Electrochemistry of chalcogen compounds: prediction of antioxidant activity.

Article abstract of DOI:10.1039/b107972g

Synthesis and characterisation of organochalcogens has demonstrated a high correlation between their electrochemical oxidation potential on the glassy carbon electrode, their activity in bioassays and an unprecedented antioxidant activity in neuronal cell

Full text of DOI:10.1039/b107972g

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Electrochemistry of chalcogen compounds: prediction of antioxidant
activity
Gregory I. Giles,a Karen M. Tasker,a Russell J. K. Johnson,a Claus Jacob,*a
Chris Peersb and Kim N. Greenb
a School of Chemistry, University of Exeter, Stocker Road, Exeter, UK EX4 4QD.
E-mail: C.Jacob@ex.ac.uk; Fax: +44 (0)1392 263434; Tel: +44 (0)1392 263462
b Institute for Cardiovascular Research, University of Leeds, Leeds, UK LS2 9JT
Received (in Cambridge, UK) 4th September 2001, Accepted 19th October 2001
First published as an Advance Article on the web 15th November 2001
Synthesis and characterisation of organochalcogens has
demonstrated a high correlation between their electro-
chemical oxidation potential on the glassy carbon electrode,
their activity in bioassays and an unprecedented antioxidant
activity in neuronal cell culture (EC50 ~ 20 nM) making
electrochemical methodology a valuable tool in drug design
for Alzheimer’s and related diseases.
standard Ag/AgCl reference electrode, a Pt counter electrode
and a GE (BAS) that was thoroughly cleaned and polished
(Al2O3) after each scan.
Most chalcogen compounds studied displayed only one
irreversible oxidation peak between 0 and +1200 mV vs. Ag/
AgCl (Table 1) with the exception of 10, which also exhibited
a reduction peak at +270 mV and a second oxidation peak at
+820 mV. The value for the first oxidation potential (Epa)
ranged from +300 to +1400 mV with a clearly observable trend,
the tellurides having the lowest potential averaging around +400
mV, increasing to approximately +900 mV for the selenides and
+1200 mV for the sulfides. Interestingly, both mono- and di-
chalcogens displayed an oxidation peak, allowing comparison
of the two species. In aqueous solution anodic oxidation of these
chalcogens is an irreversible process that involves the formation
of the intermediate radical species rapidly followed by reaction
with water to form the chalcogen- or dichalcogen oxide.4,9 The
formation of such species (e.g. disulfide S-oxides, selenoxides)
is frequently observed in biochemical studies3 and has been
implicated in catalytic antioxidation (e.g. oxidative activation of
ditellurides, selenylsulfides).4 On the assumption that the ease
of radical formation correlates with the ease of oxidation of the
Oxidative stress is a biochemical condition present in several
human disorders, including Alzheimer’s disease.1 It is the
manifestation of an imbalance in an organism’s biochemical
redox-processes that results in the production of high concentra-
tions of reactive oxygen (ROS), nitrogen and sulfur species.
Various antioxidant strategies are used to treat oxidative stress
related disorders, e.g. by supplementation with artificial ‘one
shot’ antioxidants (vitamin C, penicillamine) that react stoi-
