose a cata-
lytic mechanism for the reduction of peroxides by thiol in
the presence of diselenides 24–27, as illustrated in Scheme 8.
À
According to this mechanism, cleavage of the Se Se bond
in compounds 24–27 by thiol (PhSH) produces the corre-
sponding selenenyl sulfides 57–60 and selenols 53–56. The
selenols thus produced undergo a rapid reaction with H2O2
to produce the selenenic acids 77–80, which upon reaction
with PhSH generate the selenenyl sulfide intermediates. Ad-
dition of an excess of thiol regenerates selenols 53–56 with
the formation of PhSSPh. When the concentration of H2O2
is higher than that of thiol, the selenenic acids produced
from the selenols undergo further oxidation to produce the
corresponding seleninic acids 81–84. However, addition of
excess PhSH converts both the selenenic and seleninic acids
into the selenenyl sulfides. Although this catalytic cycle is
similar to that of the tert-amine-based diselenides,[8b,d,g] this
mechanism is completely different from that of the sec- and
tert-amide-based diselenides 17–22.[6e] Furthermore, the con-
version of sec-amide-based selenenyl sulfides into the corre-
sponding selenols is much faster than that of tert-amine-
based selenenyl sulfides. This indicates that the conversion
of selenenyl sulfides to selenols is the rate-determining step
in the catalytic cycle.
Conclusion
In this study, we have shown for the first time that sec-
amino-substituted diselenides exhibit much better GPx-like
activity than the corresponding tert-amine-based compounds.
In contrast to sec- and tert-amide-based diselenides, which
exhibit very poor reactivity toward PhSH, the amino-substi-
tuted compounds readily react with thiol to produce catalyt-
Scheme 7. Difference in the reactivity of selenenyl sulfides 45 and 57 to-
wards thiols.
9852
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Chem. Eur. J. 2009, 15, 9846 – 9854

Glutathione Peroxidase Mimics
FULL PAPER
purified by flash chromatography by using ethyl acetate and petroleum
ether as the eluent in the presence of 2% triethylamine.
ically active selenols. This study also reveals that the re-
placement of the tert-amino groups in benzylamine-based
diselenides by sec-amino moieties drastically enhances the
catalytic activity. Although the catalytic mechanism of sec-
amino-substituted diselenides is similar to that of the tert-
amine-based compounds, the sec-amino groups are better
than the tert-amino moieties in generating the catalytically
active selenols. This is due to the absence of any significant
thiol-exchange reactions in the selenenyl sulfides derived
from sec-amine-based diselenides. A comparison of the ac-
tivities of sec-amino-substituted compounds with that of sec-
amide-based diselenides indicates that the sec-amino-substi-
tuted compounds are more sensitive to the nature of the
peroxide than the sec-amide-based GPx mimics.
Compound 24: Yield 0.15 g (62%); 1H NMR (CDCl3): d=2.35 (s, 3H),
3.78 (s, 2H), 7.03–7.07 (m, 2H), 7.10–7.12 (m, 1H), 7.69–7.70 ppm (m,
1H); 13C NMR (CDCl3): d=36.1, 56.9, 127.0, 128.7, 128.9, 132.5, 133.6,
140.5 ppm; 77Se NMR (CDCl3): d=418 ppm; ESIMS: m/z calcd for
C16H20N2Se2 [M+H]+: 401.0035; found: 401.0031.
Compound 25: Yield 0.14 g (57%); 1H NMR (CDCl3): d=1.13 (t, J=
8.0 Hz, 3H), 2.69 (q, J=8.0 Hz, 2H), 3.91 (s, 2H), 7.11–7.15 (m, 2H),
7.17–7.20 ppm (m, 1H); 13C NMR (CDCl3): d=15.8, 43.9, 54.8, 127.0,
128.6, 128.7, 132.5, 133.5, 140.8 ppm; 77Se NMR (CDCl3): d=417 ppm;
ESIMS: m/z calcd for C18H24N2Se2 [M+H]+: 429.0348; found: 429.0348.
