The Study of Mg(II) Ion Influence on Catechol Autoxidation in Weakly Alkaline Aqueous Solution

Article abstract of DOI:10.1134/S0036024419130223

Abstract: High performance liquid chromatography with diode array detection (HPLC-DAD) and electron spin resonance (ESR) spectroscopy were used to study Mg(II) ion influence on the autoxidation of catechol in weakly alkaline solution. The presence of Mg(II) ions greatly enhanced the catechol autoxidation rate and probably influenced the mechanism of reaction thus enabling formation of reaction products not obtained in the absence of metal ions. Consecutive formation of 1,2-benzoquinone, 2,3-oxanthrenediol, and 2,3-oxanthrenedione with intermediate o-semiquinone anion radicals during the initial stages of catechol autoxidation was suggested based on the detailed analysis of experimental HPLC-DAD and ESR data. The results of this study may help in better understanding of autoxidation of some biologically important catecholic molecules in real systems, where Mg(II) ions are ubiquitously present. Because of the possible toxicity of simple quinone molecules, it is important that the formation of relatively stable quinoid autoxidation products were detected in this study.

Full text of DOI:10.1134/S0036024419130223

ISSN 0036-0244, Russian Journal of Physical Chemistry A, 2019, Vol. 93, No. 13, pp. 2656–2660. © Pleiades Publishing, Ltd., 2019.
PHYSICAL CHEMISTRY
OF SOLUTIONS
The Study of Mg(II) Ion Influence on Catechol Autoxidation
in Weakly Alkaline Aqueous Solution
G. M. Nikolića,*, S. C. Živanovićb, N. S. Krstićc, and M. G. Nikolićc
a Department of Chemistry, Faculty of Medicine, University of Niš, Niš, 18000 Serbia
b Research Center for Biomedicine, Faculty of Medicine, University of Niš, Niš, 18000 Serbia
c Department of Chemistry, Faculty of Sciences and Mathematics, University of Niš, Niš, 18000 Serbia
* e-mail: goranhem@yahoo.com
Received December 3, 2018; revised December 3, 2018; accepted February 12, 2019
Abstract—High performance liquid chromatography with diode array detection (HPLC-DAD) and electron
spin resonance (ESR) spectroscopy were used to study Mg(II) ion influence on the autoxidation of catechol
in weakly alkaline solution. The presence of Mg(II) ions greatly enhanced the catechol autoxidation rate and
probably influenced the mechanism of reaction thus enabling formation of reaction products not obtained in
the absence of metal ions. Consecutive formation of 1,2-benzoquinone, 2,3-oxanthrenediol, and 2,3-oxan-
threnedione with intermediate o-semiquinone anion radicals during the initial stages of catechol autoxidation
was suggested based on the detailed analysis of experimental HPLC-DAD and ESR data. The results of this
study may help in better understanding of autoxidation of some biologically important catecholic molecules
in real systems, where Mg(II) ions are ubiquitously present. Because of the possible toxicity of simple quinone
molecules, it is important that the formation of relatively stable quinoid autoxidation products were detected
in this study.
Keywords: catechol, autoxidation, magnesium ion
DOI: 10.1134/S0036024419130223
INTRODUCTION
Up to date, most studies of catechol autoxidation in
the presence of metal ions were concerned with the
reaction kinetics and less attention has been paid to the
nature of reaction products [5, 8]. In this study we
applied high performance liquid chromatography with
diode-array detection (HPLC-DAD) and electron
spin resonance (ESR) spectroscopy to study the influ-
ence of Mg(II) ions on catechol autoxidation in
weakly alkaline aqueous solution with the emphasis on
structure determination of initial reaction products.
Catechol (1,2-benzenediol) autoxidation is an
interesting research subject because catechol moiety is
found widely in many plant polyphenolic antioxidants
[1] and drugs [2] whose biological activities are tightly
connected with its ease of oxidation and autoxidation.
Autoxidation of catechol(s) was also recently investi-
gated in connection with the design of wet adhesive
materials [3].
