Synthesis and identification of 2,5-bis-(4-hydroxy-3-methoxyphenyl)- tetrahydrofuran-3,4-dicarboxylic acid, an unanticipated ferulate 8-8-coupling product acylating cereal plant cell walls

Article abstract of DOI:10.1039/b605918j

A new product implicated in cereal grain polysaccharide cross-linking has been authenticated by independent synthesis. Saponification of cereal grain fiber releases the RRRS/SSSR-isomer of 2,5-bis-(4-hydroxy-3-methoxyphenyl)- tetrahydrofuran-3,4-dicarboxylic acid. The parent ester logically derives from 8-8-coupling of ferulate followed by water addition to one of the incipient quinone methide moieties and internal trapping of the other. The finding adds complexity to the analysis of plant cell wall cross-linking, but provides clues to important polysaccharide cross-linking pathways occurring in planta. The Royal Society of Chemistry 2006.

Full text of DOI:10.1039/b605918j

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis and identification of 2,5-bis-(4-hydroxy-3-methoxyphenyl)-
tetrahydrofuran-3,4-dicarboxylic acid, an unanticipated ferulate 8–8-
coupling product acylating cereal plant cell walls†
Paul F. Schatz,a John Ralph,*b Fachuang Lu,c Ilia A. Guzeid and Mirko Bunzele
Received 25th April 2006, Accepted 24th May 2006
First published as an Advance Article on the web 15th June 2006
DOI: 10.1039/b605918j
A new product implicated in cereal grain polysaccharide cross-linking has been authenticated by
independent synthesis. Saponification of cereal grain fiber releases the RRRS/SSSR-isomer of
2,5-bis-(4-hydroxy-3-methoxyphenyl)-tetrahydrofuran-3,4-dicarboxylic acid. The parent ester logically
derives from 8–8-coupling of ferulate followed by water addition to one of the incipient quinone
methide moieties and internal trapping of the other. The finding adds complexity to the analysis of
plant cell wall cross-linking, but provides clues to important polysaccharide cross-linking pathways
occurring in planta.
dehydrodiferulates24 and dehydrotriferulates.26–29 Radical coupling
Introduction
of ferulates, via the action of wall-bound peroxidases, produces
Hydroxycinnamates have important roles in plant cell wall cross-
several regio-isomeric dehydrodiferulates with coupling occurring
linking by linking polysaccharides to each other, to lignin,
at their 4-O-, 5- or 8-carbons. After saponification, the dimeric
and to proteins.1,2 Cross-linking is involved in terminating the
products consist of 5–5-, 8–8-, 8–5-, 8–O–4- and 4–O–5-coupled
expansion of the cell growth in grasses,3,4 and in cell wall
dehydrodiferulic acids (DFAs) 5, Fig. 1. The previously assumed
stiffening.5 Important properties of forages and plant derived
foods are attributed to cross-linked polysaccharides in the wall:
limited forage degradability by ruminants,6 thermal stability of
cell adhesion and maintenance of crispness of plant-based foods
after cooking,7–9 gelling properties of sugar beet pectins and
other food compounds,10–12 and insolubility of cereal dietary
fibers.13 Hydroxycinnamates and their derivatives are also noted
as bioactive compounds with antioxidative and antimutagenic
properties.14–16
Ferulic acid, to a lesser extent p-coumaric acid, and prob-
ably sinapic acid acylate cell wall polysaccharides.17–20 Mecha-
nisms for cross-linking cell wall polymers involve photochemical
dimerization,21,22 or radical dehydrodimerization,23–25 of these
hydroxycinnamates. The dominant mechanism for cross-linking
feruloylated polysaccharides is radical coupling, leading to
DFAs were synthesized24 and identified in different samples
following alkaline hydrolysis.5,9,13,24,30,31 Isolation and identification
of dehydrodiferuloylated oligosaccharides following hydrolysis
of the plant cell walls established the linkage of DFAs to
polysaccharides.32–34
It can be assumed that the 5–5-DFA 5D and 8–O–4-DFA
5A, Fig. 1, moieties found in isolated dehydrodiferuloylated
oligosaccharides, are present as structurally analogous esters in
the plant, i.e. that the di-acid analyzed derives directly from
saponification of its ester analog. It is likely that the minor 4–
O–5-coupled DFA 5E is also present as such. Furthermore, it is
presumed that the cyclic form of the 8–5-coupled diferulate ester
of 5B1 is the natural form of the 8–5-coupled diferulate; the open
form 5B2 is produced under the alkaline saponification and GC-
derivatization conditions used. The situation is more difficult to
ascertain for the 8–8-coupled dehydrodimers (5C1) and (5C3). To
date it remains unresolved whether they are natural or if they
are formed during analysis from a common natural precursor.
As indicated in earlier studies from our groups,2,31,35 another 8–8-
coupled DFA (8–8-THF–DFA, 5C2) was preliminarily identified
via mass spectral data, leading to an even more complex situation.