Compound 26: Yield 0.15 g (54%); 1H NMR (CDCl3): d=0.93 (t, J=
8.0 Hz, 3H), 1.49–1.58 (m, 2H), 2.62 (t, J=8.0 Hz, 2H), 3.91 (s, 2H),
7.10–7.15 (m, 2H), 7.17–7.25 (m, 1H), 7.77–7.79 ppm (m, 1H); 13C NMR
(CDCl3): d=12.4, 23.6, 51.5, 55.00, 126.9, 128.6, 128.7, 132.4, 133.6,
140.8 ppm; 77Se NMR (CDCl3): d=416 ppm; ESIMS: m/z calcd for
C20H28N2Se2 [M+H]+: 457.0661; found: 457.0656.
Compound 27: Yield 0.14 g (51%); 1H NMR (CDCl3): d=1.10–1.12 (d,
6H, J=8.0 Hz), 2.82–2.92 (m, 1H), 3.90 (s, 2H), 7.10–7.14 (m, 2H), 7.15–
7.19 (m, 1H), 7.76–7.77 ppm (m, 1H); 13C NMR (CDCl3): d=23.4, 48.9,
52.7, 127.0, 128.6, 128.7, 132.4, 133.7, 141.0 ppm; 77Se NMR (CDCl3): d=
415 ppm; ESIMS: m/z calcd for C20H28N2Se2 [M+Na]+; 479.0481; found:
479.0484.
Experimental Section
General procedure: n-Butyllithium was purchased from Acros Chemical
Co. (Belgium). Tetrahydrofuran and diethyl ether were dried prior to use
over sodium metal in the presence of benzophenone. Most of the reac-
tions were carried out under a nitrogen atmosphere by using standard
vacuum-line techniques. Due to the unpleasant odors of several of the re-
action mixtures involved, most manipulations were carried out in a well-
ventilated fume hood. Thin-layer chromatography analyses were carried
out on precoated silica gel plates (Merck) and spots were visualized by
UV irradiation. Column chromatography was performed in glass columns
loaded with silica gel or with an automated flash-chromatography system
(Biotage) by using preloaded silica cartridges. 1H (400 MHz), 13C
(100.56 MHz), and 77Se (76.29 MHz) NMR spectra were obtained on a
Bruker 400 MHz NMR spectrometer. Chemical shifts are cited with re-
spect to SiMe4 as internal (1H and 13C) and Me2Se as external (77Se)
standards. A Perkin–Elmer Lambda 5 UV/Vis spectrophotometer was
used to measure the GPx-like activities. Mass spectrometry studies were
carried out on a Q-TOF micromass spectrometer or a Bruker Daltonics
Esquire 6000plus mass spectrometer with ESI mode analysis. The melting
point of compound 23 was determined in an open capillary by using an
ANALAB melting-point apparatus. Compounds 3–5 were synthesized by
following the literature method.[8g]
Synthesis of 76: Hydrogen peroxide (200 mL, 1.845 mmol, 30% aqueous
solution) was added dropwise to a solution of diselenide 23 (50 mg,
0.092 mmol) in methanol/chloroform (4:1). The reaction mixture was
stirred for 1 h at room temperature. After 1 h, the yellow color of the di-
AHCTUNGERTGsNNUN elenide disappeared completely and the solution became colorless. The
solvent was removed under reduced pressure to obtain a white solid. The
progress of the reaction was monitored by 77Se NMR spectroscopy. The
product was recrystallized from methanol to obtain colorless crystals:
1
Yield 19.4 mg (66%); H NMR ([D4]MeOH): d=1.30–1.36 (dd, 12H, J=
8.0 Hz), 3.51–3.58 (m, 2H), 4.67 (s, 2H), 7.62–7.68 (m, 2H), 7.72–7.75 (m,
1H), 8.06–8.08 ppm (m, 1H); 13C NMR ([D4]MeOH): d=17.4, 17.8, 47.1,
53.5, 128.3, 129.0, 131.4, 133.9, 135.5, 145.7 ppm; 77Se NMR ([D4]MeOH):
d=1019 ppm; ESIMS: m/z calcd for C13H21NO3Se [M+H]+: 320.0765;
found: 319.7744.
