Regioselective dimerization of ferulic acid in a micellar solution

Article abstract of DOI:10.1021/jf0014617

Dehydrodimers of hydroxycinnamates play an important role in the cross-linking of plant cell walls. An aqueous solution of quaternary ammonium salts with a long aliphatic chain is known to spontaneously organize itself into micelles with the ionic part at the outer sphere. It is shown that regioisomeric ferulic acid dehydrodimers can be obtained in one step from trans-ferulic acid after attachment to these micelles and using the biomimetic peroxidase-H₂O₂ system. The surfactant hexadecyltrimethylammonium hydroxide yielded trans-4-(4-hydroxy-3-methoxybenzylidene)-2-(4-hydroxy-3-methoxyphenyl)-5- oxotetrahydrofuran-3-carboxylic acid (25%), (E, E)-4,4′-dihydroxy-5,5′-dimethoxy-3,3′-bicinnamic acid (21%), and trans-5-[(E)-2-carboxyvinyl]-2-(4-hydroxy-3-methoxyphenyl)7-methoxy-2,3- dihydrobenzofuran-3-carboxylic acid (14%), whereas the surfactant tetradecyltrimethyl-ammonium bromide gave 4-cis, 8-cis-bis(4-hydroxy-3-methoxyphenyl)-3,7-dioxabicyclo[3.3.0]octane-2,6-dione (18%) as the main product. The use of micelles appears to be not only a new way to synthesize regioisomeric ferulic acid dehydrodimers but may also help to understand the regiospecificity of dimeric hydroxycinnamate formation in vivo.

Full text of DOI:10.1021/jf0014617

J. Agric. Food Chem. 2001, 49, 3471−3475
3471
Regioselective Dim er iza tion of F er u lic Acid in a Micella r Solu tion
Erik Larsen, Mette F. Andreasen, and Lars P. Christensen*
Department of Horticulture, Danish Institute of Agricultural Sciences,
Kirstinebjergvej 10, DK-5792 Aarslev, Denmark
Dehydrodimers of hydroxycinnamates play an important role in the cross-linking of plant cell walls.
An aqueous solution of quaternary ammonium salts with a long aliphatic chain is known to
spontaneously organize itself into micelles with the ionic part at the outer sphere. It is shown that
regioisomeric ferulic acid dehydrodimers can be obtained in one step from trans-ferulic acid after
attachment to these micelles and using the biomimetic peroxidase-H2O2 system. The surfactant
hexadecyltrimethylammonium hydroxide yielded trans-4-(4-hydroxy-3-methoxybenzylidene)-2-(4-
hydroxy-3-methoxyphenyl)-5-oxotetrahydrofuran-3-carboxylic acid (25%), (E,E)-4,4′-dihydroxy-5,5′-
dimethoxy-3,3′-bicinnamic acid (21%), and trans-5-[(E)-2-carboxyvinyl]-2-(4-hydroxy-3-methoxyphenyl)-
7-methoxy-2,3-dihydrobenzofuran-3-carboxylic acid (14%), whereas the surfactant tetradecyltrimethyl-
ammonium bromide gave 4-cis, 8-cis-bis(4-hydroxy-3-methoxyphenyl)-3,7-dioxabicyclo[3.3.0]octane-
2,6-dione (18%) as the main product. The use of micelles appears to be not only a new way to
synthesize regioisomeric ferulic acid dehydrodimers but may also help to understand the regiospeci-
ficity of dimeric hydroxycinnamate formation in vivo.
Keyw or d s: Ferulic acid; ferulic acid dehydrodimers; micelles; peroxidase; hydrogen peroxide;
surfactant
INTRODUCTION
system (5, 16, 18, 19) or a range of single-electron
oxidants (3, 5). Obviously, some kind of control must
be utilized in the cell in order to produce regiospecific
dehydrodimers. Recently it was discovered that “guid-
ing” proteins were involved in the lignin and lignan
biosynthesis (20) and a similar guiding principle might
be operating in the biosynthesis of dehydrodimers.