In this paper we describe the synthesis of one of the six di-
astereomers of compound 5C2 and its unambiguous identification
and quantification in different cereal grains. Its significance to
elucidating the nature of 8–8-coupled dehydrodiferulates in the
plant is also discussed.
aUS Dairy Forage Research Center, USDA-Agricultural Research Service,
1925 Linden Drive West, Madison, WI, 53706, USA. E-mail: schatz@chem.
wisc.edu; Fax: +1 (608) 890-0076; Tel: +1 (608) 890-0073
bUS Dairy Forage Research Center, USDA-Agricultural Research Ser-
vice, 1925 Linden Drive West, Madison, WI, 53706, USADepartment
of Forestry, U. Wisconsin-Madison, Madison, WI, 53706, USA. E-mail:
jralph@wisc.edu; Fax: +1 (608) 890-0076; Tel: +1 (608) 890-0071
cUS Dairy Forage Research Centre, USDA-Agricultural Research Ser-
vice, 1925 Linden Drive West, Madison, WI, 53706, USADepartment
of Forestry, U. Wisconsin-Madison, Madison, WI, 53706, USA. E-mail:
fachuanglu@wisc.edu; Fax: +1 (608) 890-0076; Tel: +1 (608) 890-0073
dDeptartment of Chemistry, U. Wisconsin-Madison, 1101 University Ave.,
Madison, WI, 53706, USA. E-mail: iguzei@chem.wisc.edu; Fax: +1 (608)
263-4694; Tel: +1 (608) 262-0381
eInstitut fu¨r Biochemie und Lebensmittelchemie, U. Hamburg, Grindalallee
117, 20146, Hamburg, Germany. E-mail: Mirko.Bunzel@uni-hamburg.de;
Fax: +49 (0)40-42-838-4342; Tel: +49 (0)40-42-838-4379
Materials and methods
General
† Electronic supplementary information (ESI) available: synthetic details
of the early steps of the synthesis of compound 13 (and ultimately DFA
5C2), along with the X-ray crystal structure data for compound 4C2-Ac,
are available. See DOI: 10.1039/b605918j
Heat-stable a-amylase Termamyl 120 L (EC 3.2.1.1, from Bacillus
licheniformis, 120 KNU g−1), the protease Alcalase 2.4 L (EC
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Fig. 1 Numbering system and radical dehydrodimerization of (cell wall bound) ferulates. The bond formed by radical coupling is in bold. In synthetic
compounds X represents the methyl or ethyl ester, in the cell wall X stands for arabinoxylans, arabinans or galactans.
3.4.21.62, from Bacillus licheniformis, 2.4 AU g−1) and the amy-
loglucosidase AMG 300 L (EC 3.2.1.3, from Aspergillus niger,
300 AGU g−1) were kindly donated by Novo Nordisk, Bagsvaerd,
Denmark. NMR spectra were run at 300 K on a 360 MHz Bruker
DRX-360 instrument (Bruker, Rheinstetten, Germany) using the
standard array of 1D and 2D experiments. Samples were dissolved
in acetone-d6. Chemical shifts (d) were referenced to the central
solvent signals (1H, d 2.04; 13C, d 29.8). J-Values are given in Hz.
NMR-assignments follow the numbering shown in Fig. 2. HPLC
instrumentation was from Merck/Hitachi (Darmstadt, Germany)
(L-6200 Intelligent pump, T-6300 Column thermostat) combined
with a Waters 994 programmable photodiode array detector
(Eschborn, Germany). HPLC-MS was performed using the HP
Series 1100 instrumentation: autosampler G1313A, bin pump
G1312A, degasser G1322A, mass spectrometer G1946A (ion-
source: atmospheric pressure electro-spray ionization), Hewlett
Packard (Waldbronn, Germany). GC-MS data, Fig. 3, was via
instrumentation from Thermoquest (Austin, TX, USA): Trace
2000 GC, QSC ion-trap MS. GC-FID was performed on a
Hewlett-Packard 5980 (Palo Alto, CA, USA) instrument. All fiber
samples were analyzed in triplicate. Cellulose and oat spelt xylan
used in the saponification of ester model 4C2 were from Sigma-
Aldrich.
Synthesis
Fig. 2 shows the synthetic route to the required isomer of
compound 5C2. All of the steps except the coupling step vi involve
rather standard synthetic methods. Details of the early steps are
given in the ESI.† Details of the crucial coupling reaction, and
NMR data of the important products are given here.
