The coulometric approach to the superoxide scavenging activity determination: The case of porphyrin derivatives influence on oxygen electroreduction

Article abstract of DOI:10.1142/S1088424615500807

The electrochemical and antioxidant properties of 5,10,15,20-tetrakis(3′-hydroxyphenyl)porphyrin (H₂T(m-OHPh)P) and 5,10,15,20-tetrakis(4′-hydroxyphenyl)porphyrin (H₂T(p-OHPh)P) were tested by the cyclic voltammetry (CV) method. It i

Full text of DOI:10.1142/S1088424615500807

Journal of Porphyrins and Phthalocyanines
J. Porphyrins Phthalocyanines 2015; 19: 1–10
DOI: 10.1142/S1088424615500807
Published at http://www.worldscinet.com/jpp/
The coulometric approach to the superoxide scavenging
activity determination:The case of porphyrin derivatives
influence on oxygen electroreduction
Sergey M. Kuzmin*, Svetlana A. Chulovskaya and Vladimir I. Parfenyuk◊
G.A. Krestov Institute of Solution Chemistry of the Russian Academy of Sciences, Ivanovo 153045, Russia
Received 12 May 2015
Accepted 4 July 2015
ABSTRACT: The electrochemical and antioxidant properties of 5,10,15,20-tetrakis(3′-hydroxyphenyl)
porphyrin (H2T(m-OHPh)P) and 5,10,15,20-tetrakis(4′-hydroxyphenyl)porphyrin (H2T(p-OHPh)P)
were tested by the cyclic voltammetry (CV) method. It is shown that in dimethylsulfoxide (DMSO) the
electroreduction processes of oxygen and porphyrins are under diffusion control. The electroreduction
of H2T(m-OHPh)P and H2T(p-OHPh)P with potentials about -1 V are irreversible due to the chemical
step (EC process) which leads to products with oxidation potentials about -0.5 V. Additionally in
case of oxygen and porphyrin coreduction, both H2T(m-OHPh)P and H2T(p-OHPh)P influence the
·-
O2•- electrosynthesis. The nonlinear dependence of the O2 peak current vs. porphyrin concentrations
makes the available amperometric approach unsuitable for antioxidant activity estimation. To solve
this problem, the coulometric parameters were calculated. The excellent linearity of the coulometric
response of superoxide ion vs. porphyrins concentration was demonstrated for a wide concentration
range. On the basis of coulometric responses, we constructed a parameter which separated the radical
scavenging activity and variation of oxygen electroreduction. The superoxide scavenging activities
of H2T(m-OHPh)P and H2T(p-OHPh)P were determined using the developed approach. The effect of
OH group position on the superoxide scavenge activity is shown: H2T(p-OHPh)P has a higher activity
(slope = 0.75 L.mmol-1) than H2T(m-OHPh)P (slope = 0.60 L.mmol-1).
KEYWORDS: porphyrin, cyclic voltammetry, coulometric approach, superoxide scavenging
activity.
applications [7, 8]. The phenolic fragments with a labile
INTRODUCTION
hydrogen atom in the porphyrins leads to the detection
Free radicals are a special class of chemicals which
of antioxidant properties [9]. However, the porphyrin
have an unpaired electron. As a rule, they are aggressive
structure impact on the activity and mechanisms of
oxidants which lead to pathologies of biochemical
antioxidant action are insufficiently studied up to now.
processes and damage of cells, especially cell membranes.
