A novel oxidative dimer from protocatechuic esters: Contribution to the total radical scavenging ability of protocatechuic esters

Article abstract of DOI:10.1271/bbb.80130

A novel oxidative dimer was isolated as a major product from a reaction mixture of methyl protocatechuate and DPPH radical in methanol. Its unusual benzobicyclo[3.2.1]octane structure was elucidated by extensive spectral analysis. This result suggests that the regeneration of catechol structures by the nucleophilic addition of an alcohol molecule on o-quinones and subsequent dimerization is one of the key reactions in the high radical-scavenging activity of protocatechuic esters in an alcoholic solvent.

Full text of DOI:10.1271/bbb.80130

Biosci. Biotechnol. Biochem., 72 (7), 1877–1880, 2008
A Novel Oxidative Dimer from Protocatechuic Esters:
Contribution to the Total Radical Scavenging Ability of Protocatechuic Esters
y
Shizuka SAITO and Jun KAWABATA
Laboratory of Food Biochemistry, Division of Applied Bioscience,
Graduate School of Agriculture, Hokkaido University, Kita-ku, Sapporo 060-8589, Japan
Received March 3, 2008; Accepted April 11, 2008; Online Publication, July 7, 2008
[doi:10.1271/bbb.80130]
A novel oxidative dimer was isolated as a major
product from a reaction mixture of methyl protoca-
techuate and DPPH radical in methanol. Its unusual
benzobicyclo[3.2.1]octane structure was elucidated by
extensive spectral analysis. This result suggests that the
regeneration of catechol structures by the nucleophilic
addition of an alcohol molecule on o-quinones and
subsequent dimerization is one of the key reactions in
the high radical-scavenging activity of protocatechuic
esters in an alcoholic solvent.
necessary to clarify unidentified intermediates and
oxidation products. In this paper, we report the identi-
fication of a novel dimer formed by the oxidation of
1 with DPPH radical in methanol, and we propose a
radical scavenging mechanism in alcoholic solvents.
Materials and Methods
Chemicals. Methyl protocatechuate (2) and methyl
3,4-dihydroxy-2-methoxybenzoate (4) were prepared
by methods described previously.7) DPPH radical
and other reagents were purchased from Wako Pure
Chemical Industries (Osaka, Japan). All solvents used
were of reagent grade.
Key words: antioxidant; protocatechuic acid; DPPH rad-
ical; radical-scavenging mechanism
Protocatechuic acid (3,4-dihydroxybenzoic acid) and
its esters are o-diphenols ubiquitously found in edible
plants, vegetables, and fruits. They are known to exhibit
potent antioxidant activities.1–3) It has been found that
protocatechuic acid has preventive effects in carcino-
genesis and cardiovascular diseases that are associated
with free radicals.4–6) We have reported that in non-
alcoholic solvents, methyl protocatechuate (1) scaveng-
es two equivalents of DPPH (2,2-diphenyl-1-picrylhy-
drazyl) radical and is converted to the corresponding o-
quinone (2).7) In alcoholic solvents, 1 rapidly scavenged
five equivalents of the radical to form 2, its 3-hemiacetal
(3), and further oxidized products.8) In situ NMR
analysis of the reaction mixture of 1 and DPPH radical
in methanol revealed that 2 underwent a nucleophilic
attack by a methanol molecule at C-2 to form C-2
alcohol adduct (4), which scavenged two additional
radicals to yield o-quinone (5) and its 3-hemiacetal (6).9)
Furthermore, 5 underwent a second nucleophilic attack
by a methanol molecule at C-6 to form a 2,6-bis-alcohol
adduct (7), which scavenged additional radicals to form
its o-quinone (8) and 3-hemiacetal (9).10) It has thus
been found that the nucleophilic addition of a solvent
molecule on o-quinone is a crucial reaction in the high
radical-scavenging activity of protocatechuic esters in
alcoholic solvents.9,10) However, to represent the whole
picture of the radical-scavenging mechanism, it is
Apparatus. NMR spectra were recorded on a Bruker
AMX500 spectrometer (Bruker, Tsukuba, Japan, 1H,
500 MHz; 13C, 125 MHz). Chemical shifts were ex-
pressed relative to the residual signals of methanol-d4
(ꢀH 3.30, ꢀC 49.0 ppm). Electron ionization (EI) and Fast
atom bombardment (FAB) mass spectra were obtained
with a Jeol AX500 instrument (Jeol, Tokyo, Japan).
