Sinapate dehydrodimers and sinapate-ferulate heterodimers in cereal dietary fiber

Article abstract of DOI:10.1021/jf020910v

Two 8-8-coupled sinapic acid dehydrodimers and at least three sinapate-ferulate heterodimers have been identified as saponification products from different insoluble and soluble cereal grain dietary fibers. The two 8-8-disinapates were authenticated by comparison of their GC retention times and mass spectra with authentic dehydrodimers synthesized from methyl or ethyl sinapate using two different single-electron metal oxidant systems. The highest amounts (481 μg/g) were found in wild rice insoluble dietary fiber. Model reactions showed that it is unlikely that the dehydrodisinapates detected are artifacts formed from free sinapic acid during the saponification procedure. The dehydrodisinapates presumably derive from radical coupling of sinapate-polymer esters in the cell wall; the radical coupling origin is further confirmed by finding 8-8 and 8-5 (and possibly 8-0-4) sinapate-ferulate cross-products. Sinapates therefore appear to have an analogous role to ferulates in cross-linking polysaccharides in cereal grains and presumably grass cell walls in general.

Full text of DOI:10.1021/jf020910v

J. Agric. Food Chem. 2003, 51, 1427−1434 1427
Sinapate Dehydrodimers and Sinapate−Ferulate Heterodimers
in Cereal Dietary Fiber
MIRKO BUNZEL,*,† JOHN RALPH,‡,§ HOON KIM,‡,§ FACHUANG LU,‡,§
SALLY A. RALPH,# JANE M. MARITA,‡,§ RONALD D. HATFIELD,‡ AND
†
HANS STEINHART
Institute of Biochemistry and Food Chemistry, Department of Food Chemistry,
University of Hamburg, Grindelallee 117, 20146 Hamburg, Germany; U.S. Dairy Forage Research
Center, Agricultural Research Service, U.S. Department of Agriculture, Madison, Wisconsin 53706;
Department of Forestry, University of WisconsinsMadison, Madison, Wisconsin 53706; and
U.S. Forest Products Laboratory, Forest Service, U.S. Department of Agriculture,
Madison, Wisconsin 53705
Two 8-8-coupled sinapic acid dehydrodimers and at least three sinapate-ferulate heterodimers have
been identified as saponification products from different insoluble and soluble cereal grain dietary
fibers. The two 8-8-disinapates were authenticated by comparison of their GC retention times and
mass spectra with authentic dehydrodimers synthesized from methyl or ethyl sinapate using two
different single-electron metal oxidant systems. The highest amounts (481 µg/g) were found in wild
rice insoluble dietary fiber. Model reactions showed that it is unlikely that the dehydrodisinapates
detected are artifacts formed from free sinapic acid during the saponification procedure. The
dehydrodisinapates presumably derive from radical coupling of sinapate-polymer esters in the cell
wall; the radical coupling origin is further confirmed by finding 8-8 and 8-5 (and possibly 8-O-4)
sinapate-ferulate cross-products. Sinapates therefore appear to have an analogous role to ferulates
in cross-linking polysaccharides in cereal grains and presumably grass cell walls in general.
KEYWORDS: Zizania sp.; Gramineae; wild rice; dietary fiber; hydroxycinnamic acid; sinapate; sinapic
acid; dehydrodimer; ferulate; ferulic acid; radical coupling; cross-coupling; cell-wall cross-linking; single-
electron oxidation
INTRODUCTION
lates may affect the physicochemical behavior of the fraction
and possibly also influence its physiological effects (12).
Grasses have substantial amounts of hydroxycinnamic acids
intimately associated with the cell wall, as has been detailed in
a number of reviews (1-4). Ferulate, in particular, has a
significant role in cross-linking cell-wall polymers (5-7). Poly-
saccharide-polysaccharide cross-linking is effected by ferulate
dimerization by either photochemical (8) or, more importantly,
radical coupling reactions of ferulate-polysaccharide esters (5,
7). The full range of ferulate cross-coupling products, not just
the 5-5-coupled product, can now routinely be found in a
variety of samples (5, 7, 9-13). Formation of dehydrodiferulates
in the plant cell wall is thought to terminate the expansion of
cell growth in grasses (3). Furthermore, dehydrodiferulates may
play an important role in modifying the mechanical properties
of cell walls (9) as well as in limiting polysaccharide degradation
by exogenous enzymes (14) by acting as cross-links between
polysaccharides. Dietary fiber cross-linking by dehydrodiferu-
Although sinapic acid 1S (X ) H; S, sinapate moiety) (Figure
1) has been identified in plant extracts and can be released in
small quantities from grass cell walls by base (15-18), it has
not been determined if it acylates polysaccharides or other
components. In 1969, the isolation and identification of tho-
masidioic acid (5C3SS; SS, disinapate compound, Figure 1)
from aqueous extracts of the heartwood of elm (Ulmus thomasii
Sarg.) were reported (19). Thomasidioic acid was described as
a phenolic lignan but is an analogue of the cyclic form of the
8-8-coupled dehydrodiferulic acid 5C3FF (FF, diferulate
compound), which was more recently discovered as a plant
component (5). In a study on the constituents of canola meal
(20, 21) it was discovered that thomasidioic acid was formed
when sinapic acid was air oxidized in dilute alkaline solutions.