It is known that photodimerization of cinnamic acids
can be controlled by the action of quaternary ammonium
or amine N-oxide surfactants (7, 10). These surfactants
form large aggregates, e.g. micelles, in aqueous solutions
with the ionic part at the surface to the water. In the
presence of these aggregates, the carboxylic acids will
be organized around them with the acid part pointing
inward. With trans-ferulic acid and a strongly basic
surfactant a similar organization is expected to occur
as the stronger acid moiety (Ka of carboxylic acid . Ka
of phenol) will be oriented toward the aggregates (Figure
2). With an acidic surfactant this orientation is still
expected as the undissociated carboxylic acid moiety will
have a greater polarity compared to that of a phenolic
group. The aim of this study was to investigate whether
oxidative coupling of trans-ferulic acid can be regiose-
lectively controlled using surfactants.
Hydroxycinnamates have been recognized as impor-
tant components of plant cell walls, and it has been
shown that they are present in significant quantities
mainly ester-linked to polysaccharides in Graminaceous
monocot and Caryophylaceous dicot cell walls (1, 2).
Ferulic acids esterified to polysaccharides, e.g. arabi-
noxylans, pectins, and xyloglucans, can form dimers
through a dimerization reaction. Two mechanisms have
been reported. One is an oxidative coupling reaction
which can be catalyzed by various systems, such as
peroxidase-hydrogen peroxide (3-5) or polyphenol oxi-
dase, including laccases (4), as well as some purely
chemical systems (3, 5, 6), leading to the formation of
various ferulic acid dehydrodimers. The other mecha-
nism is a photoinduced cycloaddition between the eth-
ylene carbons of two phenolic acids leading to the
formation of cyclobutane type dimers such as truxillic
and truxinic acids (7-10). Thus, ferulic acid dehy-
drodimers are then able to cross-link different polysac-
charide chains and, in some cases, polysaccharide and
lignin (11). Cross-linking of cell wall components is
expected to have a marked influence on numerous cell
wall properties such as accessibility, extensibility, plas-
ticity, digestibility, and adherence (12).
The 8-O-4-diFA, 8-5-diFA, 8-5-benzofuran-diFA, 5-5-
diFA, 8-8-aryl-diFA, and 8-8-diFA are the main dehy-
drodimers so far identified in plant materials (Figure
1), with 8-O-4-diFA often being the most abundant (3,
13-17). In contrast, the primary ferulic acid dehy-
drodimer generated in vitro from ferulic acid esters is
the 8-5-coupled product, 8-5-benzofuran-diFA, when
utilizing the biomimetic peroxidase-hydrogen peroxide
MATERIALS AND METHODS
Ch em ica ls. trans-Ferulic acid and peroxidase (EC 1.11.1.7,
type VI-A, 1000 units/mg, from horseradish) were purchased
from Sigma (Deisenhofen, Germany) and 30% aqueous hydro-
gen peroxide (H2O2), ortho-phosphoric acid (purity > 99%),
acetic acid (purity > 99.8%), sodium hydroxide, and anhydrous
sodium sulfate (Na2SO4) were purchased from Merck (Darm-
stadt, Germany). The surfactant tetradecyltrimethylammo-
nium bromide and 10% aqueous hexadecyltrimethylammoni-
um hydroxide were purchased from Aldrich (Steinheim,
Germany) and Fluka (Buchs, Switzerland), respectively. Tri-
* To whom correspondence should be addressed (tele-
phone +45 63 90 43 43; fax +45 63 90 43 95; e-mail larsp.
christensen@agrsci.dk).
10.1021/jf0014617 CCC: $20.00 © 2001 American Chemical Society
Published on Web 06/26/2001

3472 J. Agric. Food Chem., Vol. 49, No. 7, 2001
Larsen et al.
F igu r e 1. Chemical structures of the main ferulic acid dehydrodimers isolated from plants.
2:1) was added to the stirred solution followed by addition of
30% aqueous H2O2 (0.08 mL) and peroxidase (0.1 mg in 0.22
mL). The pH in the former reaction media was 7.0 before
addition of peroxidase and 8.0 after addition 5 min later. With
tetradecyltrimethylammonium bromide as surfactant the pH
remained constant at 3.0. The reactions were quenched after
5 min by addition of aqueous HCl (2.0 M, 20 mL). The reac-
tion mixtures were extracted twice with EtOAc (2 × 50 mL),
and the combined EtOAc extracts were dried over anhydrous
Na2SO4 and evaporated to dryness under reduced pressure at
35 °C.