Dimethyl 2,5-bis-(4-benzyloxy-3-methoxyphenyl)-2,3-dihydro-
furan-3,4-dicarboxylate 13. Manganese(III) acetate dihydrate
(0.75 g, 2.8 mmol) and 12 mL of acetic acid were placed into
a 50 mL round-bottom flask equipped with a reflux condenser. To
the stirred mixture was added a solution of methyl 4-benzyloxy-
3-methoxybenzoylacetate 12 (0.40 g, 1.3 mmol) and methyl 3-(4ꢀ-
benzyloxy-3ꢀ-methoxyphenyl)propenoate 9 (0.36 g, 1.2 mmol) in
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Fig. 2 Synthetic pathway for 5C2: i: BnCl, KI, K2CO3, DMF; ii: malonic acid, NH4OAc, microwave; iii: MeI, K2CO3, DMF; iv: BnCl, KI, K2CO3,
DMF; v: MeO2CO, NaH, THF; vi: Mn(OAc)3, HOAc; vii: Pd/C, H2, EtOH; viii: AcCl, pyridine; ix: 2 N NaOH.
benzyl H3, H4, H5); 7.48 (2H, br d, J = 7.2 Hz, benzyl H2, H6);
7.50 (2H, br d, J = 7.0 Hz, benzyl H2, H6); 7.59 (1H, dd, J = 2.10,
8.55 Hz, A6); 7.82 (1H, d, J = 2.10 Hz, A2). dC 51.31 (OCH3);
52.64 (OCH3); 56.30 (OCH3); 59.26 (B8); 71.18 (OCH2Ph); 71.42
(OCH2Ph); 85.50 (B7); 101.69 (A8); 110.72 (B2); 113.45 (A5);
114.65 (A2); 115.05 (B5); 118.88 (B6); 122.73 (A1); 123.87 (A6);
128.46 (benzyl C2, C6); 128.52 (benzyl C2, C6); 128.60 (benzyl
C4); 128.71 (benzyl C4); 129.22 (benzyl C3, C5); 129.27 (benzyl
C3, C5); 134.26 (B1); 138.07 (benzyl C1); 138.41 (benzyl C1);
149.69 (A3); 149.74 (B4); 151.19 (B3); 151.80 (A4); 165.17 (A9–
=
=
C O); 166.38 (A7); 173.55 (B9–C O).
Dimethyl2,5-bis-(4-hydroxy-3-methoxyphenyl)-tetrahydrofuran-
3,4-dicarboxylate 4C2. Prepared in essentially quantitative yield
by debenzylation and hydrogenation of the double bond using
10% Pd on carbon (95% EtOH, RT, H2-balloon, 24 h). The NMR
spectrum indicated that the major product was the cis–trans–trans-
isomer (approx. 96%) and the minor product was the cis–cis–
trans-product. dH 3.21 (3H, s, CO2CH3); 3.66 (3H, s, CO2CH3);
3.84 (3H, s, OCH3); 3.89 (3H, s, OCH3); 3.60 (1H, dd, J = 6.31,
8.55 Hz, B8); 3.78 (1H, dd, J = 6.31, 8.29 Hz, A8); 4.95 (1H, d,
J = 8.55 Hz, B7); 5.27 (1H, d, J = 8.29 Hz, A7); 6.80 (1H, d, J =
8.02 Hz, A5); 6.85 (1H, d, J = 8.02 Hz, B5); 6.88 (1H, dd, J =
1.97, 8.02 Hz, A6); 7.02 (1H, d, J = 1.97 Hz, A2); 7.04 (1H, dd,
J = 1.97, 8.02 Hz, B6); 7.27 (1H, d, J = 1.97 Hz, B2); 7.53 (1H, s,
phenol), 7.59 (1H, s, phenol). dC 51.84 (OCH3); 52.37 (OCH3);
55.43 (A8); 55.83 (B8); 56.21 (OCH3); 56.27 (OCH3); 83.04 (A7);
84.14 (B7); 111.25 (A2); 111.33 (B2); 115.24 (A5); 115.50 (B5);
120.48 (A6); 120.71 (B6); 130.08 (A1); 132.22 (B1); 147.26 (A4);
Fig.
3 EI mass spectrum (top) and UV spectrum (bottom, in
MeOH–1 mM aqueous trifluoroacetic acid) of 5C2.
6 mL of acetic acid. The stirred mixture was heated with a hot
water bath (75–80 C) for 90 min. The mixture was dark brown
◦
at the beginning of the heating period, but became clear and light
brown at the end of the heating period. The mixture was allowed
to cool to room temperature and was then poured into cold water.
A sticky solid formed. The mixture was extracted with 50 mL of
ether. The organic phase was washed several times with 25 mL
portions of sodium bicarbonate solution to remove the acetic
acid. Evaporation of the ether yielded 0.65 g of a sticky solid.