Literature data from many studies revealed that the
most important factors influencing catechol(s) autox-
idation are pH value [3, 4] and the presence of metal
ions [5–9]. Catechol autoxidation rate is greatly
enhanced at higher pH values [3] and in the presence
of redox active transition metal ions at very low (cata-
lytic) concentrations [5]. As opposed to transition
metal ions, alkaline earth ions, like Mg(II), exert this
effect at much higher concentrations [7, 8] but their
influence may be very important due to their ubiqui-
tous presence in the environment and all biological
systems. Indeed, the results of one recent study suggest
that human body antioxidant defenses may be partially
modulated by magnesium status as a result of Mg(II)
EXPERIMENTAL
All the chemicals used in this study were of analyt-
ical (p.a.) grade, except acetonitrile which was of
HPLC grade quality. Catechol (CAT) and trifluoro-
acetic acid were purchased from Merck (Germany).
Deionized water was obtained by using TKA
Smart2Pure water purification system (Thermo Sci-
entific, Germany).
Working solutions of CAT (0.5 mmol dm–3) were
prepared just before the use by diluting CAT stock
solution (10 mmol dm–3) obtained by dissolving
ion ability to stabilize cell membranes and oxidation exactly weighted amount of CAT in 0.1 mol dm–3 solu-
targets, such as adrenaline (a typical example of bio- tion of HCl. Autoxidation of CAT was initiated by
logically important catecholic molecule) [10].
mixing equal volumes of CAT solution and Tris buffer
2656

THE STUDY OF MG(II) ION INFLUENCE
2657
40
30
20
10
0
100
90
80
70
60
50
CAT-Mg(II)
40
30
20
0
20
40
60
80
100
ꢀ, min
10 CAT
0
Fig. 2. Catechol consumption during the autoxidation in
the absence (solid line) and presence of Mg(II) ions
(dashed line).
0
3
6
9
12
15
18
ꢀ, min
(https://www.niehs.nih.gov/research/resources/soft-
ware/).
Fig. 1. Chromatograms of reaction mixtures after 90 min
of catechol autoxidation in the absence (CAT) and in the
presence of Mg(II) ions (CAT-Mg(II)) (detection at
275 nm).
RESULTS AND DISCUSSION
Chromatograms of reaction mixtures obtained
after 90 min of catechol autoxidation at pH 8.4 in the
absence and presence of Mg(II) ions are shown in Fig. 1.
(100 mmol dm–3, pH 8.4) without salt addition or with
the addition of MgCl2 (0.2 mol dm–3). Reaction mix-
tures were kept at room temperature (22
exposed to air during the experiments.
The most prominent chromatographic peak in
both systems at retention time (RT) of 15.6 min
belongs to catechol. It is evident that catechol peak
area is much smaller in CAT-Mg(II) system in com-
parison to the system where catechol autoxidation was
performed in the absence of metal ions (CAT) which
indicates enhanced catechol autoxidation rate in the
presence of Mg(II) ions.
1°C)
HPLC analyses were performed on an Agilent
Technologies (USA) 1200 Series system equipped with
a vacuum degasser, binary pump, temperature con-
trolled column chamber, autosampler, and a diode-
array detector (DAD). Separation was achieved at
30°C by using Purospher STAR RP-18e column
(150 × 4.6 mm, 5 μm, Merck, Germany). Gradient
elution was employed with 0.1% aqueous trifluoroace-
tic acid (mobile phase A) and acetonitrile (mobile
phase B). The gradient of B was established as follows:
0–12 min from 10 to 40%, 12–15 min from 40 to 80%,
15–16 min holding 80%, and 16–20 min from 80 to
100%. The flow rate was 0.5 mL/min, and the injec-
tion volume was 10 μL. The chromatograms were
recorded in regular 30 min time intervals at detection
wavelengths of 220, 275, and 320 nm.
The dependence of relative catechol HPLC peak
area on autoxidation time is given in Fig. 2.
While only about 2% of catechol is consumed
during 90 min of autoxidation in the absence of metal
ions, almost 40% of catechol is consumed in the same
time in the presence of Mg(II) ions. Similar enhance-
ment of autoxidation rates by the presence of Mg(II)
and Ca(II) ions in weakly alkaline aqueous solutions
has been already reported for some catecholic plant
antioxidants [11–13]. Observed pattern of catechol
consumption is very similar to the patterns of oxygen
consumption during the autoxidation of catechol and
ESR spectra were recorded at room temperature two catecholic drug molecules (dopamine and iso-
proterenol) in weakly alkaline aqueous solutions in the
presence of an excess of alkaline earth metal ions [8].