Dim er iza tion of F er u lic Acid in Bu ffer ed Solu tion s.
trans-Ferulic acid (230 mg) was dissolved in either an acetate
buffer (0.1 M, 110 mL, pH 7.5) or a phosphate buffer (0.1 M,
110 mL, pH 3.0) at 60 °C. After the solution cooled (to ∼ 40
°C), aqueous 30% H2O2 (0.08 mL) was added followed by
peroxidase (0.1 mg in 0.22 mL). The reactions were quenched
after 5 min by addition of aqueous HCl (2.0 M, 20 mL). The
reaction mixtures were extracted twice with EtOAc (2 × 50
mL), and the combined EtOAc extracts were dried over
anhydrous Na2SO4 and evaporated to dryness under reduced
pressure at 35 °C.
Isola tion , Qu a n tifica tion , a n d Id en tifica tion of F er u -
lic Acid Deh yd r od im er s. The residue from the EtOAc
extract was dissolved in MeOH (10 mL) and filtered through
a
0.45-µm nylon filter (Osmonics Cameo syringe filters,
Frisenette ApS, Ebeltoft, Denmark) before separation by
preparative HPLC. For preparative HPLC a Merck L-6200
intelligent pump and a Merck L-4200 UV-Vis detector were
used. Separations were performed at 35 °C on a Develosil ODS-
HG-5 (RP-18, 250 × 20 mm i.d., Nomura Chemical Co., J apan)
column protected with a guard cartridge (50 × 20 mm i.d.)
packed with the same material as the column. The following
gradient was used: 300 mL of 32% MeOH + 1‰ TFA; 300
mL of 34% MeOH + 1‰ TFA, 300 mL of 36% MeOH + 1‰
TFA; 300 mL of 38% MeOH + 1‰TFA; 300 mL of 40% MeOH
+ 1‰ TFA, and 300 mL of 80% MeOH. Compounds were
detected at 280 nm. Flow rate was 7 mL min-1 and injection
volume was 10 mL. The purity of the isolated ferulic acid
dehydrodimers was determined by reversed-phase analytical
F igu r e 2. Expected organization of a strongly basic surfactant
and trans-ferulic acid in an aqueous solution.
fluoroacetic acid (TFA), ethyl acetate (EtOAc), and methanol
(MeOH) were all obtained from Aldrich and was of HPLC
grade.
Dim er iza tion of F er u lic Acid in Micella r Solu tion s.
trans-Ferulic acid (230 mg) was dissolved in water (110 mL)
at 60 °C. After the solution cooled (to ∼ 40 °C), aqueous
hexadecyltrimethylammonium hydroxide (0.3388 M, 3.5 mL,
molar ratio to trans-ferulic acid 1:1) or tetradecyltrimethyl-
ammonium bromide (800 mg, molar ratio to trans-ferulic acid

Regioselective Dimerization of Ferulic Acid
J. Agric. Food Chem., Vol. 49, No. 7, 2001 3473
Ta ble 1. Yield s of Regioisom er ic Deh yd r od im er s fr om
F er u lic Acid P r ep a r ed in Micella r a n d Bu ffer ed
Solu tion s
2, Rt ) 92.2 min; 3, Rt ) 103.7 min; 4, Rt ) 104.9 min; 5, Rt
) 95.6 min) using external standards of 1-5 (Table 1).
The purified ferulic acid dehydrodimers (2-5) were identi-
1
fied by UV, H and 13C NMR, and electrospray mass spectros-
compound, (yields in %)b,c
reaction
copy (ESI-MS). ESI-MS (negative ion) of all isolated dehy-
drodimers showed molecular ion peaks at m/z 385 [M-H]- in
accordance with their structure.
8-8-γ-Lactone-diFA (2): amorphous solid, UV λmax (MeOH);
235, 285 (shoulder), 334 nm and NMR (3) data in accordance
with literature values.
conditionsa
2
3
4
5
i
25 ( 3
25 ( 3
2 ( 1
0
21 ( 2
14 ( 2
38 ( 3
1 ( 1
0
0
0
ii
iii
iv
0
0
0
18 ( 3
0
a
5-5-DiFA (3): mp > 350 °C (literature, (22) > 350 °C), and
UV (13, 16) and NMR (3) data in accordance with literature
values.