Due to this observation and the fact that isolated thomasidioic
acid is optically inactive, Charlton and Lee (22) doubted that
thomasidioic acid is a natural product. They suggested that
thomasidioic acid was formed during the extraction procedure
of the elm heartwood.
* Author to whom correspondence should be addressed [telephone +49-
(0)40-42838-4379; fax +49-(0)40-42838-4342; e-mail Bunzel@lc.chemie.uni-
hamburg.de].
† University of Hamburg.
‡ U.S. Dairy Forage Research Center.
Here we describe the identification of alkali-releasable (ester-
bound) thomasidioic acid in different insoluble (IDF) and soluble
§ University of WisconsinsMadison.
# U.S. Forest Products Laboratory.
10.1021/jf020910v CCC: $25.00 © 2003 American Chemical Society
Published on Web 01/23/2003

1428 J. Agric. Food Chem., Vol. 51, No. 5, 2003
Bunzel et al.

Sinapate Dehydrodimers and Sinapate−Ferulate Heterodimers
J. Agric. Food Chem., Vol. 51, No. 5, 2003 1429
Figure 1. Radical dehydrodimerization of (cell-wall-bound) hydroxycinnamates or hetero-dehydrodimerization of sinapate and ferulate produces 8−O−4-,
8−5-, 8−8-, and 5−5-dehydrodimers via intermediates. Sinapic acid dehydrodimers 5C1SS and 5C3SS are released (following saponification) from
several cereal grain dietary fibers. The actual form of the 8−8 products in the wall is unknown. With its extra 5-OMe, sinapate cannot make 5−5- or
8−5-dehydrodimers. The numbering scheme uses the convention relating to the monomer numbering (and not the IUPAC name, which is given under
Materials and Methods for new compounds). The A-ring of 5C3SS is numbered with the condensation between carbons A6 and B7 to be consistent with
the corresponding ferulate dehydrodimer. The designation of units as arising from ferulate (F) and sinapate (S) in the dimers uses the A-ring first; e.g.,
5B1SF is from sinapate linked at its 8-position to ferulate at its 5-position. Columnar numbers in parentheses are nominal molecular masses (from
GC-MS) of the diferulate (FF), the mixed (SF), and the disinapate (SS) compounds; where such compounds are not possible, the entry is a simple dash,
e.g., 8−5 products cannot be SS. The bond formed by the radical coupling step is bolded. X ) polysaccharide in the cell-wall system, but also ) Me,
Et (and H for 1, 2) for synthetic compounds.
Saponification To Release Hydroxycinnamic Acids. Internal stan-
dards (IS) (E,E)-4-hydroxy-4′,5,5′-trimethoxy-3,3′-bicinnamic acid (5-
5-Me-diferulic acid, the monomethyl ether of 5DFF) (12) and o-cou-
maric acid dissolved in dioxane were added, and saponification with
NaOH (2 M, 5 mL) was carried out protected from light for 18 h at
room temperature. To avoid oxidative processes the NaOH solution
was degassed by bubbling N2 through for 30 min, and air in the head-
space of the screw-cap tubes was exchanged with N2. Samples were
acidified with 0.95 mL of concentrated HCl (resulting pH <2) and
extracted into diethyl ether (4 mL, three times). Extracts were combined,
evaporated under a stream of filtered air, silylated, and analyzed by
GC-MS and GC-FID.
dietary fibers (SDF) from cereal grains, as preliminarily
disclosed by Ralph et al. (23). From different extraction studies,
examination of the reactivity of model systems, the identification
of a new dehydrodisinapic acid (the open form 8-8-coupled
dehydrodisinapic acid isomer 5C1SS), and the finding of
sinapate-ferulate heterodimers, we suggest that dehydrodisi-
napic acids are natural, wall-bound, radical dimerization prod-
ucts and that sinapate functions analogously to ferulate in cell-
wall cross-linking reactions.
MATERIALS AND METHODS
Model Systems for the Saponification Process. To study sinapic
acid dimerization during saponification, sinapic acid (200 µg) as well
as ethyl sinapate 1S (X ) Et) (200 µg) were treated with 2 M NaOH
under different conditions. Cellulose (50 mg) was used to mimic the
polysaccharide matrix. 5-5-Me-diferulic acid and o-coumaric acid were
added as internal standards. Two different saponification conditions
were used: (1) nitrogen was bubbled through the NaOH solution for
30 min, and air in the headspace of the screw-cap tubes was exchanged
by N2 (conditions are identical with those used for saponification of
dietary fiber samples); (2) air was bubbled through the NaOH solution
for 30 min, and air in the headspace was not exchanged (to simulate
conditions with the highest chance of artifactual sinapate to dehydro-
disinapate conversion). Saponification was carried out protected from
light for 18 h at room temperature. Saponification products were
extracted as described above.
