Experimental and theoretical investigations of the antioxidant activity of 2,2′-methylenebis(4,6-dialkylphenol) compounds

Article abstract of DOI:10.1002/aoc.3562

The antioxidant activity of two primary antioxidants, 2,2′-methylenebis(4-methyl-6-tert-butylphenol) (MMBPH2) and 2,2′-methylenebis(4,6-di-tert-butylphenol) (MDBPH2), has been studied using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) method. The synthesized compounds have been successfully characterized systematically using elemental analyses, infrared, ¹H NMR and ¹³C NMR spectra and GC–MS. Importantly, it has been found that the compound MMBPH2 in particular is more active in DPPH radical scavenging. In addition, density functional theory calculations (B3LYP) have been used to predict the antioxidant activity and predict structural geometries of the compounds in the gas phase.

Full text of DOI:10.1002/aoc.3562

Received: 6 May 2016
DOI 10.1002/aoc.3562
Revised: 9 June 2016
Accepted: 27 June 2016
F U L L PA P E R
Experimental and theoretical investigations of the antioxidant
activity of 2,2′‐methylenebis(4,6‐dialkylphenol) compounds
1
2
Wail Al Zoubi | François Karabet | Rana Al Bandakji2 | Khansaa Hussein2
*
1 Plasticity Control and Mechanical Modeling
The antioxidant activity of two primary antioxidants, 2,2′‐methylenebis(4‐methyl‐6‐
Laboratory, School of Materials Science and
Engineering, Yeungnam University, Gyeongsan
38541, Republic of Korea
2 Chemistry Department, Faculty of Sciences,
Damascus University, Damascus, Syria
tert‐butylphenol) (MMBPH2) and 2,2′‐methylenebis(4,6‐di‐tert‐butylphenol)
(MDBPH2), has been studied using the 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH)
method. The synthesized compounds have been successfully characterized system-
1
atically using elemental analyses, infrared, H NMR and 13C NMR spectra and
Correspondence
GC–MS. Importantly, it has been found that the compound MMBPH2 in particular
is more active in DPPH radical scavenging. In addition, density functional theory
calculations (B3LYP) have been used to predict the antioxidant activity and predict
structural geometries of the compounds in the gas phase.
François Karabet, Chemistry Department, Faculty
of Sciences, Damascus University, Damascus,
Syria.
Email: info@scibd-sy.com
KEYWORDS
antioxidants, dimethoxymethane, diphenylpicrylhydrazyl, DFT calculations
1
|
INTRODUCTION
minimize oxidative damage resulting from excessive light
exposure.[11] Various methods are currently used to assess
the antioxidant activity of phenolic compounds. ABTS+•
Phenolic compounds are commonly found in both edible and
inedible plants, and they have been reported to have multiple
biological effects, including antioxidant activity.[1] Herbs are
used in many domains, including medicine, nutrition,
flavoring, beverages, dyeing, repellents, fragrances and
cosmetics.[2] Many species have been recognized to have
medicinal properties and beneficial impact on health, e.g.
antioxidant activity, digestive stimulation action, anti‐inflam-
matory, antimicrobial, hypolipidemic and antimutagenic
effects and anticarcinogenic potential.[3]
Free radicals are molecules, ions or atoms with unpaired
electrons in their outermost shell of electrons.[4,5] These
species, which are constantly formed in the human body,
can become toxic when generated in excess or in the presence
of a deficiency in naturally occurring antioxidant defenses.
High levels of free radicals can cause damage to biomole-
cules such as lipids, proteins, enzymes and DNA in cells
and tissues.[6–8]
(2,2'azinobis(3‐ethylbenzthiazoline‐6‐sulfonic acid)) or
DPPH• (1,1‐diphenyl‐2‐picrylhydrazyl) radical scavenging
methods are common spectrophotometric procedures for
determining the antioxidant activities of components.[12]
Therefore, in the study reported here, the antioxidant
activity of 2,2′‐methylenebis(4‐methyl‐6‐tert‐butylphenol)
(MMBPH2) and 2,2′‐methylenebis(4,6‐di‐tert‐butylphenol)
(MDBPH2) was investigated using the DPPH method and
using the Gaussian 03 program with density functional
theory (DFT/B3LYP) using 6‐31G(d) basis set for geometry
optimization. MMBPH2 and MDBPH2 are effective
sterically hindered phenolic primary antioxidants, which are
widely used to protect rubbers, oils, fats, adhesives and waxes
from ageing.