Data from cited study clearly indicate that enhanced
autoxidation rate of catecholic molecules is principally
caused by the lowering of their pK values in the pres-
ence of alkaline earth metal ions.
on an X-band Bruker ESR-300E spectrometer
(Bruker, Germany) by using quartz flat cell for aque-
ous samples. The main ESR settings were as follows:
microwave frequency—9.64 GHz, microwave
power—2.0 mW, modulation frequency—100 kHz,
and modulation amplitude—0.1 G. Manipulation
and computer simulation of ESR spectra were per-
formed by using epr.exe and simepr.exe DOS pro-
grams downloaded from the Web site of the National
In the chromatograms shown in Fig. 1 two com-
mon chromatographic peaks with RT values of 6.2 and
14.1 min appear and they obviously originate from the
Institute of Environmental Health Sciences, USA initial autoxidation products of catechol. Since
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2658
NIKOLIĆ et al.
10
Exp.
Sim.
5
0
200
300
400
500
600
ꢁ, nm
3435
3440
3445
H, G
Fig. 3. DAD UV–Vis spectra of catechol (solid line) and
autoxidation products with RT 6.2 min (dashed line) and
RT 14.1 min (dotted line).
Fig. 4. ESR spectrum recorded after 5 min of catechol
autoxidation at pH 8.4 in the presence of Mg(II) ions and
its computer simulation.
reversed-phase chromatographic separation was
employed, lower RT values in comparison to catechol
(RT 15.6 min) indicate that these compounds are
more polar than catechol. Both common chromato-
graphic peaks appeared in the chromatograms
recorded 30 min after catechol autoxidation was initi-
ated. However, while the intensity of these chromato-
graphic peaks steadily increased during the 90 min of
catechol autoxidation in the CAT system, the intensity
of peak with RT 6.2 min decreased and that for the
peak with RT 14.1 min increased in the CAT-Mg(II)
system.
Beside these two common chromatographic peaks,
the chromatogram of CAT-Mg(II) system contains
additional chromatographic peaks at RT values of
10.8, 13.8, and 16.8 min, not present in the chromato-
gram of CAT system. Straightforward explanation for
the appearance of these additional chromatographic
peaks would be the formation of further catechol
autoxidation products in the CAT-Mg(II) system due
to the higher reaction rate. However, two of the addi-
tional chromatographic peaks in CAT-Mg(II) system
have RT values lower than catechol (15.6 min) while
prolonged catechol autoxidation is expected to pro-
duce oligomeric and/or polymeric products with
higher RT values in comparison to catechol. There-
fore, it is highly probable that the presence of Mg(II)
ions also influences the mechanism of reaction and
causes the formation of different reaction products not
obtained in the absence of Mg(II) ions.
Autoxidation reactions of 1,2-benzenediols in alka-
line aqueous solutions are generally known to involve
free radical species which can be detected by ESR
spectroscopy [15] and the first step in catechol autox-
idation is expected to be the formation of its o-semiqui-
none anion radical. However, attempts to record use-
ful ESR spectra of solutions where catechol autoxida-
tion at pH 8.4 was performed in the absence of Mg(II)
ions were not successful. This lack of ESR signal was
perhaps the consequence of low concentration and/or
high reactivity of free radicals formed which make
them virtually undetectable by static ESR method
used in this study.
We succeeded to obtain good quality ESR spectra
for aqueous solution where catechol autoxidation at
pH 8.4 was performed in the presence of Mg(II) ions
at different time intervals from the start of reaction as
a result of the so called spin-stabilization effect [16,
17]. ESR spectrum recorded after 5 min of catechol
autoxidation at pH 8.4 in the presence of Mg(II) ions
and its computer simulation are shown in Fig. 4.