The residue was dissolved in approximately 2 mL of acetone. On
standing overnight crystals formed. The liquid was decanted and
the solid was recrystallized from methanol to afford 0.23 g of a
white solid (32%), mp. 123–124 ◦C. dH 3.62 (3H, s, CO2CH3); 3.74
(3H, s, CO2CH3); 3.83 (3H, s, OCH3); 3.86 (3H, s, OCH3); 4.21
(1H, d, J = 7.10 Hz, B8); 5.13 (2H, s, OCH2Ph); 5.20 (2H, s,
OCH2Ph); 5.71 (1H, d, J = 7.10 Hz, B7); 6.98 (1H, dd, J = 2.10,
8.29 Hz, B6); 7.06 (1H, d, J = 8.29 Hz, B5); 7.10 (1H, d, J =
8.55 Hz, A5); 7.11 (1H, d, J = 2.10 Hz, B2); 7.28–7.42 (6H, m,
=
147.54 (B4); 147.86 (A3); 148.32 (B3); 172.45 (A9–C O); 172.85
=
(B9–C O).
Dimethyl 2,5-bis-(4-acetoxy-3-methoxyphenyl)-tetrahydrofuran-
3,4-dicarboxylate 4C2-Ac. This isomeric mixture above was used
to prepare the diacetate using pyridine–acetyl chloride (82% yield
as a white solid). The solid was recrystallized from methanol to
yield 4C2-Ac as colorless prisms, mp. 137–140 ◦C. dH 2.22 (3H, s,
Ac–CH3); 2.24 (3H, s, Ac–CH3); 3.19 (3H, s, CO2CH3); 3.70 (3H, s,
CO2CH3); 3.64 (1H, dd, J = 5.39, 8.42 Hz, B8); 3.82 (3H, s, OCH3);
3.86 (1H, dd, J = 5.39, 8.16 Hz, A8); 3.88 (3H, s, OCH3); 5.10 (1H,
d, J = 8.42 Hz, B7); 5.39 (1H, d, J = 8.16 Hz, A7); 7.02 (2H, m,
A5 + A6); 7.08 (1H, d, J = 8.16 Hz, B5); 7.18 (1H, bs, A2); 7.20
(1H, dd, J = 1.84, 8.16 Hz, B6); 7.44 (1H, d, J = 1.84 Hz, B2).
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dC 20.44 (Ac–CH3); 20.48 (Ac–CH3); 51.95 (OCH3); 52.54 (OCH3);
55.43 (A8); 55.83 (B8); 56.20 (OCH3); 56.26 (OCH3); 82.99 (A7);
83.76 (B7); 111.85 (A2); 112.14 (B2); 119.55 (A6); 119.90 (B6);
123.18 (A5); 123.51 (B5); 137.27 (A1); 139.77 (B1); 140.66 (B4);
140.86 (A4); 151.83 (A3); 152.31 (B3); 168.97 (acetate), 172.37
of reflections collected, 39503; number of independent reflections,
5229 [R(int) = 0.0350]; final R indices [I > 2r(I)], R1 = 0.0453,
wR2 = 0.1220; R indices (all data), R1 = 0.0514, wR2 = 0.1280.
Identification of 8–8-THF–DFA in cereal grain fibers
=
=
(A9–C O); 172.62 (B9–C O).
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 local German suppliers. Botanical origin was
confirmed by the suppliers.
Preparation of whole grain insoluble and soluble cereal fibers
was performed according to an enzymatic, preparative isolation
procedure described previously.13 This procedure was developed
to use 10 g of sample material and involves the application of a
sequence of heat stable a-amylase, protease and amyloglucosidase.
For calculations, insoluble and soluble fibers were corrected for
residual protein and ash contents.
2,5-Bis-(4-hydroxy-3-methoxyphenyl)-tetrahydrofuran-3,4-di-
carboxylic acid 5C2. Saponification (2 N NaOH, 18 h) of 4C2-Ac
produced 5C2 as a colorless foam (54%). dH 3.81 (3H, s, OCH3);
3.87 (3H, s, OCH3); 3.61 (1H, dd, J = 6.58, 8.68 Hz, B8); 3.80
(1H, dd, J = 6.58, 8.55 Hz, A8); 4.98 (1H, d, J = 8.68 Hz, B7);
5.31 (1H, d, J = 8.55 Hz, A7); 6.77 (1H, d, J = 8.16 Hz, A5); 6.84
(1H, d, J = 8.16 Hz, B5); 6.94 (1H, dd, J = 1.97, 8.16 Hz, A6);
7.06 (1H, dd, J = 1.97, 8.16 Hz, B6); 7.09 (1H, d, J = 1.97 Hz,
A2); 7.31 (1H, d, J = 1.97 Hz, B2). dC 55.39 (A8); 55.99 (B8);
56.13 (OCH3); 56.17 (OCH3); 82.91 (A7); 84.13 (B7); 111.48 (B2);
111.51 (A2); 115.16 (A5); 115.42 (B5); 120.75 (A6); 120.80 (B6);
130.45 (A1); 132.43 (B1); 147.14 (A4); 147.44 (B4); 147.75 (A3);
=
=
148.26 (B3); 172.86 (A9–C O); 173.42 (B9–C O).