Optical absorbance was acquired using a Hitachi U-3210
spectrophotometer (Hitachi, Tokyo, Japan). Analytical
thin-layer chromatography was performed on silica
gel plates Merck 60 F254 (Merck Ltd., Tokyo, Japan,
0.25 mm thickness). Ordinary phase column chromatog-
raphy was performed with silica gel, Wakogel C-300
(Wako).
Colorimetric radical scavenging tests. DPPH radical-
scavenging activity was measured as described previ-
ously.7,9) To a solution of a test compound (12.5 mM,
4 ml) was added 1 ml of DPPH radical (500 mM) in a test
tube. The solution was immediately mixed vigorously
for 10 s with a Vortex mixer and transferred to a cuvette.
An absorbance reading at 517 nm was taken at 30 min
after initial mixing. Acetonitrile and methanol were
chosen as inert non-alcoholic and nucleophilic alcoholic
solvents respectively. A solution of DL-ꢁ-tocopherol
at the same concentration was measured as a positive
y
To whom correspondence should be addressed. Tel/Fax: +81-11-706-2496; E-mail: junk@chem.agr.hokudai.ac.jp

1878
S. SAITO and J. KAWABATA
control. A reduction in absorbance of 0.228 by the
positive control was regarded as corresponding to the
consumption of two molecules of DPPH radical. All
experiments were performed in triplicate.
Molecular orbital calculations. The electron density
and energy of LUMO were calculated by the AM1
method using the MOPAC 2000 program in the
Chem3D package (CambridgeSoft, Cambridge, MA).
Isolation of oxidation products of 1. To a solution of
compound 1 (67 mg, 0.40 mmol) in methanol (1.6 liters)
was added DPPH radical (0.95 g, 2.4 mmol, 6.0 equiv.),
and the mixture was stirred for 1 h at room temperature.
A solution of sodium dithionite (0.40 g, 2.4 mmol, 6.0
equiv.) in water (24 ml) was added to the reaction
mixture, and this was stirred for 30 min to reduce
unstable o-quinones to their catechol forms. The result-
ing mixture was concentrated under reduced pressure,
and the residue was dissolved in acetone and filtered.
The filtrate was concentrated under reduced pressure.
The residue was subjected to silica gel column chroma-
tography and eluted first with chloroform to remove
DPPH radical and its reduced hydrazine, and then with
methanol. The resulting methanol eluate was concen-
trated under reduced pressure. The crude product was
further purified by preparative HPLC (column, ODS-
Prep, 20 ꢀ 250 mm, GL-Sciences, Tokyo, Japan; mobile
phase, 40% acetonitrile containing 0.1% formic acid;
flow rate, 5.0 ml/min; detection, UV 254 nm) to afford 4
(0.9 mg, 2.1%, tR 12.4 min)7) and 13 (7.0 mg, 8.8%, tR
15.0 min). 13: a yellow oil. 1H-NMR (methanol-d4):
3.29 (3H, s, 20-OCH3), 3.53 (3H, s, 20-OCH3), 3.73
(3H, s, 70-OCH3), 3.76 (3H, s, 7-OCH3), 5.81 (1H, d,
J ¼ 9:8 Hz, H-50), 7.11 (1H, s, H-2), 7.73 (1H, d, J ¼
9:8 Hz, H-60). 13C-NMR (methanol-d4): 52.2 (7-OCH3),
52.5 (20-OCH3), 52.8 (70-OCH3), 53.9 (20-OCH3), 65.3
(C-10), 93.4 (C-30), 116.9 (C-2), 119.0 (C-20), 119.6
(C-5), 123.4 (C-1), 124.4 (C-50), 137.7 (C-6), 145.8
(C-3), 148.1 (C-4), 150.0 (C-60), 168.1 (C-7), 171.5
(C-70), 196.5 (C-40). HMBC correlation peaks: H-2/C-4,
C-6, H-50/C-10, C-30, H-60/C-6, C-20, C-40, 20-OCH3/
C-20, 7-OCH3/C-7, 70-OCH3/C-70. HR-FAB-MS m=z
ðM ꢁ HÞꢁ: calcd. for C18H17O10, 393.0822; found,
393.0827.
