Oxidative dimer produced from a 2,3,4-trihydroxybenzoic ester

Article abstract of DOI:10.1271/bbb.70139

The DPPH radical-scavenging abilities of the naturally occurring phenolic acid, 2,3,4-trihydroxybenzoic acid, and its methyl ester were evaluated. Both compounds in acetonitrile scavenged as many as four radicals compared to three or fewer radical consump

Full text of DOI:10.1271/bbb.70139

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Biosci. Biotechnol. Biochem., 71 (7), 1731–1734, 2007
Oxidative Dimer Produced from a 2,3,4-Trihydroxybenzoic Ester
y
Asuka KODAMA, Hidetoshi SHIBANO, and Jun KAWABATA
Laboratory of Food Biochemistry, Division of Applied Bioscience, Graduate School of Agriculture,
Hokkaido University, Sapporo 060-8589, Japan
Received March 12, 2007; Accepted April 2, 2007; Online Publication, July 7, 2007
[doi:10.1271/bbb.70139]
7
The DPPH radical-scavenging abilities of the natu-
rally occurring phenolic acid, 2,3,4-trihydroxybenzoic
CO2R3
1
R1
6
acid, and its methyl ester were evaluated. Both com-
pounds in acetonitrile scavenged as many as four rad-
2
5
3
R2
OH
icals compared to three or fewer radical consumption
in acetone or ethanol. Only the ester showed relatively
high ability in methanol. Oxidation with o-chloranil in
acetonitrile resulted in methyl 2,3,4-trihydroxybenzoate
giving a novel benzocoumarin-type dimer, its chemical
structure being confirmed by spectroscopic evidence.
The formation of this dimer might partly account for
the higher radical-scavenging efficiency of the ester in
acetonitrile or methanol.
4
OH
1: R1=R2=H, R3=CH3
2: R1=H, R2=OH, R3=CH3
3: R1=OH, R2=R3=H
4: R1=OH, R2=H, R3=CH3
Fig. 1. Structures of 2,3,4-Trihydroxybenzoic Acid and Related
Compounds.
Key words: 2,3,4-trihydroxybenzoic acid; phenolic acid;
oxidative coupling; DPPH radical; o-chlo-
ranil
(Fig. 1), and identified a novel oxidative dimer in the
reaction mixture with o-chloranil (3,4,5,6-tetrachloro-o-
benzoquinone) in acetonitrile.
Hydroxybenzoic acids and esters are widely distribut-
ed in plants and have attracted considerable attention for
their antioxidative activities.1–4) The radical-scavenging
reaction of catechol- and pyrogallol-type hydroxyben-
zoic acids typically comprises two distinctive phases,
initially fast and subsequently slow.5,6) The former phase
is recognized as the conversion of an o-diphenol to the
corresponding o-quinone, whereas complex reactions
including oligomerization of the resulting quinone
would occur in the latter slow phase. The difference in
total radical-scavenging efficiency of each hydroxyben-
zoic acid inherently depends on this latter phase,
although little has been found for this complex stage.
So far, a protocatechuic (3,4-dihydroxybenzoic) ester (1)
gave its quinone dimer,6) quinone acetal7) and ad-
ducts,8–11) and a gallic (3,4,5-trihydroxybenzoic) ester
(2) and its analogs also produced quinone dimers6,12,13)
and adducts.12) In contrast to protocatechuic and gallic
acids, there has been no report on the oxidation reaction
of another naturally occurring phenolic acid, 2,3,4-
trihydroxybenzoic acid (3).14–16) We have investigat-
ed the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical-
scavenging abilities and the oxidant-mediated reaction
of 2,3,4-trihydroxybenzoic acid (3) and its ester (4)
Materials and Methods
Reagents. 2,3,4-Trihydroxybenzoic acid (3) (Tokyo
Kasei Kogyo Co.), 2,2-diphenyl-1-picrylhydrazyl (DP-
PH) radical (Wako Pure Chem. Ind.) and o-chloranil
(Aldrich Chem. Co.) were purchased from each supplier.
Methyl 2,3,4-trihydroxybenzoate (4) was prepared from
3 by heating with methanol containing 10% hydrogen
chloride. All solvents used were of technical grade.