For the identification and quantification of 8–8-THF–DFA
5C2, the internal standard (E,E)-4-hydroxy-4ꢀ,5,5ꢀ-trimethoxy-
3,3ꢀ-bicinnamic acid (5–5-Me–DFA, the monomethyl ether of
5D)13 dissolved in dioxane was added to the insoluble (40–90 mg)
or soluble (60–120 mg) fiber samples weighed into a screw-cap
tube, 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 headspace of the screw-
cap tubes was exchanged with N2. Samples were acidified with
0.95 mL 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 as previously described.13 Some samples
were also analyzed as described above but spiked following the
saponification process with small amounts of the synthesized 8–
8-THF–DFA 5C2. Relative retention times of 5C2 against the
internal standard 5–5-Me–DFA: GC-MS-instrumentation: 0.691,
GC-FID-instrumentation: 0.683.
X-Ray crystal structure determination of 4C2-Ac
A colorless crystal with approximate dimensions 0.43 × 0.32 ×
0.26 mm3 was selected for crystal structure determination on
a Bruker CCD-1000 diffractometer with Mo Ka (k = 0.71073
˚
A) radiation and a diffractometer to crystal distance of 4.9 cm.
Crystallographic data (excluding structure factors) for structure
4C2-Ac, Fig. 4, in this paper have been deposited with the
Cambridge Crystallographic Data Centre as supplementary pub-
lication number CCDC 603905.‡ The compound and crystal
had the following properties and collection conditions: empiri-
cal formula, C26H28O11; formula weight, 516.48; crystal system,
orthorhombic; unit cell dimensions a = 7.3722(6), b = 25.899(2),
◦
3
˚
˚
c = 26.760(2) A, a = b = c = 90 ; unit cell volume, 5109.4(7) A ;
temperature 173(2) K; space group, Pbca; number of formula units
in unit cell, 8; linear absorption coefficient 0.106 mm−1; number
To check if the saponification procedure releases only 8–8-THF–
DFA 5C2 or if additional compounds are formed, the following
experiments were performed. The synthesized dimethyl ester of
5C2 (i.e. 4C2, 300 lg) was added to a mixture of 45 mg xylan and
30 mg cellulose simulating a typical matrix composition of cereal
insoluble fiber. In addition, the dimethyl ester of 5C2 was added
to 75 mg cellulose. Both reactions were carried out in triplicate.
To monitor interfering peaks from the carbohydrate matrix, a
mixture consisting of xylan (45 mg) and cellulose (30 mg) was
also saponified. Saponification and extraction was carried out
as described above. The extract was used for RP-HPLC analysis
with UV and MS detection. HPLC analysis was performed on
a Luna 5 l 250 × 4.6 mm phenyl–hexyl column (Phenomenex,
Aschaffenburg, Germany) at 45 ◦C, maintaining a flow rate of
1 mL min−1. The following gradient was used (eluent A: MeOH,
eluent B: acetonitrile, eluent C: 1 mM aqueous trifluoroacetic
acid): initially A 27%, B 4%, C 69 %, held isocratically for 7 min,
linear over 11 min to A 29%, B 6%, C 65%, linear over 5 min to
A 20%, B 70%, C 10%, held isocratically for 5 min, following an
equilibration step. Extracts were partially also used for GC-MS
investigations using the conditions described above.
Fig. 4 3D Structure of compound 4C2-Ac (Fig. 2) from the X-ray crystal
structure, demonstrating the assigned cis–trans–trans-stereochemistry. The
8–8-bond formed (in planta) via the radical coupling step is in bold; in the
ESI, this bond and the two original ferulates are color-differentiated. The
structure is numbered to be consistent with the structures in Fig. 2; full
numbering for the crystal structure coordinates is provided in the ESI.†
‡ CCDC reference numbers 603905. For crystallographic data in CIF or
other electronic format see DOI: 10.1039/b605918j
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DFA content in cereal fibers. In all investigated insoluble cereal
fibers 8–8-THF–DFA 5C2 was more prevalent than 4–O–5-DFA
5E. Furthermore the amounts of DFA 5C2 ranged between 50%
(in rice insoluble fiber) and 90% (in rye insoluble fiber) of the
amounts of DFA 5C1. In maize insoluble fiber DFA 5C2 was
even more prevalent than DFA 5C1 (about 180%). Due to the tiny
amounts in soluble cereal fibers, the amounts of DFA 5C2 in these
samples were not unambiguously determined; the amounts often
did not reach the determination limit. These very low amounts
were surprising because the other 8–8-coupled DFAs (5C1 and
5C3) are often determined to be the major ones in soluble cereal
fibers.
Results and discussion
Synthesis
Since the initial identification of compound 5C2 was based
on mass spectral data (see Fig. 3 for EI mass spectrum of
trimethylsilylated 5C2), we did not know which one of the six
possible diastereomers of the suspected ferulate tetrahydrofuran
“dimer” was isolated. The first synthetic path chosen produced
the correct diastereomer. The stereochemistry might logically
be the result of isomerization upon saponification; however, the
stereochemistries of the ester and the acid appear to be the same.