General. All samples were analyzed in triplicate. Heat-stable
R-amylase Termamyl 120L (EC 3.2.1.1, from Bacillus licheniformis,
120 KNU/g), the protease Alcalase 2.4 L (EC 3.4.21.62, from Bacillus
licheniformis, 2.4 AU/g), and the amyloglucosidase AMG 300 L (EC
3.2.1.3, from Aspergillus niger, 300 AGU/g) were from Novo Nordisk,
Bagsvaerd, Denmark. Cellulose (SigmaCell, noncrystalline, highly
purified) was obtained from Sigma, St. Louis, MO. NMR spectra of
synthesized samples were run at 300 K on a Bruker AMX-360
instrument (Bruker, Rheinstetten, Germany). Samples were dissolved
in [2H6]acetone. Chemical shifts (δ) were referenced to the central
solvent signals (1H, δ 2.04; 13C, δ 29.8). J values are given in hertz.
NMR assignments follow the numbering shown in Figure 1.
Plant Material. Whole grains of corn (Zea mays L.), wheat (Triticum
aestiVum L.), spelt (Triticum spelta L.), rice (Oryza satiVa L.), wild
rice (Zizania aquatica L.), barley (Hordeum Vulgare L.), rye (Secale
cereale L.), oat (AVena satiVa L.), and millet (Panicum miliaceum L.)
were obtained from a local German supplier.
GC-MS and GC-FID. Dried extracts as well as synthesized
dehydrodisinapic acids were trimethylsilylated by adding 10 µL of
pyridine and 40 µL of BSTFA and heating for 30 min at 60 °C in
sealed vials. Trimethylsilylated derivatives of phenolic acids were
separated by GC [Thermoquest (Austin, TX) Trace 2000 GC] using a
25 m × 0.2 mm i.d., 0.33 µm film, DB-1 capillary column (J&W
Scientific, Folsom, CA) and identified by their electron impact mass
data collected on a Thermoquest QSC ion-trap MS. He (0.54 mL/min)
was used as carrier gas. GC conditions were as follows: initial column
temperature, 220 °C, held for 1 min, ramped at 4 °C/min to 248 °C,
ramped at 30 °C/min to 300 °C, held for 40 min; injector temperature,
300 °C; split, 1/50 or 1/12 (determination of IDF or SDF, respectively).
Quantitative determinations were carried out by GC (Hewlett-Packard
5980, Palo Alto, CA) using the same column and GC conditions and
a flame ionization detector (detector temperature, 300 °C). He (0.4 mL/
min) was used as carrier gas. For quantitative determinations of sinapate
dimers 5-5-Me-DFS was used as internal standard. Response factors
for sinapate dimers against 5-5-Me-DFS are 0.826 (compound 5C1SS,
see below) and 0.482 (compound 5C3SS), respectively.
Preparation of Dietary Fiber. Preparation of cereal IDF and SDF
was performed according to a preparative isolation procedure described
previously (12). Briefly, milled sample material (10 g) was suspended
in phosphate buffer (pH 6.0, 0.08 M, 300 mL), and R-amylase (750
µL) was added. Beakers were heated in a boiling water bath for 20
min. The pH was adjusted to 7.5, and samples were incubated with
protease (300 µL) at 60 °C for 30 min. After the pH had been adjusted
to 4.5, amyloglucosidase (350 µL) was added and the mixture was
incubated at 60 °C for 30 min. The suspension was centrifuged, and
the supernatant was kept for isolation of SDF. The residue was washed
twice each with hot water (70 °C), 95% (v/v) ethanol, and acetone and
finally dried in a vacuum oven to give IDF. Water washings were
combined with the first supernatant for preparation of SDF. SDF was
precipitated in 80% ethanol. After centrifugation, the residue was
washed twice each with 78% (v/v) ethanol, 95% (v/v) ethanol, and
acetone and was dried in a vacuum oven. For calculations, IDF and
SDF were corrected for residual protein and ash contents.
Synthesis of Sinapate Dehydrodimers. Preparation of Compound
5C1SS [Noncyclic Form of 8-8-Coupled Dehydrodisinapic Acid-2,3-
Bis(4-hydroxy-3,5-dimethoxybenzylidene)succinic Acid] (Figure 1). To
sinapic acid (5 g) in absolute ethanol (100 mL) was slowly added acetyl
chloride (5 mL). The mixture was stirred for 16 h. TLC showed that
the esterification was not complete, so further aliquots of ethanol and
acetyl chloride were added and the mixture was stirred for another 16
h. The solution was concentrated on a rotary evaporator, and HCl (and
other solvents) was removed by coevaporation several times with
ethanol. The product, ethyl sinapate, was crystallized from MeOH. Ethyl
sinapate 1S (X ) Et) (200 mg) was dissolved in pyridine (5 mL), and
Mn(OAc)3‚2H2O (255 mg) in pyridine (5 mL) was added over a few
Additional Extraction Step of Some Wild Rice Insoluble Dietary
Fiber. To ensure that dietary fibers are free of unbound sinapic acid
and thomasidioic acid, wild rice IDF was exhaustively extracted with
ethanol for 6 h and with acetone for 8 h using Soxhlet equipment. The
combined extracts were evaporated, dissolved in dioxane, and divided
into two parts. After evaporation and addition of internal standards,
one part was directly trimethylsilylated as described below. The other
part was saponified (2 M NaOH) under N2 and protected from light
for 4 h at room temperature, silylated, and analyzed by GC-MS and
GC-FID for sinapic acid and thomasidioic acid. The extraction residue
was dried at 55 °C for 5 h, weighed, and saponified as follows.