Asymmetrical appearance of experimental ESR
spectrum indicates simultaneous presence of at least
two different radical species at the early stage of cate-
chol autoxidation in the presence of Mg(II) ions. The
primary radical formed was obviously Mg(II) ion
spin-stabilized o-benzosemiquinone anion radical
since its ESR spectrum is characterized by triplet of
triplets with 1 : 2 : 1 signal intensity ratios originating
from two pairs of two equivalent protons in its struc-
ture [16, 17]. Also, measured hyperfine coupling con-
DAD UV–Vis spectra of catechol and two com-
pounds present in both investigated systems (at RT of
6.2 and 14.1 min) are shown in Fig. 3.
Noticeable absorbance of two common autoxida- stant values (3.925 and 0.520 G) are in excellent agree-
tion products with RT of 6.2 and 14.1 min above ment with the literature data for this radical [17].
400 nm is probably the consequence of increased elec- Good computer simulation (correlation 0.9903) of
tron delocalization which may be indicative for the experimental ESR spectrum shown in Fig. 4 was
formation of quinoid compounds during catechol obtained by assuming simultaneous existence of two
autoxidation [7, 14].
radical species, one of them being the primary Mg(II)
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THE STUDY OF MG(II) ION INFLUENCE
2659
OH
O−
O
O
O
−2H+, −e−
−e−
OH
OH
O
O
O
O
OH
−2H+
+
OH
O
OH
O−
OH
OH
O
O
O
O
O
−2H+, −e−
−e−
O
O
O
Fig. 5. Initial reactions during the catechol autoxidation in weakly alkaline aqueous solution.
ion spin-stabilized o-benzosemiquinone anion radi- this is in agreement with the order of their RT values in
cal. For the secondary radical the structure containing
three groups of two equivalent protons with hyperfine
coupling constant values of 2.715, 0.505, and 0.270 G
was assumed. Based on this assumption, this second-
ary radical may be the Mg(II) ion spin-stabilized
o-semiquinone anion radical obtained by the one
reversed-phase HPLC system used in this study. As for
the 2,3-oxanthrenediol formed in the reaction
between 1,2-benzoquinone and catechol, calculated
ACD/logP value (3.16) points toward much higher RT
value in comparison to catechol and such compound
was not detected in chromatograms shown in Fig. 1.
However, the characterization of Mg(II) ion spin-sta-
electron
oxidation
of
2,3-oxanthrenediole
(dibenzo[b,e][1,4]dioxin-2,3-diol), a catechol dimer
whose quinone formation during catechol oxida- bilized secondary radical detected by ESR spectros-
tion/autoxidation under various conditions was
already reported in the literature [18–21]. Calculated
g-value of 2.0039 for the secondary radical is lower
than the g-value of Mg(II) ion spin-stabilized o-ben-
zosemiquinone anion radical (2.0042 [17]) thus indi-
cating increased unpaired electron delocalization in
comparison to the primary radical which is in agree-
ment with its proposed structure. ESR spectra
recorded during the catechol autoxidation up to the
105 min of reaction have similar appearance to the
spectrum shown in Fig. 4 with gradually increased sig-
nal intensity of secondary radical and lower signal-to-
noise ratio, especially in the central part of the spectra,
which may be indicative for the presence of small con-
centrations of some additional free radical(s).
copy can be taken as evidence for the formation of 2,3-
oxanthrenediol because only its one electron oxida-
tion can give such a radical in this system.
CONCLUSIONS
Autoxidation of catechol in weakly alkaline solu-
tion in the absence and in the presence of Mg(II) ions
was studied by using high performance liquid chroma-
tography with diode array detection (HPLC-DAD)
and electron spin resonance (ESR) spectroscopy. The
presence of Mg(II) ions greatly enhanced the catechol
autoxidation rate and possibly influenced the mecha-
nism of reaction by enabling formation of reaction
products not obtained in the absence of metal ions.
Based on the analysis of experimental results
obtained by the HPLC-DAD and ESR spectroscopy
we propose the order of initial reactions during the Detailed analysis of experimental HPLC-DAD and
catechol autoxidation in weakly alkaline aqueous solu-
tions shown in Fig. 5.