Results and Discussion
Compound 13, together with the C-2 methanol adduct
(4), was isolated as an oxidation product from the
reaction mixture of 1 and DPPH radical in methanol. Its
molecular formula was identified as C18H18O10 from the
HR-FAB-MS result, indicating that 13 was a dimeric
1
product of 1. The H-NMR spectrum showed a pair of
doublet signals at ꢀ 5.81 (H-50) and ꢀ 7.73 (H-60), with a
larger coupling constant (J ¼ 9:8 Hz) than that found in
4 (JH-5/H-6 ¼ 8:6 Hz). H-60 showed JCH HMBC corre-
3
lations with an acetal carbon at ꢀ 119.0 (C-20) and a
carbonyl carbon at ꢀ 196.5 (C-40), indicating that 13 was
a quinone acetal-like product. Additionally, HMBC
correlations between C-20 and two methoxy groups
suggested the formation of dimethyl acetal at C-20. H-50
was correlated with two sp3 carbons of C-10 and C-30.
Moreover, the HMBC spectrum showed correlations of
H-2/C-6 and H-60/C-6. These results suggest that
the two protocatechuoyl residues were linked at C-6
and C-10. The other interunit C-C linkage was between a
quaternary benzene carbon assignable to C-5 (ꢀ 119.6)
and a downfield-shifted quaternary oxycarbon of C-30 (ꢀ
1
93.4). On the basis of H and 13C-NMR, HMQC, and
HMBC spectral data, the structure of 13 is proposed to
be as depicted in Fig. 1.
From a reaction mixture of 4 and DPPH radical in
methanol, compound 14 was isolated by the procedure
described for 13. The negative FAB-MS of 14 exhibited
a ½M ꢁ Hꢂꢁ peak at m=z 423, indicating that the
molecular weight of 14 was 30 mass units larger than
that of 13. The 1H-NMR spectrum of 14 was very
similar to that of 13, except for the disappearance of a
singlet signal of H-2 and the presence of the signal of an
additional methoxy group. Furthermore, the 2D NMR
spectra were consistent with those of 13. Hence it was
concluded that the structure of 14 was the 2-methoxy
derivative of 13.
A plausible formation mechanism for 13 is outlined in
Scheme 1. First, 5 undergoes nucleophilic attack by a
methanol molecule at C-20 to yield dimethyl acetal (10).
Since the resulting dimethyl acetal (10) does not have a
proton at C-20, regeneration of a catechol structure
cannot occur. Instead, ketonization of C-40 generates an
Isolation of compound 14. Compound 14 was isolated
from a reaction mixture of 4 and DPPH radical by the
method described for 13 (15% from 4, tR 17.2 min).
Compound 14: a yellow oil. 1H-NMR (methanol-d4):
3.29 (3H, s, 20-OCH3), 3.53 (3H, s, 20-OCH3), 3.73
(3H, s, 7-OCH3), 3.76 (3H, s, 70-OCH3), 3.80 (3H, s,
2-OCH3), 5.81 (1H, d, J ¼ 9:8 Hz, H-50), 7.73 (1H, d,
J ¼ 9:8 Hz, H-60). 13C-NMR (methanol-d4): 52.1 (7-
OCH3), 52.4 (20-OCH3), 53.0 (70-OCH3), 53.8 (20-
OCH3), 62.1 (2-OCH3), 65.5 (C-10), 93.0 (C-30), 116.3
(C-1), 118.8 (C-5), 135.7 (C-3), 139.4 (C-6), 147.8
(C-4), 148.1 (C-2), 119.0 (C-20), 124.4 (C-50), 150.3
(C-60), 167.2 (C-7), 171.2 (C-70), 196.3 (C-40). HMBC
correlation peaks: H-50/C-10, C-30, H-60/C-6, C-20,
C-40, 2-OCH3/C-2, 20-OCH3/C-20, 7-OCH3/C-7, 70-
OCH3/C-70. HR-FAB-MS m=z ðM ꢁ HÞꢁ: calcd. for
C19H19O11, 423.0927; found, 423.0951.