Colorimetric radical-scavenging tests. DPPH radical
scavenging tests were conducted as described in the
previous paper.6)
HPLC analysis. A mixture of phenols (0.025 mmol)
and the DPPH radical (0.05 or 0.1 mmol) or o-chloranil
(0.025 or 0.05 mmol) in acetonitrile (0.4 ml) was ana-
lyzed 5 and 70 min after mixing. The analytical condi-
tions were as follow: column, Inertsil PREP-ODS (6:0 ꢀ
250 mm, GL Sciences); mobile phase, 4% (0–5 min), 4–
16% (5–20 min), 16–32% (20–35 min), 32–80% (35–
45 min) and 80% (45–60 min) acetonitrile in water con-
taining 2.5% acetic acid; flow rate, 1.0 ml/min; detec-
y
To whom correspondence should be addressed. Fax: +81-11-706-2496; E-mail: junk@chem.agr.hokudai.ac.jp

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1732
A. KODAMA et al.
tion, UV 260 or 400 nm. Under these conditions, 3 and 4
were eluted at tR ¼ 23:4 and 37.5 min, respectively.
compared to using radical species.12,13,19) Changing the
oxidant from the DPPH radical to o-chloranil could
simplify the chromatogram of the reaction mixture,
probably due to the mild oxidative property of the non-
radical chlorinated quinone compared to the DPPH
radical. Hence, a distinctive new peak (tR ¼ 43:5 min)
was observed in the equimolar reaction mixture of 4 and
o-chloranil in acetonitrile at 5 min, as well as a broad
peak (tR ¼ 23:6 min) detected at 400 nm. The former
peak increased in size during 70 min after the reaction
had started. These two peaks were also observed in the
corresponding reaction mixture in methanol. In addition,
a dark brown precipitate appeared in the acetonitrile
reaction solution after several hours. The precipitated
product was filtered off and directly subjected to in-
strumental analyses, which unveiled that it was a novel
benzocoumarin-type coupling product (5) derived from
two molecules of 4. The mass spectral analysis gave
a molecular peak at m=z 334, and the high-resolution
analysis indicated the molecular formula of C15H10O9,
which corresponded to a loss of CH3OH and 2H from
two molecules of 4. The 1H-NMR spectrum of 5 gave a
relatively simple peak pattern. Only two singlet signals
of aromatic protons at ꢀ 7.09 and 7.86, as well as a
methoxyl signal at ꢀ 3.94 were observed, apart from five
exchangeable hydroxyl protons in the low-field region of
ꢀ 9–11. In the 13C-NMR spectrum, 15 carbon signals,
including two ester carbonyls at ꢀ 164.0 and 168.7 and
one methoxyl at ꢀ 52.7, were observed. In the HMBC
spectrum, the lower-field aromatic proton of ꢀ 7.86
showed correlation peaks with two oxycarbons at ꢀ 142.5
and 149.4, as well as one of the ester carbonyls at 168.7
(C-7), and was thus assigned to H-6 of one unit of 4. On
the other hand, the other aromatic proton at ꢀ 7.09 had a
correlation peak with one upfield-shifted oxycarbon at ꢀ
132.7, being assignable to the central oxygenated carbon
(C-30) of a pyrogallol structure. This proton and the other
ester carbonyl of ꢀ 164.0 (C-70) gave a weak cross peak,
suggesting their W-shaped long-range relationship.
Hence, the higher-field proton could be reasonably
assigned as H-50 of the second unit of 4. The signal of
H-6 showed an additional strong three-bond cross peak
with a quaternary carbon at ꢀ 126.5, and similarly, H-50
also had a cross peak with another quaternary carbon at ꢀ
110.8. The former quaternary carbon was reasonably
assigned as C-60 (ꢀꢀ +13.2), and the latter as C-5 (ꢀꢀ
+11.0), since the chemical shifts of those carbons were
equally shifted downfield compared to unsubstituted C-6
(ꢀ 113.3) and C-50 (ꢀ 99.8), respectively, by their aryl
substitution. These HMBC correlations well support the
connectivity between C-5 of the first unit and C-60 of the
second unit. The connection of C-5 and C-60 enabled ꢀ-
lactone formation between OH-4 of the former unit and
the carboxyl of the latter. A detailed analysis of the
HMBC correlations unambiguously confirmed the total
structure of 5 (Fig. 2).
Isolation of benzocoumarin dimer 5 from the reaction
of methyl 2,3,4-trihydroxybenzoate (4) with o-chloranil.