2
| MATERIALS AND METHODS
In previous studies, reactive oxygen species, such as
superoxide radicals, hydroxyl radicals and peroxyl radicals,
have been reported as natural byproducts of the normal
metabolism of oxygen in living organisms with important
roles in signaling.[9,10] In addition, phenols with antioxidant
activity could scavenge reactive chemical species as well as
2.1 | Materials
2‐tert‐Butyl‐4‐methylphenol, 2,4‐di‐tert‐butylphenol, dime-
thoxymethane, n‐heptane, sulfuric acid 98% and ethanol were
obtained from Merck. Paraformaldehyde was obtained from
Panreac. DPPH was obtained from Sigma Aldrich.
Appl. Organometal. Chem. 2016; 1–6
wileyonlinelibrary.com/journal/aoc
Copyright © 2016 John Wiley & Sons, Ltd.
1

2
AL ZOUBI ET AL.
Melting points were determined with an Electrothermal
2.3 | Characterization
9200. Elemental analyses (C, H and N) were carried out with
a Heraeus instrument (Vario EL). Purity determination was
carried out using HPLC (Shimadzu HPLC‐SPD‐M20 A) with
MMBPH2. Anal. Found (calcd) for C23H32O2 (%): C, 80.10
(81.13); H, 8.42 (9.47). IR (cm−1): 1232.9 (C─O), 1477.6
(C═C), 3121.1 (C─H), 3393 (─OH). 1H NMR (CDCl3,
400 MHz, δ, ppm): 1.44 (s, 18H), 2.3 (s, 6H), 3.93 (s, 2H),
5.86 (s, 2H, OH‐Ar), 7 (s, 4H, Arom). 13C NMR (CDCl3,
100 MHz, δ, ppm): 150.07–126.4 (Ar‐C), 34.36 (Ar‐CH2‐
Ar), 32.14 (Ar‐CH3), 30.01 (C(CH3)3), 20.91 (CH3).
GC–MS: 177 (100%), 57 (32.1%), 105 (14.3%), 77 (7%),
106 (4%).
MDBPH2. Anal. Found (calcd) for C29H44O2 (%): C,
81.1 (82.02); H, 9.43 (10.44). IR (cm−1): 1199.6 (C─O),
1478.2 (C═C), 3121.1 (C─H), 3531.5 (─OH). 1H NMR
(CDCl3, 400 MHz, δ, ppm): 1.32 (s, 18H), 1.45 (s, 18H),
3.97 (s, 2H), 5.92 (s, 2H, OH‐Ar), 7.16 (d, J = 5.3 Hz, 2H
(near to CH2), Ar), 7.24 (d, J = 5.3 Hz, 2H, Ar). 13C NMR
(CDCl3, 100 MHz, δ, ppm): 149.9–122.57 (Ar‐C), 34.65
(Ar‐CH2‐Ar), 32.54 (C(CH3)3), 31.61 (3CH3 (near to OH)),
30.07 (3CH3). GC–MS: 177 (100%), 57 (100%), 219
(57.14%), 105 (7.14%).
a
Chromolith‐Performance HPLC column RP‐8e
(100 × 4.6 mm). The flow rate was 1 ml min−1. The
chromatograms were recorded at 280 nm (UV–visible). MS
was carried out with a Shimadzu GC–MS mass spectrometer.
The flow rate was 1 ml /min−1. The gradient profile was: 90 °
C (3 min); 280 °C (20 min). H NMR (400 MHz) and 13C
1
NMR (100 MHz) spectra were recorded with a Bruker
Advance spectrometer. Infrared (IR) spectra were obtained
with a FTIR Unicam, Galaxy Series 5000 type spectrometer
using KBr discs.
2.2 | Preparation of MMBPH2 and MDBPH2
2.2.1
| Dimethoxymethane method
Amounts of 0.025 mmol of sulfuric acid, 0.4 mmol of
dimethoxymethane and 0.5 mmol of 2‐tert‐butyl‐4‐
methylphenol (or 2,4‐di‐tert‐butylphenol) were added to a
reactor with a thermometer, a condenser and a stirrer. The
contents were stirred for 2 h at 60–70 °C. Then the stirrer
was removed and the reaction mixture was left in an ice
chest or refrigerator overnight. Then petroleum ether was
added and stirred until the complete dissolution of the
reaction mixture and then the sulfuric acid was separated.