chiometrically with stressors to form non-toxic species.2 Recent
studies have indicated that catalytic antioxidants that mimic the
action of the selenium enzyme glutathione peroxidase (GPx) are
considerably more effective. GPx catalyses the reaction of toxic
peroxides with glutathione (GSH) to form oxidised glutathione
(GSSG) and water. Most synthetic GPx mimics incorporate a Se
or Te atom within an organic framework and their catalytic
redox cycle consists of the initial oxidation of a chalcogen atom,
in the process reducing peroxide to water, followed by
regeneration of the catalyst via reduction with two equivalents
of thiols.3–5
Only a few attempts have so far been made to link the
electrochemical redox potentials of these agents with anti-
oxidant activity, among them cyclic voltammetry (CV) to
characterise tellurides and selenides in organic solvents and
polarography of diselenides on mercury electrodes.6,7 A fast,
widely applicable and reliable method to predict biochemical
and biological activity is, however, still lacking and a
requirement for an accurate prediction is that antioxidants are
compared under conditions closely resembling a biological
environment. The results presented in this communication show
that oxidation potentials can be obtained in aqueous media on
the glassy carbon graphite electrode (GE) for both mono- and
di-chalcogens and that there is a good correlation between these
potentials and biochemical activity in oxidation assays. This
data can be used to predict antioxidant activity in cell culture
experiments indicative of Alzheimer’s disease.
Table 1 Correlation between Epa and the biochemical activity of
compounds 1–10. Cyclic voltammograms were recorded at a scan rate of
200 mV s21. MT (0.5 mM) was incubated with t-BuOOH (500 mM) and
catalyst (200 nM, 100 nM for 7 to allow for the presence of two tellurium
catalytic sites) in the presence of PAR (100 mM) in 20 mM HEPES-Na+, pH
7.5 at 25 °C (N2 purged). The reaction was continuously monitored for 60
min at 500 nm (Cary50/Varian). Initial rates (r) were calculated between 0
and 10 min
r
Comp.
X
n
Y
Epa/mV
3 10210 M s21
1
2
S
S
1
2
1
2
1
2
1
1
1
1
H
+1366
+1176
+753
+1000
+368
+578
+527
+448
+694
+299
1.0
1.3
0.1
3.8
4.3
9.3
11.6
5.5
6.7
14.1
OCH3
OCH3
OCH3
OCH3
3
Se
Se
Te
Te
Te
Te
Te
Te
4
5
6
OCH3
a
7
The redox-behaviour of a number of structurally related
mono- and di-chalcogens has been investigated (Table 1).
Compounds 1 and 2 were purchased from Aldrich and 3–6 and
10 were synthesised according to established procedures.8 7 and
9 were synthesised by the reduction of 6 with sodium
borohydride followed by the addition of either a stoichiometric
amount of 1,3-dibromopropane (7) or an excess of the reagent
(9). Compound 8 was synthesised by stirring 10 in excess acetic
anhydride over 20 h. Cyclic voltammograms of organochalco-
gens (50 mM) were recorded in 50 mM potassium phosphate
buffer (pH 7.4) containing 30% methanol (due to the limited
solubility of the organochalcogens in aqueous media) using a
8
O(O)CCH3
a
9
10
OH
Exp. error 10%. a Structures for 7 and 9 given below.
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Chem. Commun., 2001, 2490–2491
This journal is © The Royal Society of Chemistry 2001
DOI: 10.1039/b107972g