The high absorption coefficients of porphyrins [10] lead
Differentantioxidantspreventthedamagingeffectsoffree
to difficulties in spectrometric measurements [11] of
radicals on living cells [1, 2]. It is known that heterocyclic
antioxidant activity. Therefore, the electrochemical assay
compounds [3, 4] and some porphyrin ligands [5, 6]
was preferred. The determination of radical scavenging
have a significant antioxidant activity. The complexes
properties of compounds is based on the monitoring of
of porphyrins with transition metals exhibit superoxide
superoxide amperometric response in the antioxidant
scavenging activity and are promising for pharmaceutical
presence [12–16]. Previously [17], we have shown
the antioxidant properties of 5,10,15,20-tetrakis(4′-
aminophenyl)porphyrin and the essential influence of
◊ SPP full member in good standing
the substituent position on the superoxide scavenging
activity. This paper is focused on the development of
*Correspondence to: Sergey M. Kuzmin, email: smk@isc-ras.ru,
tel: +7 915-824-9554
Copyright © 2015 World Scientific Publishing Company

2
S. M. KUZMIN ET AL.
The mixture was stirred at room temperature for 2 h, and
then 5 mL of methanol was added. Fifteen minutes later,
2.5mLofammoniasolutionwasdropaddeduntilthecolor
changed from green to red. Chloroform was distilled off,
the residue dried, dissolved in 5% potassium hydroxide
solution, filtered and precipitated by diluted hydrochloric
acid. The precipitate was filtered off, washed with water
and dried in air at room temperature. The deposit was
dissolved in ethyl acetate and purified on silica gel by
eluting with ethyl acetate. The eluate was evaporated and
precipitated with petroleum ether. Yield 1.35 g (97.5%).
R1
R2
OH
R
OH
N
NH
R
R
HN
N
R
1
Rf = 0.88 (ethyl acetate). H NMR (500 MHz, CDCl3,
Me4Si): dH, ppm -2.45 (2H, s, pyrrole-NH), 7.33 and 7.85
(16H, m, H-Ph), 8.52 (8H, s, pyrrole-H). UV-vis (CHCl3):
Fig. 1. Chemical structures of porphyrins
l
max, nm (log e) 645 (3.71), 588 (3.88), 550 (3.92), 514
a coulometric approach to the superoxide scavenging
activity determination in case of antioxidants influence
on the superoxide electrosynthesis process.
(4.31), 419 (5.62).
5,10,15,20-Tetrakis(4′-methoxyphenyl)porphine
{intermediate compound}. A solution of 5.0 mL
(72.2 mmol) of pyrrole and 8.8 mL (72.2 mmol) of
anisaldehyde was drop added to a boiling solution of 16 g
of chloroacetic acid in 300 mL of an isomeric
xylenes mixture for 20 min. The resulting mixture
was refluxed with air bubbling for one more hour.
After xylene steaming, the deposit was filtered off,
washed with water and dried in air at 80°C. The
deposit was dissolved in chloroform and purified on
aluminum oxide (Brockmann Activity III) eluting
with chloroform. The first red zone was collected,
the eluate was evaporated, and the porphyrin was
precipitated with methanol, filtered and dried at room
temperature in air. The yield was 5.6 g (42%). Rf =
EXPERIMENTAL
Procedure of synthesis
H2T(m-OHPh)P and H2T(p-OHPh)P (Fig. 1) were
synthesized by the two-step method [18, 19] via
demethylation of methoxyphenylporphines that was
obtained in high yield by condensation of benzaldehydes
withpyrrole[20].Thepurifiedproductswerecharacterized
by thin-layer chromatography (silufol plates), UV-vis
spectrometry (Varian Cary 50 spectrometer) and 1H
NMR spectrometry (Bruker AVANCE-500 spectrometer)
methods.
1
0.33 (CHCl3). H NMR (500 MHz, CDCl3, Me4Si): dH,
ppm -2.79 (2H, s, pyrrole-NH), 4.03 (12H, s, –O–CH3),
7.22 (8H, d, m-H-Ph), 8.06 (8H, d, o-H-Ph), 8.79 (8H,
s, pyrrole-H 8H). UV-vis (CHCl3): lmax, nm (log e) 652
(3.87), 595 (3.78), 557 (4.07), 520 (4.25), 423 (5.69).