The petroleum ether and unreacted dimethoxymethane were
distilled off from the reaction mixture. The residue was
purified with 280 ml of an ethanol–water mixture (3:1 v/v)
at 75 °C, with stirring for 1 h. After that the resulting mix-
ture was cooled to 20 °C, filtered, washed with 160 ml of
ethanol–water mixture and filtered. The residue was dried
for 3 h at 85 °C.
3
| RESULTS AND DISCUSSION
MMBPH2 and MDBPH2 were prepared by two referential
methods: the first one uses dimethoxymethane as condensing
agent for 2,4‐dialkylphenols[10] and the second one uses
paraformaldehyde.[11] The reaction for both methods was
carried out in the presence of sulfuric acid (Scheme 1).
As evident from Table 1, the compound MMBPH2
(Fig. 1) is an effective antioxidant comparing with gallic acid
at 1 mmol l−1 concentration, and it is more effective by 35%
than MDBPH2 (Fig. 1), even when MDBPH2 is used at a
concentration of more than 20 mmol l−1. From the theoretical
study which used density functional theory (DFT/B3LYP) it
is found that the two compounds are stable and have asym-
metric stereochemical structures. The tert‐butyl groups at
para position in MDBPH2 lead to stereochemical hindrance
preventing the compound from making an intramolecular
hydrogen bond which means this compound has less ability
MMBPH2: yield 70%; m.p. 127–130 °C; purity ≥99%
(HPLC). MDBPH2: yield 84%; m.p. 147–150 °C; purity
≥99% (HPLC).
2.2.2
| Paraformaldehyde method
Amounts of 0.006 mmol of sulfuric acid, 200 ml of n‐hep-
tane, 0.5 mmol of 2‐tert‐butyl‐4‐methylphenol (or 2,4‐di‐
tert‐butylphenol) and 0.275 mmol of paraformaldehyde were
added to a reactor with a thermometer, a condenser and a
stirrer. The contents of the reactor were stirred for 2 h at
85–93 °C. Then the obtained reaction mixture was cooled
to 20 °C and the sulfuric acid separated. The n‐heptane was
distilled off from the reaction mixture. The residue was
purified with an ethanol–water mixture (3:1 v/v) in the same
way as described for the dimethoxymethane method.[11]
MMBPH2: yield 69.7%; m.p. 127–129 °C; purity ≥99%
(HPLC). MDBPH2: yield 85%; m.p. 149–150 °C; purity
≥99% (HPLC).
SCHEME 1 Synthesis of MMBPH2 and MDBPH2.

AL ZOUBI ET AL.
3
TABLE 1 Results of DPPH tests
MDBPH2
23.6
MMBPH2
1.18
Gallic acid
Concentration (mmol l−1
DPPH (%)
)
28.3
21.2
1.47
1.12
1
57.9 0.5
54.5 0.25
50.7 0.5
82.9 0.45
76.6 0.61
75.76 0.38
74.89
calculated. The free radical scavenging activity is expressed
as follows:
AB−AA
AB
DPPH scavenging activity ð%Þ ¼
×100
where AB is the absorbance of the blank (EtOH) and AA is the
absorbance of the sample. All experiments were performed in
triplicate.
FIGURE 1 Chemical structures of MMBPH2 and MDBPH2
to donate a hydrogen radical to free radicals. For this reason,
MMBPH2 is more effective than MDBPH2 as an antioxidant.
Three solutions with different concentrations of the two
compounds were prepared using ethanol as diluent. An
amount of 6 ml of 45 μg ml−1 DPPH solution was added to
100 μl of each of the three solutions of the two compounds.
The mixed solution was incubated at room temperature for
30 min in the dark, and then the absorbance of the reaction
mixture was measured at 517 nm. Table 1 shows the results
of the DPPH test, and the difference in the antioxidant
activity between the two compounds.
3.1 | Study of Antioxidant Activity using DPPH
Method
Gallic acid was used as phenolic reference to study the anti-
oxidant activity of the two compounds MMBPH2 and
MDBPH2.
A series of standards of gallic acid were prepared
(Table 2). Five normal solutions with different concentrations
(0.2, 0.4, 0.6, 0.8, 1 mmol l−1) (Fig. 2) were prepared from a
10 mmol l−1 solution of gallic acid, using ethanol as diluent.