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Fig. 2 Effect of antioxidant concentration on current augmentation of PC12
cells with AbP(1–40). The plot shows the preventive effect of different
concentrations of antioxidants. The lower dashed line indicates current
density in control cells, whilst the upper dashed line represents currents
Fig. 1 Correlation between Epa and % zinc transfer from MT (experimental
details given in legend of Table 1). A value for the maximum zinc release
was obtained by incubation with 10 mM ebselen. The value for zinc release
after 60 min was expressed as a percentage of the maximum zinc release and
plotted as a function of the catalyst’s (separately determined) oxidation
potential. Experiments were repeated (N = 3) and error bars (SD) applied
for zinc release as well as Epa.
observed following 24 h incubation with 100 nM AbP(1–40).
~ 20 nM). The diselenide 4 was also found to be highly active
in cell culture, albeit considerably less active than 10, in line
with its higher oxidation potential (+ 1000 mV) and lower
bioassay activity (EC50 ~ 160 nM). In accordance with a high
oxidation potential (Epa = +1155 mV) the commonly studied
Se-compound selenocystamine proved rather inactive (EC50
~ 10 mM).
chalcogen in vivo Epa has the potential to be used as a predictor
of biological activity.
These experiments have demonstrated that antioxidants
selected on the basis of electrochemical redox-behaviour and
their activity in bioassays are also likely to be highly active in
cell cultures. Compound 10 exhibited the lowest Epa and the
most promising antioxidant activity in the in vitro bioassays. It
also showed a remarkable activity when compared to ebselen
and melatonin in cell culture while showing little cell toxicity,
demonstrating that this compound is among the best anti-
oxidants available to date in cultured cells mimicking Alzhei-
mer’s disease. It is expected that this combination of chalcogen-
electrochemistry with organoselenide and organotelluride
catalysts will provide a new methodology for innovative
antioxidant research in Alzheimer’s disease.
Organochalcogens were evaluated for GPx-like activity via
their ability to catalyse the reaction of tert-butyl hydrogen
peroxide (t-BuOOH) with the thiol protein metallothionein
(MT) (this redox assay is increasingly used since it provides
excellent reproducibility).10,11 Zinc release from MT occurs
upon oxidation of the thiol ligands and is detected spec-
trophotometrically in the presence of the chromophoric dye
4-(2-pyridylazo)resorcinol (PAR). Agents catalysing this proc-
ess can be evaluated according to both the initial rate and the
total extent of zinc release. Maximum zinc release from 0.5 mM
MT was calculated by incubation with 10 mM ebselen
(2-phenyl-1,2-benzisoselenazol-3[2H]-one) and the amount of
zinc release for different antioxidants compared to this
theoretical maximum.11 Table 1 and Fig. 1 indicate that Epa
correlated well with the activity in the bioassay, with the lowest
potentials associated with the highest activity. A similar
correlation was observed in a standard GPx assay monitoring
thiophenol oxidation by hydrogen peroxide in methanol (data
not shown).12 Among the agents tested, 10 had the lowest Epa
and also one of the highest activities. It was therefore tested in
PC12 cell culture indicative of Alzheimer’s disease and
compared with other antioxidants.
This work was financially supported by the EPSRC (GR/
N22007), the MRC, an Alzheimer’s Society Innovation Grant,
the Wellcome Trust, the Royal Society and the University of
Exeter.
Notes and references
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PC12 cells were cultured as previously described.13,14 24 h
prior to study aliquots were exposed to 100 nM amyloid beta
peptide (residues 1–40) (AbP(1–40)) and varying concentrations
of antioxidants. AbP(1–40) causes dramatic and selective en-
hancement of Ca2+ channel activity in these cells via a
mechanism which involves increased production of ROS.13,14
Ca2+ channel currents were recorded using the whole-cell patch
clamp technique, with 20 mM Ba2+ as charge carrier. Currents
were evoked by applying 200 ms ramps (2100 mV to +100mV,
0.2 Hz) from 280 mV holding potential, measured at the peak
of the current–voltage relationship (+20 mV) and corrected for
cell size to yield current densities (pA/pF, means sem).14 Cell
viability was determined using a standard 3-(4,5-dimethylth-
iazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and
at this dose of AbP(1–40) does not result in cell kill. The
antioxidants tested reversed the current augmentation induced
by AbP(1–40) without any detectable loss of cell viability (Fig.
2). The naturally occurring antioxidant melatonin exhibited the
least potency (EC50 ~ 30 mM), whereas the commonly used
selenium based anti-inflammatory agent ebselen (Epa deter-
mined as +1044 mV) showed increased potency (EC50 ~ 1 mM).
In line with a variation in Epa of approximately 700 mV,
compound 10 by far exceeded the activity of ebselen and,
astonishingly, was active in nanomolar concentrations (EC50
9 B. Svensmark and O. Hammerich, in Organic Electrochemistry, eds. H.
Lund and M. M. Baizer, Marcel Dekker, Inc., New York, 3rd edn., 1991,
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Toxicol., 2000, 13, 3; C. Jacob, W. Maret and B. L. Vallee, Biochem.
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13 S. C. Taylor, T. F. C. Batten and C. Peers, J. Biol. Chem., 1999, 274,
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14 K. N. Green and C. Peers, J. Neurochem., 2001, 77, 953.
Chem. Commun., 2001, 2490–2491
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