To a solution of 4 (23 mg, 0.13 mmol) in acetonitrile
(1 ml) was added o-chloranil (31 mg, 0.13 mmol) in
acetonitrile (1 ml). After 16 hr at room temperature, a
dark brown precipitate appearing in the reaction mixture
was separated and washed with acetonitrile to give a
pure dimer (5) as dark brown powder (4 mg, 18%). 5.
Mp >300 ꢁC; FD-MS m=z (%): 334 (100); EI-HR-MS
m=z: 334.0334 ([M]þ, calcd for C15H10O9, 334.0325);
1H-NMR ꢀ (DMSO-d6) ppm: 3.94 (3H, s, H–OCH3),
7.09 (1H, s, H-50), 7.86 (1H, s, H-6), 9.17 (1H, s, OH-30),
9.86 (1H, s, OH-3), 10.57 (1H, s, OH-2), 10.58 (1H, s,
OH-40), 10.98 (1H, s, OH-20); 13C-NMR ꢀ (DMSO-d6)
ppm: 52.7 (C–OCH3), 98.0 (C-10), 99.8 (C-50), 110.7 (C-
1), 110.8 (C-5), 113.3 (C-6), 126.5 (C-60), 132.7 (C-30),
133.6 (C-3), 142.5 (C-4), 149.4 (C-2), 150.0 (C-20),
154.6 (C-40), 164.0 (C-70), 168.7 (C-7); HMBC corre-
lation: H–OCH3/C-7, H-50/C-5, 10, 30, 40 and 70, H-6/C-
2, 4, 7 and 60, OH-30/C-20, OH-3/C-2 and 4, OH-2/C-1,
2 and 3, OH-40/C-30 and 50, OH-20/C-10, 20 and 30.
Results and Discussion
The DPPH radical-scavenging abilities of 2,3,4-
trihydroxybenzoic acid (3) and methyl 2,3,4-trihydrox-
ybenzoate (4) were determined by the colorimetric
method. After 30 min, the relative radical-scavenging
equivalence of each compound, when that of ꢁ-toco-
pherol in ethanol as standard was designated as 2,17,18)
was as follows: 3, 4.3 and 4, 3.7 in acetonitrile; 3, 3.1
and 4, 2.1 in acetone; 3, 2.9 and 4, 4.7 in methanol; and
3, 2.7 and 4, 3.2 in ethanol. The radical-scavenging
ability of 3 was highest in acetonitrile and decreased in
the order of acetonitrile > acetone > methanol > etha-
nol, whereas methyl ester 4 showed the order of meth-
anol > acetonitrile > ethanol > acetone. Unlike the
protocatechuic esters, the DPPH radical-scavenging
equivalence of 2,3,4-trihydroxybenzoic acid (3) and its
ester (4) did not show any marked solvent dependence,
without any difference in alcoholic and non-alcoholic
solvents.9) The radical-scavenging equivalence of 3 and
4 in acetonitrile exceeded three, whereas nearly three or
fewer radicals were consumed in other solvents, except
with 4 in methanol. These results indicate that a further
oxidative reaction might have occurred in acetonitrile,
since only three radicals were expected to be scavenged
during the simple quinone-oxy radical formation from
pyrogallols.
An HPLC analysis of the reaction mixture of 3 or 4
with the DPPH radical in acetonitrile, however, gave
no significant peak of an oxidation product. Oxidation
mechanism studies on polyphenols have frequently used
o-chloranil as an oxidant from a catechol to the cor-
responding quinone, since the reaction proceeds cleanly
Benzocoumarin 5 comprises two molecules of 4 by
connecting between C-5 of one unit and C-6 of the other.