5,10,15,20-Tetrakis(4′-hydroxyphenyl)porphine. A
solution of 0.5 mL (5.29 mmol) of boron tribromide in
10 mL of methylene chloride was added to the stirring
and cooling solution of 1.0 g (1.36 mmol) of 5,10,15,20-
tetrakis(4′-methoxyphenyl)porphyrin in 200 mL of dried
methylene chloride. The mixture was stirred at room
temperature for 2 h, and then 5 mL of methanol was
added. The mixture was neutralized by ammonia until
the color changed from green to dark cherry, washed
with water, dried over sodium sulfate and evaporated to
dryness. The deposit was dissolved in ethyl acetate and
purified on silica gel by eluting with ethyl acetate. The
eluate was evaporated and precipitated with petroleum
ether.Yield 0.9 g (98%). Rf = 0.33 (CHCl3). 1H NMR (500
MHz; CDCl3, Me4Si): dH, ppm -2.92 (2H, s, pyrrole-NH),
7.70 (8H, d, m-H-Ph), 8.08 (8H, d, o-H-Ph), 8.79 (8H, s
pyrrole-H). UV-vis (CHCl3): lmax, nm (log e) 650 (3.72),
595 (3.71), 556 (3.90), 519 (4.06), 423 (5.43).
5,10,15,20-Tetrakis(3′-methoxyphenyl)porphine
{intermediate compound}. A solution of 5.4 g
(0.04 mol) of 3-methoxybenzaldehyde and 2.7 mL
(0.04 mol) pyrrole in 50 mL xylene was drop added to a
boiling solution of 5.0 g of chloroacetic acid in 150 mL
of a mixture of isomeric xylenes during 20 min. The
resulting mixture was refluxed with air bubbling for
one additional hour. After that, xylene was steamed, the
deposit was filtered off, washed with water and dried in
air at 70°C. The deposit was dissolved in chloroform
and purified on aluminum oxide (Brockmann Activity
III) eluting with chloroform. The first red zone was
collected, the eluate was evaporated, and the porphyrin
was precipitated with methanol, filtered and dried at
room temperature in air. The yield was 2.0 g (27.2%).
1
Rf = 0.45 (CHCl3 — hexane, 1:1). H NMR (500 MHz,
CDCl3, Me4Si): dH, ppm -2.85 s (2H, s, pyrrole-NH), 3.92
(12H, s, –O–CH3), 7.28, 7.53 and 7.71 (16H, m, H-Ph),
8.85 (8H, s, pyrrole-H). UV-vis (CHCl3): lmax, nm (log e)
648 (3.72), 590 (3.83), 550 (3.89), 516 (4.31), 420 (5.70).
5,10,15,20-Tetrakis(3′-hydroxyphenyl)porphine.
4.5 g of boron tribromide was added to 1.5 g (2.04 mmol)
of 5,10,15,20-tetrakis(3′-methoxyphenyl)porphyrin solu-
tion being stirred and cooled in 50 mL of dry chloroform.
The porphyrins characterized by standard analytical
and spectroscopic techniques agree quite well with the
reported data [21, 22].
Copyright © 2015 World Scientific Publishing Company
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THE COULOMETRIC APPROACH TO THE SUPEROXIDE SCAVENGING ACTIVITY DETERMINATION
3
Electrochemical procedure
equals 2.1 mM [23, 24]. A free convection mode was
formed within 3 min after removing the capillary from
the solution. Thereafter the first cycles of voltammograms
were recorded at scan rates from 0.005 to 1 V/sec. The
CV data were corrected for Ohmic (iR) losses using a
current interruption technique [25].
Intensity and position of UV-vis absorption bands did
not change in the presence of supported electrolyte, which
indicates that there was no interaction between the ions
of the supported electrolyte and the studied porphyrins.