The crucial step in the synthetic pathway chosen involves the
coupling of cinnamate ester 9 with a b-ketoester 12 (Fig. 2) via
Mn(OAc)3.36 The stereochemistry of the resulting dihydrofuran
ring in 13 is assigned as trans. The coupling constant between the
ring protons is 7 Hz which compares favorably with the coupling
constant reported for the trans-isomer of diethyl 2-chloro-5-
phenyl-4,5-dihydrofuran-3,4-dicarboxylate.37 The coupling con-
stant of the cis-isomer of the same compound is reported to
be 11 Hz. The regio- and stereoselectivity for dihydrofuran 13
is consistent with that demonstrated previously for Mn(OAc)3-
mediated coupling of cinnamates and b-ketoesters.36,38–41 Pal-
ladium catalyzed cis-hydrogenation and debenzylation of the
dihydrotetrahydrofuran 13 resulted in the tetrahydrofuran product
4C2, largely as the cis–trans–trans-isomer, with a small amount of
the cis–cis–trans-isomer. Assignments are based on the assumption
that cis-hydrogenation has occurred from the least hindered face of
dihydrofuran 13. Saponification of this product, or of the derived
acetate 4C2-Ac, gave the required di-acid 5C2. After confirming
this to be the same isomer released from the natural plant sources
(see below), the isomeric form was elucidated by NMR and,
on 4C2-Ac, unambiguously validated by X-ray crystal structure,
Fig. 4 (and ESI†).
Nature of dehydrodiferulates in the plant
We have already established that saponification of esters 4C1 and
4C3 yield only their corresponding acids 5C1 and 5C3 respectively;
for example, the ester 4C1 does not produce acid 5C3. Therefore,
if these esters are in the wall, both forms need to be present to
explain the saponification products. A more appealing situation
that needed testing was that all three 8–8-DFAs 5C1-3 result from
a single ester precursor in the wall such as the tetrahydrofuran
4C2. To test the plausibility of a common precursor, the formerly
synthesized methyl ester of 5C2 (4C2, X = Me, Fig. 1, or the
ethyl analog) was saponified in a cell-wall-like matrix consisting
of cellulose–xylan 2 : 3 or in pure cellulose. These experiments
yielded predominantly the tetrahydrofuran 5C2. The non-cyclic
8–8-DFA 5C1 was not detected in any saponification experiment,
thus excluding 4C2 as precursor of the non-cyclic 8–8-DFA
5C1. The situation was different with the cyclic 8–8-DFA 5C3.
Although in all saponification experiments the 8–8-THF 5C2
clearly dominated the HPLC-UV and GC-FID-chromatograms,
in the HPLC runs two small peaks were detected showing UV-
spectra and mass peaks consistent with cyclic 8–8-DFAs. Based
on peak areas at 280 nm the single peaks were typically 0.4 to 5.8%
of the peak area of 5C2. However, some unwelcome variability is
noted—in a single saponification experiment the peak areas were
9 and 19% of the peak area of 5C2. In a further saponification
experiment the alkaline hydrolysate was investigated using GC-
MS. Chromatograms showed two additional peaks, one of them
having the same retention time and mass spectrum as the cyclic
8–8-DFA 5C3. The second, slightly bigger peak with the same
mass spectrum elutes very close to 5C3. It is assumed that this
compound is an isomer of 5C3 (Fig. 1), presumably having
alternate ring-B C7 and C8 stereochemistry. Re-evaluation of the
GC-MS chromatograms that were used for the identification of
ferulate dimers in cereal grains13 revealed that this assumed isomer
of 5C3 was not produced during the saponification of plant cell
walls. This indicates that saponification produces no detectable
conversion from arabinoxylan esters of 5C2 to the cyclic 8–8-DFA
products. Furthermore, the saponification experiments using the
methyl ester of 5C2 showed that 5C2 is always the predominant
product. The small amounts of cyclic 8–8-DFA 5C3 formed upon
saponification cannot explain the determined amounts of 5C3
from the wall.
Identification and quantification of 8–8-THF–DFA in cereal grain
fibers
In all extracts of investigated saponified insoluble cereal fibers,
8–8-THF–DFA (5C2, Fig. 1) was identified by comparison of
its mass spectrum and its relative GLC retention time with that
of the genuine compound. Identity was further confirmed by
spiking some samples with the synthesized compound. Although
the identification of 8–8-THF–DFA in the corresponding soluble
fiber fraction was difficult due to the tiny amounts detected,
it was found in all investigated cereals. Fig. 3 shows the MS-
spectrum of trimethylsilylated 8–8-THF–DFA obtained via GC-
MS; with originally two phenolic and two acid groups and, in
comparison to the other DFAs, an additional water, its nominal
molecular mass is 692. As HPLC-analysis with diode array
detection of monomeric, dimeric42 and trimeric hydroxycinnamic
acids is becoming attractive, the UV-spectrum of 8–8-THF–DFA
in MeOH–1 mM aqueous trifluoroacetic acid is provided in Fig. 3.