1430 J. Agric. Food Chem., Vol. 51, No. 5, 2003
Bunzel et al.
minutes. The reaction mixture was stirred for a further 5 min. Na2SO3
(solid, 120 mg) was added, and stirring of the mixture was continued
for 10 min. To acetylate the phenols acetic anhydride (10 mL) was
added. The mixture was stirred for 2 h. After evaporation of solvents
on a rotary evaporator, the mixture was partitioned between CH2Cl2
and saturated aqueous NaCl. The organic layer was washed consecu-
tively with 2 M HCl, 0.4 M NaHCO3, and saturated aqueous NaCl,
then dried over MgSO4 and evaporated to dryness to provide 198 mg
of product (yield 85%), which was crystallized from acetone/petroleum
ether as red needles: mp 104 °C. Diacetate of compound 4C1SS [X )
Et, diethyl 2,3-bis(4-acetoxy-3,5-dimethoxybenzylidene)succinate]: 1H
NMR δ 1.11 (6H, t, J ) 7.1, CH3-CH2-O), 2.20 (6H, s, OCOCH3),
3.73 (12H, s, OMe), 4.14 (4H, q, J ) 7.1, CH3-CH2-O), 6.90 (4H, s,
H-2/6), 7.85 (2H, s, H-7); 13C NMR δ 14.4 (CH3-CH2-O), 20.2
(OCOCH3), 56.4 (OMe), 61.7 (CH3-CH2-O), 107.3 (C-2/6), 129.1 (C-
8), 130.8 (C-4), 133.9 (C-1), 142.5 (C-7), 153.2 (C-3/5), 167.0 (C-9),
168.3 (OCOCH3).
123.3 (A C-1), 123.9 (A C-8), 124.6 (A C-6), 134.2 (B C-1), 135.8 (B
C-4), 138.4 (A C-7), 142.8 (A C-4), 146.4 (A C-5), 148.49 (B C-3/5),
148.59 (A C-3), 167.5 (A C-9), 172.8 (B C-9).
Compound 4C3SS (X ) Me) (100 mg) was dissolved in 2 M NaOH
(20 mL) and hydrolyzed at room temperature for ∼16 h. The solution
was acidified with 2 M HCl to pH 2 and extracted into EtOAc. The
organic layer was dried over MgSO4, evaporated, and submitted to
preparative silica TLC in EtOAc/CHCl3/formic acid (2:1:0.1). EtOAc
was used to extract products from the scraped-off TLC silica. The
slower moving material was identified as compound 5C3SS (56 mg,
yield 60%). Compound 5C3SS was crystallized from acetone/petroleum
ether as gray granules, mp 230 °C. Compound 5C3SS: 1H NMR δ
3.40 (1H, d, J ) 1.3, B H-8), 3.62 (3H, s, A5-OMe), 3.69 (6H, s,
B3/5-OMe), 3.88 (3H, s, A3-OMe), 5.05 (1H, bs, B H-7), 6.40 (2H, s,
B H-2/6), 6.95 (1H, s, A H-2), 7.68 (1H, s, A H-7); 13C NMR δ 40.3
(B C-7), 47.3 (B C-8), 56.58 (A3-OMe), 56.64 (B3/5-OMe), 60.4 (A5-
OMe), 106.1 (B C-2/6), 108.9 (A C-2), 124.07 (A C-1), 124.11 (A
C-8), 125.0 (A C-6), 134.6 (B C-1), 135.7 (B C-4), 138.3 (A C-7),
142.7 (A C-4), 146.4 (A C-5), 148.45 (B C-3/5), 148.52 (A C-3), 168.6
(A C-9), 173.4 (B C-9); high-resolution MS, found M+ 446.1219,
C22H22O10 requires M 446.1213.