Dimethylsulfoxide (DMSO ≥ 99.5, ALDRICH) was
purified by the zone melting method and then stored over
molecularsievesinadryboxbeforeuse.Tetrabutylammo-
nium perchlorate (TBAP ≥ 98.0,ALDRICH) was purified
by recrystallization from ethanol. The concentrated
solutions of porphyrins containing 0.02 M TBAP were
prepared as supported electrolyte by the gravimetric
method using electronic analytical balance «Sartorius»
ME215S. The lower concentrations of porphyrins were
prepared by serial dilutions. DMSO was selected as the
solvent in electrochemical studies because porphyrins are
RESULTS AND DISCUSSION
Electrochemical behavior of porphyrins
CV curves for H2T(m-OHPh)P and H2T(p-OHPh)
P 31 on Pt electrode at negative vs. SCE potentials are
shown in Fig. 2. It is evident that the substituent position
significantly influences the electrochemical behavior of
hydroxyphenyl porphyrins.
·-
In case of H2T(m-OHPh)P, three separate reduction
waves with a current maximum at potentials around -0.77,
-1.16 and -1.55 V are observed. In contrast, in case of
H2T(p-OHPh)P, there is one more intense oxidation wave.
The oxidation peaks are weak at a low scan rate for both
porphyrins. The increasing of potential scan rate leads to
shifts of reduction peak positions to the negative region
(Table 1). The peak currents of the electroreduction waves
are proportional to the concentration of both porphyrins.
At the same time, the peak current density is proportional
to the square root of scan rates of up to 0.05 V/s for both
porphyrins as well as oxygen. It indicates the diffusion
nature of the electrode processes [26].
In addition, in case of H2T(p-OHPh)P the increasing
of potential scan rate invokes the appearing of wide
oxidation peak in the potential range from -0.8 to -0.2 V.
At the same time, there is no redox response if the
cycling was in the range from 0.1 to -0.9 V (Fig. 3,
curve 2). Thus, we can conclude that the peak is caused
by electrooxidation of products formed by the chemical
reaction (or structure reorganization) of porphyrin anions.
readily soluble in the solvent, O2 is stable and can be
electrochemically generated.
The electrochemical cell and measurement procedure
aredescribedelsewhere[6,17].Thecellwasatemperature-
controlled (25 0.5°C) vessel which was separated by
a porous glass plug into two volumes to segregate the
working and auxiliary electrodes. The saturated calomel
electrode (SCE) inserted into the electrochemical cell
through the Luggin capillary was used as the reference
electrode. Before measuring, the working electrode
(the polishing platinum strip with an active area of
1.2 cm2) was pretreated by mechanical polishing,
degreased with ethanol, and etched by a chromic mixture
for 20 min, carefully washed out by distilled water and
test solution. The working electrode was immersed in
the cell with the test solution where the potential of the
working electrode reached a steady value in 10 min. The
atmospheric pressure of argon or oxygen was used for
solution degassing or oxygenation. The oxygen saturation
condition was verified using the cyclic voltammetry (CV)
method. In this case, the dissolved oxygen concentration
Fig. 2. CV response of H2T(m-OHPh)P (a) and H2T(p-OHPh)P (b) in DMSO during the first cycle at potential scan rates of 0.02,
0.05, 0.1, 0.2, 0.5, 1.0 V/s. The porphyrins concentrations are 1 mM
Copyright © 2015 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2015; 19: 3–10

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S. M. KUZMIN ET AL.
Table 1. Scan rate influence on the reduction waves of H2T(m-
OHPh)P and H2T(p-OHPh)P
The CV curves recorded under the low scan rate (Fig. 4)
show that the voltammograms become quite steady
during first three cycles. Moreover, the some common
features were detected in the shape of CV curves. There
are three oxidation waves (A, B, C) on the CV cathodic
branch which has complementary waves (A′, B′, C′) on
the CV anodic branch. The distance between the cathodic
and anodic maximums is in the range of 0.07–0.13 V
(Table 2) that indicates the quasi reversibility of the
charge transfer on the interface in the studied condition.