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Oxidative Dimer from 2,3,4-Trihydroxybenzoic Ester
1733
compared to catechol-type ester 1. The rate of the
addition could directly reflect the radical-scavenging
equivalence of 3 and 4 in methanol or ethanol. The
highest equivalence (4.7) obtained with the combination
of 4 and methanol well corresponds to the result from
the high reactivity in methyl protocatechuate (1) and
methanol.9) The relatively low reactivity of 3 (2.9)
compared to 4 can also be accounted for by conversion
to the less reactive carboxylate form of the quinone, as
has been seen in the case of protocatechuic acid.20)
On the other hand, in non-alcoholic solvents, any
direct participation of the solvent molecule in the
reaction must be negligible. Hence, the high radical-
scavenging equivalence of 3 (4.3) and 4 (3.7) in
acetonitrile could be due to the ability to produce a
coupling product such as 5. This type of dimeric product
might easily undergo a further oxidation reaction by
consuming more radicals to give complex products. The
higher dielectric constant of acetonitrile (36.6) com-
pared to less polar acetone (20.7) could be favorable for
the effective molecular stacking of hydrophobic 4 and
4q. Unlike the reactivity in alcoholic solvents, free acid
3 showed higher radical-scavenging activity than its
ester 4 in non-alcoholic solvents. Although the reason is
unknown at this stage, the coupling reaction of 3 and its
quinone may be rapid enough to give polymeric
products, this being supported by the failure to identify
the acid derivative of 5 from the reaction mixture of 3 in
methanol.
O
OCH3
7
1
4
2
OH
OH
H
6'
6
H
5'
HO
HO
3
5
4'
O
1'
3'
7'
2'
OH
O
Fig. 2. HMBC Correlations in 5.
First, 4 is oxidized to give the corresponding 3,4-
quinone (4q), since OH-2 could hardly be oxidized by
intramolecular hydrogen bonding with an ester carbonyl.
Then, nucleophilic attack on C-6 of 4q with C-5 of 4,
which is a doubly activated position by para-OH-2 and
ortho-OH-4, occurs and subsequent deprotonation and
lactonization would afford 5 (Fig. 3). The sharp peak
eluted at tR ¼ 43:5 min in the HPLC analysis could be
assigned as benzocoumarin 5, and the broad peak at
tR ¼ 23:6 min detected at the longer wavelength of
400 nm was probably due to quinone 4q. When
increasing the molar ratio of o-chloranil against 4 to
2:1, the peak of 5 diminished after 70 min of reaction,
although it was visible at 5 min, which suggests that
further oxidation of 5 to the corresponding quinone or
equivalents might occur with excess oxidant. In the case
of 3, similar oxidative coupling could occur in the
reaction with o-chloranil, although the detection of any
corresponding HPLC peak failed and no such precipitate
as that found in the reaction of 4 appeared in the reaction
mixture.
In conclusion, benzocoumarin dimer 5 produced in
the reaction mixture of methyl 2,3,4-trihydroxybenzoate
4 with o-chloranil in acetonitrile or methanol is a novel
type of oxidative coupling product of phenolic acids,
and a similar coupling reaction by other polyphenols
may proceed in suitable reaction media.
Acknowledgments
In alcoholic solvents, the nucleophilic addition of an
alcohol molecule to the resulting quinone would
predominate, although no methanol adduct was actually
identified from the reaction mixture of 4 with the DPPH
radical or o-chloranil in methanol due to the complexity
of the oxidation reaction of pyrogallol-type ester 4
We are grateful to Mr. Kenji Watanabe and Dr. Eri
Fukushi of our school for their skilful measurement of
the mass spectra. This work was supported partly by the
Japan Food Chemical Research Foundation.
OH
HO
OH
MeO2C
OH
OH
4
HO
O
HO
O
O
O
OMe
OH
CO2Me
CO2Me
OH
OH
[O]
MeO2C
OH
OH
H
MeO2C
H
OH
O
O
OH
O
OH
OH
4
4q
5
Fig. 3. Plausible Oxidation Pathway of 4.

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1734
A. KODAMA et al.
11) Saito, S., Gao, H., and Kawabata, J., DPPH (=2,2-di-
phenyl-1-picrylhydrazyl=2,2-diphenyl-1-(2,4,6-trinitro-
phenyl)hydrazyl) radical-scavenging reaction of proto-
catechuic acid esters (=3,4-dihydroxybenzoates) in
alcohols: formation of bis-alcohol adduct. Helv. Chim.
Acta, 89, 821–831 (2006).
References
1) Cuvelier, M.-E., Richard, H., and Berset, C., Compar-
ison of the antioxidative activity of some acid-phenols:
structure-activity relationship. Biosci. Biotechnol. Bio-
chem., 56, 324–325 (1992).
2) Rice-Evans, C. A., Miller, N. J., and Paganga, G.,
Structure-antioxidant activity relationships of flavonoids
and phenolic acids. Free Radic. Biol. Med., 20, 933–956
(1996).