General synthesis of compounds 49–52 and 61–64: Thiophenol (2 equiv)
was added to a solution of the appropriate diselenide (3–5 and 23–27) in
CDCl3 in an NMR tube. The yellow color of the solution immediately in-
tensified due to the formation of the corresponding selenols. The reaction
mixture was treated with iodoacetic acid (2 equiv) to produce the mono-
selenides 49–52 and 61–64. The progress of the reactions was monitored
by 77Se NMR and ESIMS spectrometric analyses (see the Supporting In-
formation). As the iodoacetic acid treatment was used mainly to confirm
the formation of the selenols, no attempts were made to isolate and
purify the monoselenides.
Synthesis of 23: nBuLi (1.2 mL; 1.6m in hexane) was added dropwise
with stirring to a cooled (À788C) solution of 2-bromo-N,N-diisopropyl-
benzylamine (0.40 g, 1.48 mmol) in dry Et2O (15 mL). The reaction mix-
ture was slowly allowed to reach room temperature. After 1.5 h, the n-
butyl bromide produced in the reaction was removed under reduced
pressure. After removal of most of the solvent under reduced pressure,
freshly distilled dry Et2O (15 mL) was added. Selenium powder (0.12 g,
1.48 mmol) was then added at 08C. After stirring of the reaction mixture
for an additional 3 h at room temperature, the reaction mixture was
poured into an ice-cooled saturated sodium bicarbonate solution. Diethyl
ether (ꢀ50 mL) was added, and oxygen gas was passed slowly through
the solution for 15 min. The compound was extracted with diethyl ether
and dried over sodium sulfate. The solvent was evaporated under re-
duced pressure to obtain a yellow liquid, which crystallized upon standing
Determination of GPx-like activity:
HPLC assay: The GPx-like activity was measured by using high-perfor-
mance liquid chromatography apparatus consisting of a 2695 separation
module,
a 2996 photodiode-array detector, and a fraction collector.
Assays were performed in 1.8 mL sample vials, and a built-in autosam-
pler was used for sample injection. In this assay, a mixture containing a
1:2 molar ratio of PhSH and peroxide in methanol at room temperature
(228C) was used as the model system. Runs with and without catalysts
(selenium compounds) were carried out under identical conditions. Peri-
odically, aliquots were injected into the reversed-phase column (Lichro-
sphere 60, RP-select B, 5 mm) and eluted with methanol/water (95:5). The
amount of product (PhSSPh) formed in the reactions was determined at
various time intervals by comparison with the calibration plot obtained
from known concentrations of pure PhSSPh. The chromatograms were
extracted at 254 nm.
1
for 2 h: Yield 0.49 g (61%); m.p. 102–1048C; H NMR (CDCl3): d=1.07–
1.09 (d, 12H, J=8.0 Hz), 3.06–3.13 (m, 2H), 3.85 (s, 2H), 7.06–7.13 (m,
2H), 7.27–7.29 (m, 1H), 7.71–7.72 ppm (m, 1H); 13C NMR (CDCl3): d=
21.1, 48.8, 51.9, 126.4, 128.1, 129.1, 131.4, 133.1, 141.1 ppm; 77Se NMR
(CDCl3): d=406 ppm; ESIMS: m/z calcd for C26H40N2Se2 [M+H]+:
541.1600; found: 541.1613.
General synthesis for sec-amine-based compounds 24–27: 2,2’-Diseleno-
bis(bromomethylbenzene) (0.6 mmol) in THF (20 mL) was added drop-
wise by using an addition funnel to a THF solution (10 mL) of the appro-
priate primary amine (6.0 mmol) over a period of 3–4 h, and the reaction
mixture was stirred for 12 h at room temperature. The solvent was evapo-
rated under reduced pressure to obtain a yellow-colored oil, which was
GSH–GSSG coupled assay:[8a] The GPx activity was followed spectropho-
tometrically. The test mixture contained GSH (2.0 mm), EDTA (1 mm),
glutathione disulfide reductase (1.7 unitsmLÀ1), and NADPH (0.4 mm) in
0.1m potassium phosphate buffer at pH 7.5. GPx samples (80 mm) were
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9853

G. Mugesh and K. P. Bhabak
added to the test mixture at room temperature, and the reaction was
started by the addition of peroxide (1.6 mm). The initial reduction rates
were calculated from the rate of NADPH oxidation at 340 nm in the
GSH assay. Each initial rate was measured at least 3 times and calculated
from the first 5–10% of the reaction by using 6.22 mmÀ1 cmÀ1 as the
molar extinction coefficient for NADPH. For the peroxidase activity, the
rates were corrected for the background reaction between peroxide and
thiol.