ESR data indicated that successive formation of
1,2-benzoquinone, 2,3-oxanthrenediol, and 2,3-
oxanthrenedione with intermediate o-semiquinone
Successive formation of 1,2-benzoquinone, 2,3-
oxanthrenediol, and 2,3-oxanthrenedione with inter- anion radicals occurred during the initial stages of cat-
mediate o-semiquinone anion radicals is proposed
during the initial stages of catechol autoxidation. Two
quinones, 1,2-benzoquinone and 2,3-oxanthrenedi-
one, are compounds with RT values of 6.2 and
14.1 min, respectively, in chromatograms shown in
Fig. 1. Calculated ACD/LogP values (data taken from
www.chemspider.com) for 1,2-benzoquinone (‒0.60)
and 2,3-oxanthrenedione (0.63) indicate that these
echol autoxidation. The results of this study may help
in better understanding of autoxidation of some bio-
logically important catecholic molecules in real sys-
tems with ubiquitous presence of Mg(II) ions. Addi-
tional point to note is the formation of relatively stable
quinoid autoxidation products, which may be very
important if we consider the reactivity and possible
compounds are more polar than catechol (0.88) and toxicity of simple quinone molecules [22, 23].
RUSSIAN JOURNAL OF PHYSICAL CHEMISTRY A Vol. 93 No. 13 2019

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NIKOLIĆ et al.
ACKNOWLEDGMENTS
10. C. P. Monteiro, C. N. Matias, M. Bicho, H. Santa-
Clara, and M. J. Laires, Magnes. Res. 29, 161 (2016).
This work was partially supported by the Ministry for
Science and Technological Development of the Republic of
Serbia (grant no. 172044).
11. S. C. Živanović, R. S. Nikolić, and G. M. Nikolić, Acta
Fac. Med. Naiss. 33, 163 (2016).
12. S. C. Živanovic, R. S. Nikolić, D. A. Kostic, and
G. M. Nikolić, Oxid. Commun. 40, 581 (2017).
REFERENCES
13. S. C. Živanović, A. M. Veselinović, Ž. J. Mitić, and
G. M. Nikolić, New J. Chem. 42, 6256 (2018).
1. S. Quideau, D. Deffieux, C. Douat-Casassus, and
14. L. V. Volod’ko, A. I. Komyak, A. A. Min’ko, B. A. Ta-
tarinov, and P. A. Matusevich, J. Appl. Spectrosc. 24,
717 (1976).
15. R. Hoskins, J. Chem. Phys. 23, 1975 (1955).
16. D. R. Eaton, Inorg. Chem. 3, 1268 (1964).
L. Pouységu, Angew. Chem. Int. Ed. 50, 586 (2011).
2. D. P. Yang, H. F. Ji, G. Y. Tang, W. Ren, and H. Y. Zhang,
Molecules 12, 578 (2007).
3. G. P. Maier, C. M. Bernt, and A. Butler, Biomater. Sci.
6, 332 (2018).
17. C. C. Felix and R. C. Sealy, J. Am. Chem. Soc. 104,
4. M. Friedman and H. S. Jürgens, J. Agric. Food Chem.
1555 (1982).
48, 2101 (2000).
18. W. G. C. Forsyth, V. C. Quesnel, and J. B. Roberts,
5. J. Balla, T. Kiss, and R. F. Jameson, Inorg. Chem. 31,
Biochim. Biophys. Acta 37, 322 (1960).
58 (1992).
19. M. B. McBride and F. J. Sikora, J. Inorg. Biochem. 39,
6. P. García, C. Romero, M. Brenes, and A. Garrido,
247 (1990).
J. Agric. Food Chem. 44, 2101 (1996).
20. A. Naidja, P. M. Huang, and J.-M. Bollag, Soil Sci.
7. G. M. Nikolić, P. I. Premović, and R. S. Nikolić, Spec-
Soc. Am. J. 62, 188 (1998).
trosc. Lett. 31, 327 (1998).
21. F. Solano, New J. Sci. 2014, 498276 (2014).
8. A. V. Lebedev, M. V. Ivanova, A. A. Timoshin, and
E. K. Ruuge, ChemPhysChem 8, 1863 (2007).
22. J. L. Bolton and T. Dunlap, Chem. Res. Toxicol. 30, 13
(2017).
9. E. Nikhili, M. Loonis, S. Mihai, H. El Hajji, and
O. Dangles, Food Funct. 5, 1186 (2014).
23. T. M. Penning, Toxicol. Res. 6, 740 (2017).
RUSSIAN JOURNAL OF PHYSICAL CHEMISTRY A
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