An amount of 6 ml of 45 μg ml−1 DPPH solution was added
to 100 μl of each of the normal solutions of gallic acid. The
mixed solution was incubated at room temperature for
30 min in the dark, and then the absorbance of the reaction
mixture was read at 517 nm and the remaining DPPH was
3.2 | Theoretical Study
The theoretical treatment of MMBPH2 and MDBPH2
included in this work was performed using the DFT/B3LYP
approach implemented in the Gaussian 03 series of pro-
grams.[13,14] It aimed to find a scientific explanation for the
results of the DPPH test. Standard pseudopotentials devel-
oped in Toulouse were used to describe the atomic cores.
3.2.1
| Optimized geometries
TABLE 2 Series of standards of gallic acid
The geometries of MMBPH2 and MDBPH2 were optimized
using analytic gradients. These results (Figs 3 and 4; Table 3)
show that the two compounds have C1 symmetry. Hydroxyl
group O(1)─H(1) (which is connected to carbon atom C(4))
and hydroxyl group O(2)─H(2) (which is connected to
Absorbance
1.374
1.142
0.927
0.707
0.523
0.345
Concentration
0
0.2
0.4
0.6
0.8
1
(mmol l−1
)
FIGURE 2 Series of standards of gallic acid
FIGURE 3 DFT/B3LYP optimized geometry of MMBPH2

4
AL ZOUBI ET AL.
compound MDBPH2 has tert‐butyl groups at the para posi-
tion in each of the two aromatic rings. The tert‐butyl groups
cause stereochemical hindrance and force the two parts of
MDBPH2 (the two aromatic rings) to turn around the methy-
lene group (CH2) just to retain the hybrid sp3 orbital for
carbon atom C(1) which makes the distance between H(1)
and O(2) (Fig. 4) to be 4.188 Å (instead of the 1.800 Å in
MMBPH2).[15] The optimized geometrical parameters are
summarized in Table 3. According to the previous study we
can state the following reasons as to why MMBPH2 is more
effective than MDBPH2 as an antioxidant:
1. Stereochemical structure. Figure 4 shows the bulky
stereochemical structure of the tert‐butyl groups at para
position to the hydroxyl groups in MDBPH2 decreases
the ability of the hydroxyl groups to donate a hydrogen
radical to the free radicals in oxidation reactions and
decreases the ability of these free radicals to reach the
hydrogen radical of the hydroxyl group; this situation
does not occur for MMBPH2.[15]
2. Intramolecular hydrogen bond.[14] The intramolecular
hydrogen bond in MMBPH2 (Fig. 3) stabilizes the
phenoxy radical of the compound. This will make the
hydroxyl groups in this compound have more of an
ability to donate a hydrogen radical to free radicals than
the hydroxyl groups in MDBPH2 which does not have
an intramolecular hydrogen bond, it just having an
intermolecular hydrogen bond (Fig. 5).
FIGURE 4 DFT/B3LYP optimized geometry of MDBPH2
TABLE 3 Selected geometrical parameters (bond lengths, Å; angles, °) for
MMBPH2 and MDBPH2 optimized at the DFT/B3LYP level (see Figs 3 and
4 for labeling of the atoms)
MMBPH2
C1
MDBPH2
C1
Geometrical
parameters
1.106
1.110
1.535
1.521
1.380
1.379
0.971
0.976
4.188
120.1
118.6
109.3
108.8
119.6
119.6
102.4
23.8
1.101
1.101
1.531
1.531
1.373
1.393
0.981
0.970
1.800
121.3
113.7
112.3
110.3
121.9
121.1
169.3
38.2
C(1)–H(3)
C(1)–H(4)
C(1)–C(2)
C(1)–C(3)
C(4)–O(1)
C(7)–O(2)
O(1)–H(1)
O(2)–H(2)
O(2)–H(1)
C(2)–C(4)–O(1)
C(3)–C(7)–O(2)
C(4)–O(1)–H(1)
C(7)–O(2)–H(2)
C(1)–C(2)–C(4)
C(1)–C(3)–C(7)
O(1)–H(1)–O(2)
O(1)–H(1)–O(2)–H(2)
O(1)–C(4)–O(2)–C(7)
3. Inductive effect of the alkyl groups.[14] The positive
inductive effect of the tert‐butyl groups (+I) is stronger