The faster moving fraction (14 mg, yield 27%) was identified by
NMR and GC-MS as compound 5C3′S (6-hydroxy-5,7-dimethoxy-
naphthalene-2-carboxylic acid). Compound 5C3′S: 1H NMR δ 3.99
(3H, s, A5-OMe), 4.01 (3H, s, A3-OMe), 7.30 (1H, s, A H-2), 7.91
(1H, dd, J ) 8.7, 1.6, B H-8), 8.00 (1H, d, J ) 8.7, B H-7), 8.48 (1H,
d, J ) 1.6, A H-7); 13C NMR δ 56.4 (A3-OMe), 60.9 (A5-OMe), 104.1
(A C-2), 121.6 (B C-7), 124.2 (B C-8), 126.8 (A C-4), 127.8 (A C-6),
128.2 (A C-8), 130.6 (A C-7), 141.05 (A C-5), 141.11 (A C-1), 150.8
(A C-3), 168.1 (A C-9); high-resolution MS, found M+ 248.0692,
C13H12O5 requires M 248.0685. Methods for the synthesis of compound
5C3′S described in the literature (27, 28) are time-consuming, requiring
five or six steps. To produce small amounts, the procedure described
here seems to be more convenient.
The diacetate of 4C1SS (X ) Et) (80 mg) was dissolved in dioxane
(5 mL), and degassed 2 M NaOH (20 mL) and additional dioxane (5
mL) were added. Deacetylation was carried out at room temperature
for 16 h. The solution was acidified (pH 2) with 2 M HCl and parti-
tioned between EtOAc and saturated aqueous NaCl. The organic layer
was dried over MgSO4 and evaporated. After degassed 2 M NaOH
(20 mL) had been added, ester hydrolysis was carried out for 24 h at
room temperature in the dark. This two-step saponification was neces-
sary because the acetylated derivative of diethyl ester 4C1SS (X )
Et) was not completely dissolved in NaOH without dioxane and
dissolved in dioxane/NaOH, but these conditions removed only the
acetate groups. Following the saponification process, the solution was
acidified with 2 M HCl and extracted into EtOAc. The organic layer
was dried over MgSO4, evaporated, and submitted to preparative silica-
TLC in EtOAc/CHCl3/formic acid (1:1:0.1) to afford compound 5C1SS
as a red solid (19 mg, yield 30%). The residual material still contained
the ester, which was therefore surprisingly difficult to saponify.
Compound 5C1SS: 1H NMR δ 3.75 (12H, s, OMe), 7.01 (4H, s, H-2/
6), 7.85 (2H, s, H-7); 13C NMR δ 56.5 (OMe), 108.8 (C-2/6), 126.4
(C-8), 126.6 (C-1), 138.8 (C-4), 142.6 (C-7), 148.5 (C-3/5), 168.6 (C-
9); high-resolution MS, M+ not sufficiently strong, seen in low-
resolution spectrum; M+ - H2O 428.1103, C22H20O9 requires M
428.1107; M+ - H2O - CO 400.1143, C21H20O8 requires M 400.1158.
Preparation of 5C3SS [Cyclic Form of 8-8-Coupled Dehydrodi-
sinapic Acid, Thomasidioic Acid, trans-7-Hydroxy-1-(4-hydroxy-3,5-
dimethoxyphenyl)-6,8-dimethoxy-1,2-dihydronaphthalene-2,3-dicarbox-
ylic Acid] (Figure 1). Ahmed et al. (24) described the synthesis of
thomasidioic acid in two steps starting from sinapic acid. Oxidation of
methyl sinapate 1S (X ) Me) with ferric chloride in aqueous acetone
gave the dimethyl ester of thomasidioic acid (4C3SS, X ) Me) as a
byproduct (25), whereas oxidative coupling of methyl sinapate in
aqueous acetone at pH 4 with H2O2 and horseradish peroxidase gave
the dimethyl ester of thomasidioic acid in 41% yield (26). Changing
the oxidation conditions of the methyl sinapate reaction with ferric
chloride gave the dimethyl ester of thomasidioic acid in good yields.
To sinapic acid (5 g) in methanol (150 mL) was slowly added acetyl
chloride (15 mL) over 15 min, and the mixture was stirred for 16 h,
producing methyl sinapate as determined by TLC. Water (30 mL) and
FeCl3‚6H2O (45 g) were added, and the mixture was stirred continuously
for another 48 h, by which time the methyl sinapate had been consumed,
as indicated by TLC. After methanol was removed by evaporation under
reduced pressure, the mixture was poured into EtOAc and washed with
3% HCl and saturated aqueous NH4Cl. The EtOAc fraction was dried
over MgSO4 and evaporated. Crude products were applied to flash
chromatography on silica gel in CHCl3/EtOAc (5:1). The dimethyl ester,
compound 4C3SS (X ) Me), was obtained in 52% yield along with
∼5% of a minor isomer, which had a cis configuration between carbons
B7 and B8. Compound 4C3SS (X ) Me, thomasidioic acid dimethyl
ester): 1H NMR δ 3.60 (3H, s, B-COOCH3), 3.62 (3H, s, A5-OMe),
3.68 (6H, s, B3/5-OMe), 3.68 (3H, s, A-COOCH3), 3.88 (3H, s, A3-
OMe), 4.02 (1H, s, B H-8), 5.00 (1H, s, B H-7), 6.36 (2H, s, B H-2/6),
6.96 (1H, s, A H-2), 7.66 (1H, s, A H-7); 13C NMR δ 40.3 (B C-7),
47.5 (B C-8), 51.9 (A-COOCH3), 52.4 (B-COOCH3), 56.58 (A3-OMe),
56.64 (B3/5-OMe), 60.4 (A5-OMe), 106.1 (B C-2/6), 109.0 (A C-2),
RESULTS AND DISCUSSION
Possible Pathways for Dimerization of Sinapate Esters.