[5] a) H. J. Reich, C. P. Jasperse, J. Am. Chem. Soc. 1987, 109, 5549–
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X-ray crystallography: X-ray crystallographic studies were carried out on
a Bruker CCD diffractometer with graphite-monochromatized MoKa ra-
diation (l=0.71073 ꢁ) controlled by a Pentium-based PC running on the
SMART software package.[21] Single crystals were mounted at room tem-
perature on the ends of glass fibers, and data were collected at room tem-
perature. The structures were solved by direct methods and refined by
using the SHELXTL software package.[22] All non-hydrogen atoms were
refined anisotropically, and hydrogen atoms were assigned idealized loca-
tions. Empirical absorption corrections were applied to all structures by
using SADABS software.[23] The structures were solved by direct methods
(SIR-92) and refined by a full-matrix least-squares procedure on F2 for
all reflections (SHELXL-97).[24]
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Crystal data for 5:[25] C26H40N2Se2; Mr =538.5; triclinic; space group P1;
¯
a=11.3408(3), b=12.1700(4), c=12.6738(6) ꢁ; a=96.4748, b=108.6328,
g=117.6728; V=1395.07(23) ꢁ3; Z=2; 1calcd =1.28 gcmÀ3; MoKa radia-
tion (l=0.71073 ꢁ); T=291(2) K; R1 =0.038, wR2 =0.089 (I>2s(I));
R1 =0.076, wR2 =0.10 (all data).
Crystal data for 23:[25] C26H40N2Se2; Mr =538.5; triclinic; space group P1;
¯
a=10.360(19), b=12.499(23), c=12.658(23) ꢁ; a=101.4258, b=105.8728,
g=113.4498; V=1356(39) ꢁ3; Z=2; 1calcd =1.32 gcmÀ3; MoKa radiation
(l=0.71073 ꢁ); T=291(2) K; R1 =0.036, wR2 =0.069 (I>2s(I)); R1 =
0.072, wR2 =0.075 (all data).
Crystal data for 76:[25] C13H21NO3Se; Mr =319.0; orthorhombic; space
group P212121; a=8.3416(5), b=12.3004(6), c=16.5098(9) ꢁ; a=b=g=
90.008; V=1693.99(2) ꢁ3; Z=4; 1calcd =1.39 gcmÀ3; MoKa radiation (l=
0.71073 ꢁ); T=291(2) K; R1 =0.036, wR2 =0.079 (I>2s(I)); R1 =0.055,
wR2 =0.086 (all data).
[17] It is known that selenium in organoselenium compounds can inter-
act with nearby heteroatoms, such as nitrogen or oxygen atoms, to
produce hypervalent compounds having Se···N or Se···O interactions.
These interactions generally lead to downfield shifts in the
77Se NMR chemical shift values.[6a,b,8]
Acknowledgements
This study was supported by the Department of Science and Technology
(DST), New Delhi, India. We are grateful to the Alexander von Hum-
boldt Foundation, Bonn, Germany, for the donation of an automated
flash-chromatography system. G.M. acknowledges the DST for the award
of Ramanna and Swarnajayanti fellowships and K.P.B. thanks the Council
of Scientific and Industrial Research (CSIR), India, for a research fellow-
ship.
[18] R. Kaur, H. B. Singh, R. P. Patel, J. Chem. Soc. Dalton Trans. 1996,
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[23] G. M. Sheldrick, SADABS, A Program for Absorption Correction
with the Siemens SMART Area Detection System, University of
Gçttingen, Gçttingen (Germany), 1996.
[24] G. M. Sheldrick, SHELX-97, Program for the Refinement of Crystal
Structures, University of Gçttingen, Gçttingen (Germany), 1997.
[25] CCDC 724870 (5), 724870 (23), and 724871 (76) contain the supple-
mentary crystallographic data for this paper. These data can be ob-
tained free of charge from The Cambridge Crystallographic Data
Centre via www.ccdc.cam.ac.uk/data_request/cif.
[1] a) L. Flohe, E. A. Gꢃnzler, H. H. Schock, FEBS Lett. 1973, 32, 132–
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Received: March 30, 2009
Published online: June 23, 2009
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