3) Natella, F., Nardini, M., Di Felice, M., and Saccini, C.,
Benzoic and cinnamic acid derivatives as antioxidants:
structure-activity relation. J. Agric. Food Chem., 47,
1453–1459 (1999).
4) Tyrakowska, B., Soffers, A. E. M. F., Szymusiak, H.,
Boeren, S., Boersma, M. G., Lemanska, K., Vervoort, J.,
and Rietjens, I. M. C. M., TEAC antioxidant activity of
4-hydroxybenzoates. Free Radic. Biol. Med., 27, 1427–
1436 (1999).
5) Kimura, T., Yamamoto, S., Ogawa, I., Miura, H., and
Hasegawa, M., Antioxidant ability of chicoric acid and
its analogous compounds. Nippon Kagaku Kaishi, 739–
750 (1999).
6) Kawabata, J., Okamoto, Y., Kodama, A., Makimoto, T.,
and Kasai, T., Oxidative dimers produced from proto-
catechuic and gallic esters in the DPPH radical scaveng-
ing reaction. J. Agric. Food Chem., 50, 5468–5471
(2002).
7) Saito, S., Okamoto, Y., Kawabata, J., and Kasai, T.,
Quinone hemiacetal formation from protocatechuic acid
during DPPH radical scavenging reaction. Biosci. Bio-
technol. Biochem., 67, 1578–1579 (2003).
8) Sayre, L. M., and Nadkarni, D. V., Direct conversion of
phenols to o-quinones by copper (I) dioxygen. Questions
regarding the monophenolase activity of tyrosinase
mimics. J. Am. Chem. Soc., 116, 3157–3158 (1994).
9) 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).
12) Feldman, K. S., Quideau, S., and Appel, H. M., Galloyl-
derived orthoquinones as reactive partners in nucleo-
philic additions and Diels-Alder dimerizations: a novel
route to the dehydrodigalloyl linker unit of agrimoniin-
type ellagitannins. J. Org. Chem., 61, 6656–6665 (1996).
13) Quideau, S., and Feldman, K. S., Ellagitannin chemistry.
The first synthesis of dehydrohexahydroxydiphenoate
esters from oxidative coupling of unetherified methyl
gallate. J. Org. Chem., 62, 8809–8813 (1997).
14) Bhatia, I. S., Nagpal, M. L., Singh, P., Kumar, S., Singh,
N., Mahindra, A., and Parkash, O., Chemical nature of
the pigment of the seed coat of guar (cluster bean,
Cyamopsis tetragonolobus L. Taub). J. Agric. Food
Chem., 27, 1274–1276 (1979).
15) Gorinstein, S., Weisz, M., Zemser, M., Tilis, K., Stiller,
A., Flam, I., and Gat, Y., Spectroscopic analysis of
polyphenols in white wines. J. Ferment. Bioeng., 75,
115–120 (1993).
16) Li, G., Min, B.-S., Zheng, C., Lee, J., Oh, S.-R., Ahn,
K.-S., and Lee, H.-K., Neuroprotective and free radical
scavenging activities of phenolic compounds from
Hovenia dulcis. Arch. Pharm. Res., 28, 804–809 (2005).
17) Blois, M. S., Antioxidant determinations by the use of a
stable free radical. Nature, 181, 1199–1200 (1958).
18) Brand-Williams, W., Cuvelier, M. E., and Berset, C.,
Use of a free radical method to evaluate antioxidant
activity. Lebensm. Wiss. Technol., 28, 25–30 (1995).
19) Quideau, S., Feldman, K. S., and Appel, H. M.,
Chemistry of galloyl-derived o-quinones: reactivity
toward nucleophiles. J. Org. Chem., 60, 4982–4983
(1995).
20) Saito, S., and Kawabata, J., DPPH (=2,2-diphenyl-1-
picrylhydrazyl) radical-scavenging reaction of protoca-
techuic acid (=3,4-dihydroxybenzoic acid): difference in
reactivity between acids and their esters. Helv. Chim.
Acta, 89, 1395–1407 (2006).
10) Saito, S., and Kawabata, J., Synergistic effects of thiols
and amines on antiradical efficiency of protocatechuic
acid. J. Agric. Food Chem., 52, 8163–8168 (2004).