Along with ferulic acid, the main hydroxycinnamic acid in the
plant cell wall, alkali-extractable p-coumaric acid and sinapic
acid [1S (X ) H), Figure 1] have been identified from grasses
(1-4, 18). p-Coumaric acid may cross-link cell-wall polymers
by [2+2]-cyclodimerization; radical coupling products have not
been identified (29). Radical dehydrodimerization of sinapate
esters 1S can produce only two primary products, 8-O-4- and
8-8-dehydrodimers (Figure 1). Ferulates additionally produce
8-5-, 5-5-, and 4-O-5-coupled products (5). Following the
radical coupling step, which produces intermediates 3 (quinone
methides for the 8-coupled dimers 3A-3C), re-aromatization
is achieved during subsequent reactions. For the 8-O-4-
coupled intermediate 3A re-aromatization is via elimination of
the acidic 8-proton (5). 8-5-Coupling leads to the formation
of phenylcoumaran structures 4B; with no 5-position available
for coupling, there is obviously no disinapate 8-5-dehydro-
dimer, although sinapate-ferulate cross-coupling can give 8-5-
coupled products. Saponification of the phenylcoumaran ester
4B gives variable partitioning between the phenylcoumaran acid
5B1 and the eliminated open form 5B2. As with ferulate
dimerization, the pathway from the 8-8-coupled product is less
clear. Elimination of both 8-protons from quinone methide 3C
can occur to produce the conjugated noncyclic product 4C1.
However, this cannot be the only path occurring in vivo; such
a product cannot give rise to the observed cyclic 8-8-product
5C3 or the more recently discovered diferulate tetrahydrofuran
5C2FF involving water addition to the quinone methide (23).
The finding of the cyclic product 5C3SS, complementing its
ferulate analogue 5C3FF, implies that either 4C2 or 4C3 (or
both, or possibly an alternate intermediate) are the in vivo
products. As is the case with ferulates, the nature of the 8-8-

Sinapate Dehydrodimers and Sinapate−Ferulate Heterodimers
J. Agric. Food Chem., Vol. 51, No. 5, 2003 1431
Figure 2. (A) GC-MS total-ion chromatogram of saponified extracts of wild rice IDF showing ferulate dehydrodimers (cyan) and the two new sinapate
dehydrodimers 5C1SS and 5C3SS (red), as well as (B, C) their mass spectra. Structures are from Figure 1. c, cyclic form; nc, noncyclic (open) form;
dc, decarboxylated form.
or from SDF from oats, corn, or millet (Table 1). Furthermore,
compound 5C3′S, which arises in synthesis during the saponi-
fication step of the dimethyl ester 4C3SS (X ) Me), was
generally not detected. The compound 5C2SS, an analogue of
the recently identified diferulate tetrahydrofuran compound
5C2FF, was not detected. Quantification (Table 1) was carried
out using internal standards. Although identified unequivocally,
quantification of compound 5C1SS was not carried out in IDF
of wheat and spelt due to some peak impurities recognized by
GC-MS. The highest amounts of dehydrodisinapates were
detected in wild rice IDF, with 355 µg/g IDF of 5C1SS and
126 µg/g IDF of 5C3SS. Interestingly, wild rice IDF is the only
sample from which higher amounts of 5C1SS than of 5C3SS
were detected. Although the saponification procedure was car-
ried out identically, other dietary fibers showed higher amounts
of 5C3SS compared to 5C1SS. As shown in Table 1, amounts
of dehydrodisinapic acids in some cereal dietary fibers are
substantial, but their amounts are rather low in comparison to
dehydrodiferulates (12). The sum of dehydrodisinapic acids,
where quantification of at least one compound was possible, in
cereal IDF (wheat, spelt, and rice) is ∼1% of the total de-
hydrodiferulates level. Wild rice IDF differs significantly from
other cereal IDFs investigated: dehydrodisinapates amount to
17% of the dehydrodiferulates level. In wild rice IDF the di-
sinapate/sinapate ratio (∼40%) is slightly lower than the di-
ferulate/ferulate ratio at ∼60%. In SDF of wheat, spelt, and
rye the relative amounts of dehydrodisinapates are a little higher
than in the corresponding IDF at roughly 4-7% of the de-
hydrodiferulates level. Contrary to the dehydrodisinapates, the
dehydrodiferulate level in wild rice SDF was below the
determination limit (12).