MeO OMe
OH
OH
2'
4
3'
HO
3
5
O
4'
5'
2
1'
1
6
6'
CO2Me
CO2Me
Fig. 1. Key HMBC Correlations of 13.

Novel Oxidative Dimer from Protocatechuic Esters
1879
-2H+
-2
OH
O
2
MeO OH
MeOH
OH
O
O
MeO2C
MeO2C
MeO2C
1
3
MeOH
a
-2H+
-2
MeOH
O
OH
4
O
MeOH
MeO
MeO OH
3'
2'
MeO
MeO
OH
O
4'
5'
MeO2C
MeO2C
1'
MeO2C
6'
b
5
6
b
MeOH
-2H+
-2
a
OH
O
MeO OH
MeO
O
MeOH
MeO
MeO
OH
O
MeOH
MeO2C
MeO2C
MeO2C
OMe
OMe
OMe
7
8
9
OMe
2'
OMe
OMe
MeO
MeO
MeO
O
O
H
-2H+
-2
O
6'
OH
O
O
4
OH
4
OH
OH
OH
MeO 2'
MeO
3'
MeO
MeO
3'
4' OH
5'
3
2
O
R
O
O
3
HO
R
5
O
O
O
O
R
O
5
4'
5'
1'
1'
6
6
R
MeO2C
R
6'
2
1
1
CO2Me
H
7'CO2Me
CO2Me
CO2Me
CO2Me
CO2Me
CO2Me
7 CO2Me
CO2Me
10
2: R = H
5: R = OMe
11: R = H
12: R = OMe
13: R = H
14: R = OMe
15: R = H
16: R = OMe
Scheme 1. Possible Formation Mechanism for Compounds 13 and 14.
anion at C-10, which attacks C-6 of 2 to form 11 via
Michael addition. Then intramolecular aldol condensa-
tion and subsequent aromatization give 13. Similarly,
dimerization of 5 and 10 forms 14.
6
5
4
3
2
1
0
To substantiate this preference in the positions of the
nucleophilic attacks, the electron density of LUMO of 5
was calculated by the semi-empirical method. The
electron densities of the carbons of 5 were as follows:
C-10, 0.20; C-20, 0.38; C-30, 0.29; C-40, 0.16; C-50, 0.14;
and C-60, 0.17. Since the LUMO electron density of C-20
of 5 was much higher than that of C-60, the nucleophilic
addition on C-20 of 5 to form 10 (reaction a, Scheme 1)
should be a kinetically preferred reaction. However, we
have found that 5 underwent a nucleophilic attack at
C-60 (reaction b, Scheme 1), rather than at C-20, by a
second methanol molecule to yield the 2,6-bis-methanol
adduct (7) in methanol.10) Considering that reaction a
is reversible and that dimerization occurs only when
a reactive C-10 anion produced is caught by a strong
electrophile such as an o-quinone, that exists in the near
environment, a nucleophilic addition on C-60, a less
likely but irreversible reaction, should occur in part
and then rapidly convert to a thermodynamically stable
catechol (7). It is interesting that a similar benzobi-
cyclo[3.2.1]octane dimer was isolated as a coupling
product between 4-methyl-o-benzoquinone and 5-meth-
ylpyrogallol.11) This dimerization reaction might be a
common oxidation mechanism of polyphenol molecules.
Indeed, HPLC analysis of reaction mixtures of 1 and 4
0
5
10
15
Time (min)
20
25
30
Fig. 2. Time Courses of DPPH Radical Scavenging Activity of
Methyl Protocatechuate (1, ), and Its Oxidative Dimers 13 ( ) and
14 ( ) in Methanol.
Equivalence is expressed as values relative to that of DL-ꢁ-
tocopherol as 2.0.
with DPPH radical in methanol revealed that these
dimers, 13 and 14 respectively, were the predominant
products.
The DPPH radical scavenging activity of isolated 13
and 14 was examined in methanol (Fig. 2). Both 13
and 14 scavenged approximately two equivalents of
the radical in 30 min, indicating that 13 and 14 were
converted to the corresponding o-quinones (15 and 16).

1880
S. SAITO and J. KAWABATA
2866–2887 (2003).