It should be noted that low intensive waves A, A′
probably occurred due to rearrangement phenomena
in the adsorbed layer [27, 28]. The slight difference
between the voltage of waves in case of para- and meta-
substitutions allows us to make a conclusion about
the similarity of charge transfer energy for H2T(m-
OHPh)P and H2T(p-OHPh)P in DMSO. Therefore, the
distinction in the electrochemical behavior of the studied
porphyrins should be associated with different kinetics
of ion-molecule reactions. Two waves of single electron
reductions which accompanied the proton transfer were
proposed under negative potentials vs. Fco/Fc+ [29]. The
presence of irreversible oxidation peaks under positive
potentials vs. SCE was described elsewhere [30].
Vscan, V/s
H2T(m-OHPh)P*
Ered, V
jred, mA/cm2
H2T(p-OHPh)P
Ered, V
jred, mA/cm2
0.01
0.02
0.05
0.1
-1.07
-1.09
-1.10
-1.11
-1.12
-1.15
-1.16
-0.04
-0.05
-0.06
-0.08
-0.10
-0.16
-0.23
-1.09
-1.11
-1.13
-1.16
-1.18
-1.20
-1.24
-0.07
-0.09
-0.12
-0.15
-0.19
-0.26
-0.38
0.2
0.5
1.0
*Only the most resolved wave is shown.
For waves B (Fig. 4), the number of electrons involved
in the electrochemical step was determined using
Equation 1 [31]:
∂Ep
1.15RT
naF
=
(1)
∂(lg(n))
where n is the scan rate; Ep is the peak voltage, F is
the Faraday constant, R is the gas constant, n is the
number of electrons involved in electrochemical step, a
is the transfer coefficient. The calculations showed that
the value of na was around 0.81 for H2T(m-OHPh)P
Fig. 3. Cycling solution of H2T(p-OHPh)P in the range of:
(1) from -1.6 to +0.1; (2) from -0.9 to +0.1 V. The scan rate is
0.1 V/s. The porphyrin concentration of 1 mM
Fig. 4. CV response of H2T(m-OHPh)P (a) and H2T(p-OHPh)P (b) during 5 cycles at potential scan rates of 0.01 V/s. The porphyrins
concentrations are 1 mM
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THE COULOMETRIC APPROACH TO THE SUPEROXIDE SCAVENGING ACTIVITY DETERMINATION
5
Table 2. Voltage of cathodic and anodic maxima for the 5th cycle at scan rates of 0.01 V/s. The
porphyrin concentration is 1 mM
Peak label
Potential, V
A
A′
B
B′
C
C′
D
F
H2T(m-OHPh)P
H2T(p-OHPh)P
-0.75
-0.7
-0.63
-0.63
-1.06
-1.08
-0.99
-0.95
-1.48
-1.49
-1.37
-1.37
-0.25
+0.44
+0.48
+0.2
(wide)
_
and 0.52 H2T(p-OHPh)P and that the first electron
transfer controls the overall reaction rate. In case of
H2T(m-OHPh)P, the gaps between peaks B and B′ were
measured at a different scan rate. After approximation to
the zero scan rate the gap between the peaks was about
60 mV. The obtained value confirms the one-electron
nature of the redox process. In case of H2T(p-OHPh)P,
relation (2) was used.
•-
O2 + e
O2
Scheme 2. Electrochemical formation of superoxide ion.
|Ep – Ep/2| = 47.7/na
(2)
where Ep/2 is the half wave voltage.
The calculated na value was around 0.5, which also
confirms the one-electron nature of wave B.
The reduction of compounds including phenolic
fragments is often realized via multistep proton coupled
electron transfer mechanisms [32], so the reduction of the
porphyrin under study can be similar. On the other hand,
the disproportionation reaction (Scheme 1) is possible
because the first reduction peaks (labeled B) demonstrate
high current densities [33, 34]. Disproportionation, like
most chemical reactions, passes through a transition state
(intermediate — two anions are located close enough for
the electron transfer (P2)2-). That intermediate oxidation
can be one of possible reasons for peaks appearance in
the region from -0.8 to -0.2 V (Fig. 3). The intermediates
oxidation will be observed if step II is slow on the
voltammetric time scale. One electron waves C (Fig. 4)
indicate that step I is slow too. Within this framework, the
disparity between H2T(p-OHPh)P and H2T(m-OHPh)P
(Fig. 4) current densities indicates a different dispropor-
tionation rate of porphyrins.