Table 1. Amounts of 8−8-Coupled Sinapic Acid Dehydrodimers
(5C3SS and 5C1SS) with Corresponding Standard Deviations (SD) (n
) 3) Released by Saponification from Soluble and Insoluble Cereal
Grain Dietary Fiber Fractions
µg/g of IDF
5C3SS SD 5C1SS SD 5C3SS SD 5C1SS SD
Insoluble Dietary Fiber Soluble Dietary Fiber
µg/g of SDF
a
wheat
spelt
rye
barley
oat
corn
millet
rice
33
16
tr
tr
nd
tr
nd
44
126
4
1
+
8
15
3
1
6
1
4
1
+
trb
tr
ndc
nd
nd
nd
nd
nd
355
tr
nd
nd
nd
nd
tr
nd
nd
nd
nd
2
10
16
wild rice
49
3
1
1
a +, compound identified but not possible to quantify due to peak impurities.
b tr, traces, compounds identified by GC-MS, but levels below their determination
limit by GC-FID of ∼2 µg/g. c nd, not detected.
coupled product in vivo remains to be elucidated. Finally, 5-5-
coupling is only possible between ferulates to yield, after
saponification, 5DFF.
Identification and Quantification of Dehydrodisinapates
in Cereal Grain Dietary Fibers. As seen in Figure 2, two
8-8-dehydrodimers, compounds 5C1SS (the noncyclic isomer)
and 5C3SS (thomasidioic acid, the cyclic isomer) of 8-8-
coupled dehydrodisinapic acid, result following saponification
of wild rice IDF. The dehydrodisinapates were authenticated
by comparison of their mass spectra (Figure 2) and their relative
GC retention times with those of the synthesized compounds.
The 8-8-coupled dehydrodisinapates are analogues of the
corresponding ferulate dimers (5). They were also detected in
other cereal grains (Table 1) but not in IDF from oats or millet
GC-MS chromatograms were screened for 8-O-4-coupled
dehydrodisinapates, 5ASS, by looking for typical masses
expected from its structure by analogy with the dehydrodiferulic

1432 J. Agric. Food Chem., Vol. 51, No. 5, 2003
Bunzel et al.
Figure 3. (A) GC-MS total-ion chromatogram of saponified extracts of spelt SDF showing ferulate (cyan) and sinapate (red) dehydrodimers as well as
the sinapate−ferulate crossed dimers (magenta). (B) Selected-ion chromatogram (m/z 704) revealing three of the SF cross-dimers. (C−E) Mass spectra
of sinapate−ferulate cross products. Structures are from Figure 1. c, cyclic form; nc, noncyclic (open) form; dc, decarboxylated form.
acid analogue (12). The 8-O-4-dimer was not seen at any
significant level. Sinapates are known to predominantly 8-8-
coupled (25). Analogously, sinapic acid gives high yields of
the 8-8-coupled dilactone (30), and sinapyl alcohol coupled
under peroxidase-H2O2 gives some 95% syringaresinol, the
8-8-coupled product, and only ∼5% 8-O-4-coupling (31).
Dehydrodisinapic Acids)Natural Products or Air Oxida-
tion Artifacts from Sinapic Acid? In 1968-1969 three papers
were published about the occurrence of lignans in wood of the
elm U. thomasii Sarg. (19, 32, 33). Besides thomasic acid,
racemic lyoniresinol and (+)-lyoniresinol-2R-O-rhamnoside,
thomasidioic acid (5C3SS), and 6-hydroxy-5,7-dimethoxynaph-
thalene-2-carboxylic acid (compound 5C3′S) were isolated and
identified from an aqueous extract of the heartwood. Rubino et
al. (20, 21) described the conversion of sinapic acid to thom-
asidioic acid in the presence of oxygen predominantly under
alkaline conditions, but on a smaller scale also under neutral
conditions. Charlton and Lee (22) also noted the conversion of
thomasidioic acid to compound 5C3′S under strong alkaline
conditions (pH 13) in the presence of oxygen, an observation
we exploited for synthesizing compound 5C3′S. The air oxi-
dation process of sinapic acid caused Charlton and Lee (22) to
question whether thomasidioic acid and compound 5C3′S are
really natural products.
Sinapic acid is a common constituent of many plants. Dietary
fibers investigated are washed with water, ethanol, and acetone
after their isolation. One can imagine that thomasidioic acid
could be formed during dietary fiber preparation from free
sinapic acid and that identified thomasidioic acid is just an
extraction residue after washing. This is not the case as we
showed by exhaustive extraction of wild rice IDF with ethanol
and acetone. Exhaustively extracted wild rice IDF and normally
isolated wild rice IDF showed quantitatively comparable
amounts of thomasidioic acid 5C3SS and compound 5C1SS
following saponification. Neither thomasidioic acid nor sinapic
acid was detected in the combined ethanol/acetone extracts.