8-5-Benzofuran-diFA (4): mp 166-170 °C with decomposi-
tion (literature, (3) 169-174 °C), and UV (13, 16) and NMR
(3) data in accordance with literature values.
8-8-Bis-lactone-diFA (5): mp 211-213 °C (literature, (23)
213.8-214.7 °C), and UV (24) and NMR (23, 24) data in
accordance with literature values.
(i) Hexadecyltrimethylammonium hydroxide (molar ratio to
trans-ferulic acid 1:1), peroxidase, and H2O2 (pH 7.0-8.0); (ii)
trans-ferulic acid, peroxidase, and H2O2 in 0.1 M acetate buffer
solution (pH 7.5); (iii) tetradecyltrimethylammonium bromide
(molar ratio to trans-ferulic acid 2:1), peroxidase, and H2O2 (pH
3.0); (iv) trans-ferulic acid, peroxidase, and H2O2 in 0.1
M
b
phosphate buffer solution (pH 3.0). Means ( standard deviation
(n ) 2). c Compound 2, 8-8-γ-lactone-diFA; compound 3, 5-5-diFA;
compound 4, 8-5-benzofuran-diFA; compound 5, 8-8-bis-lactone-
diFA.
RESULTS AND DISCUSSION
HPLC on a LiChrospher 100 RP-18 (5 µm; 244 × 4 mm i.d.,
Merck, Darmstadt, Germany) column according to the method
of Andreasen et al. (15, 21). trans-Ferulic acid (1), 8-8-γ-
lactone-diFA (2), 5-5-diFA (3), 8-5-benzofuran-diFA (4), and
8-8-bis-lactone-diFA (5) were quantified directly in the reaction
mixtures by reversed-phase analytical HPLC (1, Rt ) 69.7 min;
The reaction of ethyl ferulate with H2O2 and peroxi-
dase according to the method of Ralph et al. (19) gave
primarily the 8-5-benzofuran-diFA and 8-O-4-diFA, 8-5-
diFA, and 5-5-diFA (Figure 1) as minor products (16),
F igu r e 3. Dimerization of trans-ferulic acid in
a micellar solution with hexadecyltrimethylammonium hydroxide and
tetradecyltrimethylammonium bromide as the surfactants, respectively, and the corresponding dimerizations in nonmicellar
buffered solutions. (i) Hexadecyltrimethylammonium hydroxide, peroxidase, and H2O2 (pH 7.0-8.0). (ii) Peroxidase, H2O2, and
0.1 M acetate buffer solution (pH 7.5). (iii) Tetradecyltrimethylammonium bromide, peroxidase, and H2O2 (pH 3.0). (iv) Peroxidase,
H2O2, and 0.1 M phosphate buffer solution (pH 3.0).

3474 J. Agric. Food Chem., Vol. 49, No. 7, 2001
Larsen et al.
whereas we observed no reaction in an unbuffered
solution using trans-ferulic acid as the substrate. On
the other hand, we found that the combination of trans-
ferulic acid and the strongly basic surfactant hexade-
cyltrimethylammonium hydroxide in a molar ratio of 1:1
gave a complete reaction within 5 min. The distribution
of products was quantified by analytical HPLC and was
25% 8-8-γ-lactone-diFA (2), 21% 5-5-diFA (3), and 14%
8-5-benzofuran-diFA (4) (Figure 3 and Table 1). During
the reaction, the pH of the solution changed from 7.0 to
8.0. Repeating this experiment in a buffered solution
(pH 7.5) but without surfactant gave a different product
distribution, as no 5-5-diFA (3) was produced. The major
products were 8-8-γ-lactone-diFA (2, yield 25%) and 8-5-
benzofuran-diFA (4, yield 38%). Compounds 2-4 were
obtained in a pure state by preparative HPLC (see
Materials and Methods). Unidentified polymeric or
higher oxidized products accounted for the remainder
in both the micellar and buffered solutions, as an almost
complete conversion of trans-ferulic acid was observed
by analytical HPLC. 8-8-γ-Lactone-diFA (2) probably
arises from an 8-8-coupled dehydrodimer followed by
an intramolecular attack of one of the carboxylic acid
moieties, whereas 5-5-diFA (3) and 8-5-benzofuran-diFA
(4) are the well-known 5-5- and 8-5-coupled ferulic
acid dehydrodimers, respectively (3). With other sub-
strates (trans-p-coumaric and trans-sinapic acid) a
similar reaction pattern was observed yielding a com-
plex mixture of dimers (data not shown).