Fig. 5. Cyclic voltammograms: background current (1), H2T(m-
OHPh)P (2) and H2T(p-OHPh)P (3) in degassed solution; O2/O2·-
redox couple without porphyrin (4), with H2T(m-OHPh)P (5),
with H2T(p-OHPh)P (6) in oxigen saturated solution. The scan
rate is 0.02 V/s. The porphyrin concentrations are 1 mM
good agreement with the data obtained under similar
conditions [23, 36–38].
·-
The current density peak of O2 oxidation shows
very good reproducibility and it is significantly higher
than the current density peak of porphyrins. The cyclic
voltammograms of porphyrins in oxygen-saturated
DMSO solutions are shown in Fig. 5, curves 5 and 6. It
is clearly seen that porphyrins evoke significant changes
in both reduction and oxidation currents. Therefore, the
studied porphyrins influence both the synthesis and the
decomposition of superoxide ions. The onset potentials
for oxygen reduction shift toward the positive region
in the porphyrins presence. At the same time, the onset
superoxide oxidation in the presence and absence of
porphyrin is almost the same. This may indicate that the
oxygen reduction is catalyzed by porphyrins, whereas the
superoxide oxidation is not catalyzed effectively.
2P-
(P2)2-
P2- + P
Step I
Step II
Scheme 1. Disproportionation of porphyrins via possible
intermediates.
Effects of porphyrins on O2/O2·- redox
The redox response of oxygen in DMSO was described
elsewhere [17]. The results (Fig. 5, curve 4) designate
the one-electron superoxide (O2·-) ion formation due to
well-proven electroreduction reaction in aprotic solvents
(Scheme 2) [23, 35]. The calculated values of O2/O2·-
redox potential and oxygen diffusion coefficient are in
The O2/O2·- redox processes were analyzed by the
coulometric approach. An example of current vs. time
plots obtained in the first cycles of voltammograms
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S. M. KUZMIN ET AL.
The gray colored area between the curves labeled 1
and 2 (Fig. 6) corresponds to the quantity of electricity
associated with H2T(p-OHPh)P electroreduction in the
degassing solution (Q1red in Table 3). The quantity of
electricity Q2red associated with oxygen electroreduction
in the oxygenated solution and Q3red — porphyrins and
oxygen electroreduction in the oxygenated solution
was determined in a similar way. The gray colored area
between the curves labeled 3 and 4 shows the Q3red
–
Q2red value that should be equal to Q1red in case of two
independent processes. In other words. the Q3red–Q1red
value (the area between the curves labeled 2 and 4)
should be equal to Q2red. The coulometric characteristics
of the reduction processes are presented in Table 3.
According to Table 3, simultaneous reduction processes
of porphyrins and oxygen lead to an increase in Qred
values. So, the charges of individual oxygen reduction
(Q2red) differ by more than 15% from the Q3red–Q1red values
estimated the charges of oxygen reduction in porphyrins
presence if the processes are assumed additive.
Fig. 6. The redox currents vs. time plots: (1) background;
(2) 1 mM H2T(p-OHPh)P in degassing solution; (3) oxygen in
oxygenated solution; (4) porphyrins and oxygen in oxygenated
solution. The scan rate is 0.02 V/s
Figure 7 illustrates the effects of H2T(m-OHPh)P
and H2T(p-OHPh)P on the redox process of the O2/O2•-
couple. In general, the porphyrins evoke a similar
transformation of CV curves: the shift of positions of
cathodic and anodic maxima as well as a decrease in
the reduction onset potentials. The decrease in the gaps
between the cathodic and anodic maxima indicates the
increasing rate constants of the interfacial electron
transfer [31]. The influence of (aminophenyl)porphyrins
was similar as described elsewhere [17]. Obviously,
are shown in Fig. 6. The electroreduction process is
accompanied by an electron transfer from the electrode to
the electroactive species in the solution, so they dominate
if the current value is below the zero current line. The
quantity of reduction species produced during the first
CV cycle is proportional to the quantity of electricity (Q)
of reduction processes. The Q values were determined
by integrating the experimental i(t) dependence after
correcting the current value using background current.