Because both sidechains are saturated, there is no maximum at
∼325 nm, typical of ferulic acid and the DFAs.
The amounts of 8–8-THF–DFA in insoluble cereal fibers were
determined as follows [lg g−1]: maize 692 39; wheat 84 8; spelt
109 8; rye 132 5; barley 133 3; oats 149 34; millet 242 13;
rice 109 11; wild rice 115 8. Regarding the DFA distribution,13
8–8-THF–DFA contributes between 2.5 and 4.9% to the absolute
It therefore now appears that 8–8-coupling in the plant cell
wall leads to three distinct 8–8-diferulates (4C1, 4C2, 4C3, X =
arabinoxylan, Fig. 1) that are the precursors for the detected acids
5C1, 5C2 and 5C3. There is an urgent need to investigate the
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Org. Biomol. Chem., 2006, 4, 2801–2806 | 2805
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nature of the dehydrodiferulates in vivo and not only in alkaline
hydrolysates.
3 K. S. Tan, T. Hoson, Y. Masuda and S. Kamisaka, Plant Cell Physiol.,
1992, 33, 103–108.
4 S. Kamisaka, S. Takeda, K. Takahashi and K. Shibata, Physiol. Plant.,
1990, 78, 1–7.
5 I. Zarra, M. Sanchez, E. Queijeiro, M. J. Pena and G. Revilla, J. Sci.
Food Agric., 1999, 79, 416–420.
6 R. D. Hatfield, J. Ralph and J. H. Grabber, Crop Sci., 1999, 39, 27–
37.
7 K. W. Waldron, A. C. Smith, A. J. Parr, A. Ng and M. L. Parker, Trends
Food Sci. Technol., 1997, 8, 213–221.
8 A. J. Parr, K. W. Waldron, A. Ng and M. L. Parker, J. Sci. Food Agric.,
1996, 71, 501–507.
9 C. C. Parker, M. L. Parker, A. C. Smith and K. W. Waldron, J. Agric.
Food Chem., 2003, 51, 2034–2039.
10 H. U. Markwalder and H. Neukom, Phytochemistry, 1976, 15, 836–837.
11 A. Oosterveld, J. H. Grabber, G. Beldman, J. Ralph and A. G. J.
Voragen, Carbohydr. Res., 1997, 300, 179–181.
12 A. Ng, R. N. Greenshields and K. W. Waldron, Carbohydr. Res., 1997,
303, 459–462.
13 M. Bunzel, J. Ralph, J. M. Marita, R. D. Hatfield and H. Steinhart,
J. Sci. Food Agric., 2001, 81, 653–660.
14 L. R. Ferguson and P. J. Harris, Lancet, 2003, 361, 1487–1488.
15 L. R. Ferguson, I. F. Lim, A. E. Pearson, J. Ralph and P. J. Harris,
Mutat. Res., 2003, 542, 49–58.
16 M. F. Andreasen, P. A. Kroon, G. Williamson and M. T. Garcia-
Conesa, Free Radical Biol. Med., 2001, 31, 304–314.
17 E. Allerdings, J. Ralph, H. Steinhart and M. Bunzel, Phytochemistry,
2006, DOI: 10.1016/j.phytochem.2006.04.018.
18 L. Saulnier and J.-F. Thibault, J. Sci. Food Agric., 1999, 79, 396–402.
19 T. Ishii, Plant Sci., 1997, 127, 111–127.
As with any chemical reaction that produces multiple products,
the ratio of products is dependent upon reaction conditions. Some
hint as to what might be driving post-coupling reactions in the cell
wall comes from more detailed recent unpublished examinations
of ferulate coupling. [Partitioning among all the various coupling
modes is also condition-dependent, but it is reasonable to assume
that once radical coupling regiochemistry has been determined
(e.g. as 8–8, for example) it is the various rearomatization
possibilities that control the final product distribution]. Lu (2005,
unpublished) has found that low pH favors the tetrahydrofuran
product. This is a mechanistically reasonable observation. The
intermediate quinone methide 3C, produced directly following the
8–8-coupling step, will more readily add water nucleophilically
at low pH resulting in tetrahydrofuran 5C2; such addition is
protonation-dependent. At high pH, the elimination reactions
producing 5C1 are more likely.
Cursory mass spec analysis indicates no other isomers of 5C2 at
a significant level (trace amounts of one more isomer are possible
from the selected ion mode in the GC-MS) although the synthesis
and coupling reactions with methyl ferulate appear to produce
other isomers. Determining whether other isomers of the ester
4C2 produce the same acid isomer 5C2 following saponification
is beyond the current study, which sought to identify the product
liberated from natural sources.
20 M. Bunzel, E. Allerdings, V. Sinwell, J. Ralph and H. Steinhart, Eur.
Food Res. Technol., 2002, 214, 482–488.