than the (+I) of the methyl groups. This will make the
hydroxyl groups in MMBPH2 have more of an ability
to donate a hydrogen radical to free radicals than the
−98.2
22.4
carbon atom C(7)) are asymmetric, which is because com-
pound MMBPH2 has an intramolecular hydrogen bond
between H(1) and O(2), with a length of 1.800 Å, whereas
FIGURE 5 Intramolecular hydrogen bond in MMBPH2
TABLE 4 Calculated harmonic vibrational frequencies and IR intensities (Km mol−1) at the DFT/B3LYP level for MMBPH2 and MDBPH2
MDBPH2
MMBPH2
Experimental (IR)
B3LYP
Intensity (Km mol−1
Experimental (IR)
B3LYP
Vibration (cm−1
)
Vibration (cm−1
38013078
)
)
Vibration (cm−1
)
Intensity (Km mol−1
)
Vibration (cm−1
)
3393
2953
—
93325
3531.5
2955.6
—
297920
38123593
─OH
310030873087
30202951
5073166
187176
575878
300730183083
308830883044
─CH aliphatic
751223
─CH aliphatic
(methylene
group)
—
1642163616251622
1256
3021
111
—
2119
1650164616311626
1273
Benzene
─C═O
1232.9
—
1199.6
—
58
3161318032143245
14483
17151711
3143315431673194
H─benzene

AL ZOUBI ET AL.
5
[3] K. Aaby, E. Hvattum, G. Skrede, J. Agric. Food Chem. 2004, 52, 4595–4603.
hydroxyl groups in MDBPH2 which has tert‐butyl
groups in para and ortho positions.
[4] S. F. Barbuceanu, D. C. Ilies, G. Saramet, V. Uivarosi, C. Draghici, V.
Radulescu, Int. J. Mol. Sci. 2014, 15, 10908–10925.
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45, 1068–1077.
3.2.2
| Vibrational frequency calculations
The harmonic vibrational frequencies of the different station-
ary points of the potential energy surface have been calcu-
lated at the same level of theory in order to identify the
local minima.
The vibration frequency of the ─OH bond in MDBPH2
has two theoretical values (3812 and 3593 cm−1). The theo-
retical value which has the high intensity is in agreement with
the experimental value obtained from IR analysis: 3531.5 cm
−1 (Table 4).[15,16]
[7] V. P. Patil, V. L. Markad, K. M. Kodam, S. B. Waghmode, Bioorg. Med.
Chem. Lett. 2013, 23, 6259–6263.
[8] P. Muniandy, A. B. Shori, A. S. Baba, Food Packag. Shelf Life 2016, 8, 1–8.
[9] L. Cavas, K. Yurdakoc, J. Exp. Mar. Biol. Ecol. 2005, 325, 189–200.
[10] B. N. Ames, Science 1983, 221, 1256–1264.
[11] I Gulçin. D. Berashvili, A. Gepdiremen, J. Ethnopharmacol.. 2005, 101,
287–293.
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3, 43–53.
[13] Y. A. Gurvich, S. Bulvar, A. A. Grinberg, J. A. Grigorievich, L. I. Michurov,
O. F. Starikova, V. A. Yanshevsky, S. T. Kumok, E. L. Styskin, G. I. Rutman,
US Patent US 4087469, 1997.
4
| CONCLUSIONS
[14] T. Zhanz, Y. Zhang, Z. Wang, CN Patent 159‐65, 2008.
[15] A. A. S. Al‐Hamdani, W. Al Zoubi, Spectrochim. Acta A 2015, 137, 75–89.
We synthesized MMBPH2 and MDBPH2. In this study, the
two compounds were studied experimentally and theoreti-
cally. Also, it has been found that MMBPH2 is more effective
than MDBPH2 as an antioxidant because of the tert‐butyl
groups at para position in MDBPH2 which lead to stereo-
chemical hindrance.
[16] H. Veisi, A. Rashtiani, V. Barjasteh., Appl. Organometal. Chem. 2016, 30,
231–235.
How to cite this article: Al Zoubi, W., Karabet, F.,
Al Bandakji, R., and Hussein, K. (2016), Experimental
and theoretical investigations of the antioxidant activ-
ity of 2,2′‐methylenebis(4,6‐dialkylphenol) com-
pounds, Appl. Organometal. Chem., doi: 10.1002/
aoc.3562
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