Table 3. Coulometric characteristics of reduction processes (scan rate of 0.02 V/s)
Porphyrin concentration,
1 mM
Background
(Q0red, mC)
Q2red, mC
Q1red, mC
Q3red, mC
Q3red-Q1red, mC
H2T(m-OHPh)P
0.00
0.05
0.10
0.25
0.50
1.00
1.50
0.76
0.76
0.76
0.76
0.76
0.76
0.76
13.19
13.19
13.19
13.19
13.19
13.19
13.19
—
—
13.19
12.83
13.19
14.36
13.95
16.15
17.73
0.69
1.11
1.24
1.82
2.77
3.61
13.52
14.3
15.6
15.77
18.92
21.34
H2T(p-OHPh)P
0.00
0.05
0.10
0.25
0.50
1.00
1.50
0.76
0.76
0.76
0.76
0.76
0.76
0.76
13.19
13.19
13.19
13.19
13.19
13.19
13.19
—
—
13.19
15.38
14.63
14.55
16.09
17.59
16.18
1.24
1.37
1.86
2.78
4.39
6.26
16.62
16.00
16.41
18.87
21.98
22.44
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THE COULOMETRIC APPROACH TO THE SUPEROXIDE SCAVENGING ACTIVITY DETERMINATION
7
Fig. 7. H2T(m-OHPh)P (a) and H2T(p-OHPh)P (b) influence on the O2/O2·- redox process. The scan rate is 0.02 V/s. The porphyrin
concentration of 0.00, 0.05, 0.10, 0.25, 0.50, 1.0, 1.5 mM
P2- + O2
•-
P- + O2
Scheme 3. Electron transfer reaction between superoxide and
porphyrin anions.
porphyrins act as redox mediators, most probably, due
·-
…
…
to Por O2 and Por O2 adducts formation because of
the significant interaction of (hydroxyphenyl)porphyrins
with oxygen and superoxide ions that was confirmed by
the transfer of excitation [22, 39] and complexation [40]
phenomena. The greater values of reduction currents in
porphyrins presence can be explained by the reaction
described in Scheme 3.
The dimensionless current density j/jo of O2·- oxidation
was calculated as a ratio of current densities measured
at potentials of peak with (j) and without (jo) porphyrin.
As Fig. 8 shows, the increase in porphyrins concentration
leads to nonlinear variation of O2·- dimensionless current
density. According to [14], the only linear part of such
dependences is used to evaluate antioxidant activity. In
case of H2T(p-OHPh)P, the concentration range from
0.05 to 1 mM can be used. In case of H2T (m-OHPh)P,
it is difficult to offer a linear part of the dependence.
Using the logarithmic coordinates [16] leads to certain
linearization of data. So, the estimation of superoxide
scavenging activities of tetra(aminophenyl)porphyrins
[17] was successful in terms of binding constants [16].
The influence of antioxidants [13] and porphyrin [41]
on the oxygen reduction was also reported. Hence, the
noticeable effects of antioxidant on the oxygen reduction
and superoxide oxidation currents as well as nonlinearity
of current vs. concentration dependences are quite typical.
Fig. 8. H2T(m-OHPh)P (1) and H2T(p-OHPh)P (2) influence
on dimensionless peak current density of O2·- electrooxidation.