21 R. D. Hartley, W. H. Morrison, III, F. Balza and G. H. N. Towers,
Phytochemistry, 1990, 29, 3699–3703.
22 R. D. Hartley, W. H. Morrison, III, D. S. Himmelsbach and W. S.
Borneman, Phytochemistry, 1990, 29, 3705–3709.
23 M. Bunzel, J. Ralph, H. Kim, F. Lu, S. A. Ralph, J. M. Marita, R. D.
Hatfield and H. Steinhart, J. Agric. Food Chem., 2003, 51, 1427–1434.
24 J. Ralph, S. Quideau, J. H. Grabber and R. D. Hatfield, J. Chem. Soc.,
Perkin Trans. 1, 1994, 3485–3498.
25 T. Geissmann and H. Neukom, Helv. Chim. Acta, 1971, 54, 1108–1112.
26 C. Funk, J. Ralph, H. Steinhart and M. Bunzel, Phytochemistry, 2005,
66, 363–371.
27 M. Bunzel, J. Ralph, C. Funk and H. Steinhart, Eur. Food Res. Technol.,
2003, 217, 128–133.
28 X. Rouau, V. Cheynier, A. Surget, D. Gloux, C. Barron, E. Meuded, J.
Louis-Montero and M. Criton, Phytochemistry, 2003, 63, 899–903.
29 M. Bunzel, J. Ralph, C. Funk and H. Steinhart, Tetrahedron Lett., 2005,
46, 5845–5850.
30 K. W. Waldron, A. Ng, M. L. Parker and A. J. Parr, J. Sci. Food Agric.,
1997, 74, 221–228.
31 J. H. Grabber, J. Ralph and R. D. Hatfield, J. Agric. Food Chem., 2000,
48, 6106–6113.
32 T. Ishii, Carbohydr. Res., 1991, 219, 15–22.
33 E. Allerdings, J. Ralph, P. Schatz, D. Gniechwitz, H. Steinhart and M.
Bunzel, Phytochemistry, 2005, 66, 113–124.
34 L. Saulnier, M. J. Crepeau, M. Lahaye, J. F. Thibault, M. T. Garcia-
Conesa, P. A. Kroon and G. Williamson, Carbohydr. Res., 1999, 320,
82–92.
35 J. Ralph, M. Bunzel, J. M. Marita, R. D. Hatfield, F. Lu, H. Kim,
J. H. Grabber, S. A. Ralph, G. Jimenez-Monteon and H. Steinhart,
Polyphe´nols Actual., 2000, 13–17.
36 F. Z. Yang, M. K. Trost and W. E. Fristad, Tetrahedron Lett., 1987, 28,
1493–1496.
Conclusions
Alkaline hydrolysates of cereal grain and grass walls contain
a previously unauthenticated component derived from ferulate.
Strictly DFA 5C2 is not a true dehydrodimer since an additional
oxygen (from water) is incorporated. Its higher molecular mass
is responsible for its being missed as a dimeric ferulate product
previously. It is still formed via radical coupling, but the post-
coupling steps are different from those in the previously identified
DFAs.2 DFA 5C2 is a substantial component that should also
be quantified as resulting from 8–8-dehydrodimerization. Only
one of the six potential diastereomers of 5C2, the cis–trans–trans-
isomer, was detected among the products from saponified plant
cell walls. The finding of this 8–8-tetrahydrofuran DFA 5C2 along
with the previously identified 5C1 and 5C3 implicates at least three
8–8-coupling products of ferulate in the cell walls of these plants
and suggests that cell wall cross-linking may occur under acidic
conditions.
Acknowledgements
We thank Carola Funk and Jane Marita for experimental support.
We gratefully acknowledge partial funding through the USDA-
CSREES National Research Initiatives (Food Characterization
#2003-35503-13820).
37 H. S. Gill and J. A. Landgrebe, J. Org. Chem., 1983, 48, 1051–1055.
38 F. Garzino, A. Meou and P. Brun, Tetrahedron Lett., 2000, 41, 9803–
9807.
39 F. Garzino, A. Meou and P. Brun, Eur. J. Org. Chem., 2003, 1410–1414.
40 F. Garzino, A. Meou and P. Brun, Synthesis, 2003, 598–602.
41 F. Garzino, A. Meou and P. Brun, Tetrahedron Lett., 2002, 43, 5049–
5051.
References
1 M. Piber and P. Koehler, J. Agric. Food Chem., 2005, 53, 5276–5284.
2 J. Ralph, M. Bunzel, J. M. Marita, R. D. Hatfield, F. Lu, H. Kim,
P. F. Schatz, J. H. Grabber and H. Steinhart, Phytochem. Rev., 2004, 3,
79–96.
42 K. W. Waldron, A. J. Parr, A. Ng and J. Ralph, Phytochem. Anal., 1996,
7, 305–312.
2806 | Org. Biomol. Chem., 2006, 4, 2801–2806
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The Royal Society of Chemistry 2006
©