As shown in Scheme 1, two molecules of 1 should
scavenge eight equivalents of the radical to afford 15
and 10 equivalents to yield 16, indicating consumption
of four and five equivalents respectively per molecule of
1. The fact that more than five equivalents of the radical
are scavenged by 1 in methanol cannot be explained
only by the formation of these dimers. The radical-
scavenging reactions of polyphenols are quite complex,
and hence a complete elucidation of the total reaction
profile appears to be impracticable. However, in this
case, considering that 13 and 14 were the predominant
isolable oxidation products from 1 and 4 respectively,
even in the diluted solution, the formation of these
dimers evidently contributes to the total radical scav-
enging activity of 1.
In conclusion, compound 13, a novel oxidation dimer,
was isolated from a mixture of 1 and DPPH radical
in methanol. The result suggests that regeneration of
catechol structures by the nucleophilic addition of a
solvent molecule on o-quinones and subsequent dime-
rization are key reactions in the high radical-scavenging
activity of protocatechuic esters in alcoholic solvents.
Further investigation is needed to determine whether the
radical-scavenging reaction of protocatechuic esters
found in the present study occurs in aqueous biological
systems.
2) Natella, F., Nardini, M., Felice, M. D., and Scaccini, C.,
Benzoic and cinnamic acid derivatives as antioxidants:
structure-activity relation. J. Agric. Food Chem., 47,
1453–1459 (1999).
3) Mattila, P., and Kumpulainen, J., Determination of
free and total phenolic acids in plant-derived foods by
HPLC with diode-array detection. J. Agric. Food Chem.,
50, 3660–3667 (2002).
4) Nakamura, H., Nishikawa, A., Furukawa, F., Kasahara,
K., Miyauchi, M., Son, H. Y., and Hirose, M., Inhibitory
effects of protocatechuic acid on the post-initiation
phase of hamster pancreatic carcinogenesis induced by
N-nitrosobis(2-oxopropyl)amine. Anticancer Res., 20,
3423–3427 (2000).
5) Tseng, T. H., Hsu, J. D., Lo, M. H., Chu, C. Y., Chou,
F. P., Huang, C. L., and Wang, C. J., Inhibitory effect
of Hibiscus protocatechuic acid on tumor promotion in
mouse skin. Cancer Lett., 126, 199–207 (1998).
6) Tanaka, T., Kojima, T., Kawamori, T., and Mori, H.,
Chemoprevention of digestive organs carcinogenesis
by natural product protocatechuic acid. Cancer, 75,
1433–1439 (1995).
7) Saito, S., Okamoto, Y., and Kawabata, J., Effects of
alcoholic solvents on antiradical abilities of protocate-
chuic acid and its alkyl esters. Biosci. Biotechnol.
Biochem., 68, 1221–1227 (2004).
8) Saito, S., Okamoto, Y., Kawabata, J., and Kasai, T.,
Quinone hemiacetal formation from protocatechuic
acid during the DPPH radical scavenging reaction.
Biosci. Biotechnol. Biochem., 67, 1578–1579 (2003).
9) Saito, S., and Kawabata, J., Effects of electron-with-
drawing substituents on DPPH radical scavenging
reactions of protocatechuic acid and its analogues in
alcoholic solvents. Tetrahedron, 61, 8101–8108 (2005).
10) Saito, S., Gao, H., and Kawabata, J., DPPH radical
scavenging reaction of protocatechuic esters in alcohols:
formation of bis-alcohol adduct. Helv. Chim. Acta, 89,
821–831 (2006).
Acknowledgments
We are grateful to Mr. Kenji Watanabe and Dr. Eri
Fukushi of the GC-MS and NMR Laboratory of our
school for measuring mass spectra. This work was
supported by research fellowships from the Japan
Society for the Promotion of Science for Young
Scientists (to S.S.).
11) Yanase, E., Sawaki, K., and Nakatsuka, S., The isolation
of a bicyclo[3.2.1] intermediate during formation of
benzotropolones, a common nucleus found in black
tea pigments: theaflavins. Synlett, 2661–2663 (2005).
References
1) Robbins, R. J., Phenolic acids in foods: an overview of
analytical methodology. J. Agric. Food Chem., 51,