·-
The scan rate is 0.02 V/s. jo is the peak current density of O2
electrooxidation without porphyrins
complicated cases. The quantity of electricity of the
oxidation processes was calculated by integrating the
experimental i(t) curves of the CV anodic branch. The
calculation method was the same as described above:
the quantity of electricity of O2•- oxidation in porphyrins
presence was estimated as Q3ox–Q1ox where the quantity
of electricity Q3ox associated with electrooxidation in
the oxygenated solution of porphyrins and Q1ox — with
electrooxidation in the degassed solution of porphyrins.
The results are shown in Fig. 9.
As it follows from Fig. 9, the coulometric response of
superoxide ion vs. porphyrins concentration (line 2) is
linear.ThelinesofH2T(m-OHPh)P(a)andH2T(p-OHPh)P
(b) are extrapolated to the similar zero concentration
point. The slopes (-2.24 CM-1 for H2T(m-OHPh)P and
-2.95CM-1 forH2T(p-OHPh)P)indicatemorepronounced
Coulometric approach to the superoxide
scavenging activity determination
In our opinion, using of coulometric parameters
improves the antiradical activity determination in
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S. M. KUZMIN ET AL.
·-
Fig. 9. H2T(m-OHPh)P (a) and H2T(p-OHPh)P (b) influence on the quantity of electricity of O2 electroreduction (1) and O2
electrooxidation (2)
Fig. 10. Dimensionless parameters of H2T (m-OHPh)P (1) and H2T (p-OHPh)P (2) superoxide scavenging activity (explanations
are shown in the text)
antioxidant properties of the para-substituted compound.
But the involvement of porphyrins in the processes of
radical synthesis is not considered if only lines 2 are
analyzed. As it follows from line 1 Fig. 9 and Table 3,
the quantity of electricity of oxygen electroreduction
increases almost linearly with porphyrins concentration.
So, the factor of superoxide electrosynthesis can be
included into the scavenging activity determination.
The dimensionless parameter v was calculated using
Equation 3:
scavenging activity determination. The change in the
v values was described using parameter k (Equation 4,
Fig. 10b).
k = (v0–v)/v0
(4)
where v0 is the v value extrapolated to the zero
concentration point.
The slopes of k value vs. concentration plots should be
considered as a superoxide scavenging characteristic. The
rise of the antioxidant activity leads to an increase in the
plot slope. The slopes (0.6 L.mmol-1 for H2T(m-OHPh)P
and 0.76 L.mmol-1 for H2T(p-OHPh)P) show the higher
superoxide scavenging properties of H2T(p-OHPh)P.
v = (Q3ox–Q1ox)/(Q3red–Q1red
)
(3)
where (Q3ox–Q1ox) is the quantity of electricity
•-
associated with O2 oxidation in porphyrins presence,
(Q3red–Q1red) is the quantity of electricity associated
with O2 reduction in porphyrins presence. The v values
normalize the coulometric response of superoxide
(Fig. 10a) and, consequently, improve the radical
CONCLUSION
The electrochemical assay of superoxide scavenging
activity was adopted to porphyrins having an intricate
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THE COULOMETRIC APPROACH TO THE SUPEROXIDE SCAVENGING ACTIVITY DETERMINATION
9
electrochemical behavior and a significant effect on the
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redox of the O2/O2•- couple. The factor of superoxide
electrosynthesis included into the scavenging activity
determination allows us to obtain the more careful
parameter of the radical scavenging activity. The new
method was used to demonstrated the hydroxy-group
positions influence on the superoxide scavenging activity;
it was found to be identical to amino-group positions
influence described elsewhere [17]. This fact indicates the
similar (hydrogen atom transfer) antioxidant mechanism.
Therefore, predominantly effect of bond dissociation
energy (BDE) for labile H-atom in phenyl substituents
on the antioxidant activities of tetraphenyl porphyrins
should be expected.
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The authors thank Professor Semeikin A. S. (Ivanovo
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developing methods of porphyrins synthesis and
assistance. The research was financially supported by
the Russian Foundation for Basic Research, grant no.
13-03-00087.
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