Changing the surfactant to tetradecyltrimethylam-
monium bromide gave 18% 8-8-bis-lactone-diFA (5) and
only small amounts of 8-8-γ-lactone-diFA (2) and 8-5-
benzofuran-diFA (4) (Figure 3 and Table 1) but with a
substantial amount of polymeric or higher oxidized
compounds. The pH remained constant at 3.0 during
the reaction. Repeating this experiment in a buffered
solution (pH 3.0) but without surfactant gave no reac-
tion. Similar surfactants such as hexadecyltrimethyl-
ammonium hydrogencarbonate or hexadecyltrimethyl-
ammonium bromide gave almost identical reaction
yields compared to that from tetradecyltrimethylam-
monium bromide (data not shown). The ratio of these
surfactants to trans-ferulic acid was found to be impor-
tant as a ratio close to 1 gave rise to additional amounts
of byproducts. A molar excess of surfactant (50-100%)
gave the cleanest reactions and with similar yields. 8-8-
Bis-lactone-diFA (5) closely resembles the lignan pi-
noresinol, which has recently been stereoselectively
prepared from coniferyl alcohol by means of a “guiding”
protein (20). Compound 5 probably arises from an 8-8-
coupled dehydrodimer followed by intramolecular attack
of both of the carboxylic acid moieties, and it was
obtained in a pure state by preparative HPLC (see
Materials and Methods). With other substrates (trans-
p-coumaric or trans-sinapic acid) only trace amounts of
dimer products were observed (data not shown).
rable to those obtained by existing synthetic methods
(16, 22, 25).
From the present results it can be concluded that the
outcome of radical dimerization of hydroxycinnamic
acids is dependent on both the geometric and electronic
nature of a controlling agent, and this seems to offer a
new way to produce dehydrodimers of hydroxycinnamic
acids on a preparative scale. Although micelles might
not be involved in the biosynthetic assembly of these
dehydrodimers, the present results indicate that a
controlling agent might be present in vivo, e.g. “guiding”
proteins as in the case of the biosynthesis of lignins and
lignans.
ABBREVIATIONS USED
8-O-4-diFA, (Z)-â-{4-[(E)-2-carboxyvinyl]-2-methoxy-
phenoxy-4-hydroxy-3-methoxycinnamic acid; 8-5-diFA,
(E,E)-4,4′-dihydroxy-3,5′-dimethoxy-â,3′-bicinnamic acid;
8-5-benzofuran-diFA, trans-5-[(E)-2-carboxyvinyl]-2-(4-
hydroxy-3-methoxyphenyl)-7-methoxy-2,3-dihydroben-
zofuran-3-carboxylic acid; 5-5-diFA, (E,E)-4,4′-dihydroxy-
5,5′-dimethoxy-3,3′-bicinnamic acid; 8-8-aryl-diFA, trans-
7-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-6-methoxy-
1,2-dihydronaphthalene-2,3-dicarboxylic acid; 8-8-γ-
lactone-diFA, trans-4-(4-hydroxy-3-methoxybenzylidene)-
2-(4-hydroxy-3-methoxyphenyl)-5-oxotetrahydrofuran-
3-carboxylic acid; 8-8-bis-lactone-diFA, 4-cis,8-cis-bis(4-
hydroxy-3-methoxyphenyl)-3,7-dioxabicyclo[3.3.0]octane-
2,6-dione; 8-8-diFA, 4,4′-dihydroxy-3,3′-dimethoxy-â,â′-
bicinnamic acid.
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Received for review December 7, 2000. Revised manuscript
received April 24, 2001. Accepted May 1, 2001.
J F0014617