Furthermore, model reactions showed that sinapate esters will
hydrolyze with only minor levels of contaminating artifacts
under the conditions used in this study for the saponification
of dietary fibers. As the conversion of free sinapic acid to
thomasidioic acid depends on oxygen, two different saponifica-
tion conditions were tested on both ethyl sinapate and free
sinapic acid: (1) nitrogen was bubbled through the NaOH
solution for 30 min, and air in the headspace of the screw-cap
tubes was exchanged with N2 (“normal” saponification condi-

Sinapate Dehydrodimers and Sinapate−Ferulate Heterodimers
J. Agric. Food Chem., Vol. 51, No. 5, 2003 1433
tions); (2) air was bubbled through the NaOH solution for 30
min, and air in the headspace was not exchanged (“oxygen”
saponification conditions). In accordance with the saponification
process of dietary fiber samples, screw-cap tubes were closed
and the mixture was neither shaken nor stirred. Under normal
saponification conditions 2.8% of initial sinapic acid dimerized
to thomasidioic acid and 2.8% of hydrolyzable sinapic acid
(from ethyl sinapate) was converted to thomasidioic acid.
Oxygen conditions converted 5.1% of sinapic acid to thoma-
sidioic acid and 2.5% of hydrolyzable sinapic acid from ethyl
sinapate were dimerized. Under normal conditions compound
5C3′S was not detected, and oxygen conditions produced only
trace amounts of it. These results show that a small percentage
of liberated sinapic acid may be converted to thomasidioic acid,
but this cannot explain the relatively high amounts of thoma-
sidioic acid, for example, in wild rice IDF (126 µg of
thomasidioic acid/g of IDF, 445 µg of sinapic acid/g of IDF).
The identification of the noncyclic isomer 5C1SS provides
a strong indication that the dehydrodisinapates are naturally
occurring. Only the condensed structure of thomasidioic acid
results from the air oxidation mechanism described by Rubino
et al. (20, 21) and Charlton and Lee (22) and in our own
experiments. The release here of 5C1SS from the fiber samples
provides compelling evidence for the presence in the dietary
fibers of sinapate esters and their resultant dimers.
counterpart 5B1SF was not detectable. However, the decar-
boxylated product 5B2′SF was detectable at 31.1 min, with a
mass spectrum (not shown, M+, m/z 588) analogous to that of
the diferulate counterpart (12).
The cross-coupling products are considerably more evident
in the spelt sample (Figure 3A) than in the wild rice (Figure
2A), which has a higher proportion of sinapate versus ferulate
dehydrodimers. Presumably there are either spatial or temporal
mechanisms occurring in the wall, or perhaps in the organelles
that form the sinapate esters, that limit cross-coupling reactions
in the wild rice. In a purely chemical sense, sinapates may have
a higher propensity for homocoupling reactions, as do their
alcohol analogues in lignification; homocoupling products are
usually the prevalent products in laboratory reactions attempting
to produce cross-coupled dehydrodimers.
Sinapates in some cereal grains, like ferulates in all grasses,
dimerize via radical coupling reactions to produce sinapate
dehydrodimers. Following saponification, two 8-8-dehydrodi-
sinapic acids are released and can be detected by GC-MS in
the dimer region where ferulate-ferulate-dehydrodimers and
ferulate-monolignol crossed products also elute. The levels
found, and the presence of noncyclic 8-8-dehydrodisinapate,
provide evidence that dehydrodisinapates are not simply artifacts
derived from sinapate during the saponification. Sinapate-
ferulate cross-products from 8-8-, 8-5-, and possibly 8-O-
4-coupling reactions were also observed. The findings suggest
that oxidative dehydrodimerization of available polysaccharide
hydroxycinnamate esters is a general mechanism for cross-
linking plant cell wall polysaccharides.
Sinapate-Ferulate Crossed Dehydrodimers. The major
dimeric components in these cereal dietary fiber fractions, as
was described more fully elsewhere (12), were the dehy-
drodimers from ferulates. As shown above, dehydrodimers from
sinapates are significant in some cereal grains. More minor were
“dimers” resulting from cross-coupling of ferulate and coniferyl
alcohol (unpublished results). Small amounts of sinapate-
ferulate crossed dehydrodimers could be detected here in some
cereal dietary fibers [IDF of wheat, spelt, rye, and wild rice
and SDF of wheat, spelt (Figure 3A), and rice]. The sinapate-
ferulate crossed dehydrodimers were identified from their mass
spectra, by their fragmentation similarity with their diferulate,
and, when possible, disinapate analogues but have not been
unambiguously validated.
ABBREVIATIONS USED
IDF, insoluble dietary fiber; SDF, soluble dietary fiber; IS,
internal standard; 5-5-Me-diferulic acid, (E,E)-4-hydroxy-
4′,5,5′-trimethoxy-3,3′-bicinnamic acid; F, ferulate moiety; S,
sinapate moiety; FF, diferulate compound; SF, mixed sinapate-
ferulate compound; SS, disinapate compound.
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Received for review August 23, 2002. Revised manuscript received
November 15, 2002. Accepted November 18, 2002. This work was
supported by the Deutsche Forschungsgemeinschaft. We are grateful
to the H. Wilhelm Schaumann-Stiftung and the Josef-Schormu1ller-
Geda1chtnisstiftung for funding M.B.’s visits to the U.S. Dairy Forage
Research Center and to the USDA-National Research Initiatives for
Competitive Grants 96-35304-3864 and 94-02764) for partially funding
the diferulates studies that formed the basis for this work.
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