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J Sci Food Agric 79 :408–410 (1999)
Journal of the Science of Food and Agriculture
Structure-specific functionality of plant cell wall
hydroxycinnamates¹
Wendy R Rus s ell,1* Mark J Burkitt,2 Gordon J Provan1 and Andrew Ches s on1
1 Rowett Res earch Ins titute , Greenburn Road , Bucks burn , Aberdeen AB21 9SB , UK
2 Lund Univers ity , Department of Biochemis try , Center for Chemis try and Chemical Engineering , PO Box 124 , S -221 00 , Sweden
Abstract : Within the plant cell wall, 4-hydroxy-3-methoxycinnamic acid (ferulic acid) has a clear role
in polymer cross-linking. However, why this function appears largely restricted to the monomethoxylated compound and not to other hydroxycinnamates appearing in the wall is less evident.
Since radical coupling is the main mechanism by which hydroxycinnamate cross-linking occurs, the
ease of parent radical formation and distribution of the unpaired electron were investigated. Ease of
oxidation increased with increased substrate methoxylation, as did the amount of positive spindensity residing on the phenolic oxygen. The properties of the dimethoxylated hydroxycinnamate
indicated that when esteriüed to the wall, coupling would result in C–O bond formation. This form of
bonding is weaker and more ýexible than the C–C bonding which would result from coupling of 4hydroxy-3-methoxycinnamate and would not be desirable as a cross-link. Although the nonmethoxylated compound could also couple via C–C bonds, ESR measurements of phenoxyl radical
formation suggested that this compound would not easily participate in coupling reactions.
( 1999 Society of Chemical Industry
Keywords : phenylpropanoid ; ESR ; AM1; peroxidase ; hydroxycinnamic acid ; ferulic acid ; hydroxycinnamate ; lignin ; oxidative coupling
INTRODUCTION
Substituted hydroxycinnamic acids are products of
the phenylpropanoid pathway and, in addition to
being precursors to many other secondary metabolites, are often found associated with the plant cell
wall. Within the Poaceae, 4-hydroxy-3-methoxycinnamic acid (ferulic acid) is esteriüed to structural
polysaccharides and may provide cross-links to other
polysaccharide chains or to lignin.1h4 However, the
more abundant 4-hydroxycinnamic acid appears to
have a diþerent function, as most is deposited in
parallel with lignin to which it appears covalently
linked.5 The structurally similar 3,4-dihydroxycinnamic acid, 4,5-dihydroxy-3-methoxycinnamic acid
and 3,5-dimethoxy-4-hydroxycinnamic acid, while
occurring as metabolic intermediates in the formation of lignin precursors, are found only in trace
quantities in cell wall preparations.
Speciüc roles for hydroxycinnamic acids within
the plant are dependant on the chemical and physical
properties imparted by structural variation. Crosslinking requires the coupling of monomers and, with
the exception of cycloaddition, this reaction proceeds
via a radical mechanism. The reaction is controlled
by a complex interplay of polar, steric and bondstrength terms. It has been demonstrated that the
synthetic coupling of phenylpropanoid radicals is
controlled by the distribution of the unpaired electron.6 Once generated, coupling of the unpaired electrons will occur at the position of highest electron
density unless there is steric compression in the
product.
Generation of the parent radical is also a major
factor contributing to the reactivity of hydroxycinnamic acids and this reaction is catalysed in situ
by peroxidases and, possibly, monooxygenases.7h9
When recorded at low temperature and neutral pH,
the electron spin resonance (ESR) spectrum of
horseradish peroxidase exhibits a broad signal at
g \ 6.277 characteristic of high spin FeIII.10 Addition
of hydrogen peroxide results in the formation of a
signal at g \ 2.003 indicative of the formation of
compound I and the concomitant loss of the signal at
g \ 6.277 as FeIII is oxidised to FeIV.11 It is the free
radical at g \ 2.003 that initially accepts an electron
from a suitable reducing substrate yielding the substrate free radical and the oxyferryl haem intermediate, compound II, which is subsequently reduced
to the original ferric form of peroxidase. Reduction
in the signal observed at g \ 2.003 can be used as a
guide to the ease of phenoxyl radical formation.
Since hydroxycinnamic acids are most commonly
found esteriüed to polysaccharides, we sought to
determine the regiochemistry and reactivity of ester-
¹ Bas ed on a paper pres ented at Ferulate ’98, IFR, Norwich, 8–11
July 1998.
* Corres pondence to : Wendy R Rus s ell, Rowett Res earch
Ins titute, Greenburn Road, Bucks burn, Aberdeen, AB21 9SB, UK
Contract/grant s pons or : The Scottis h Office, Agriculture, Environment and Fis heries Dept
(Received 9 July 1998 ; revis ed vers ion received 2 September
1998 ; accepted 15 October 1998 )
( 1999 Society of Chemical Industry. J Sci Food Agric 0022-5142/99/$17.50
408
Functionality of plant cell wall hydroxycinnamates
bound hydroxycinnamic acids as a guide to their
possible function within the plant.
EXPERIMENTAL
General laboratory reagents and substituted 4hydroxycinnamic acids were obtained from Aldrich
(UK). The ethyl esters were prepared by dissolving
the substituted cinnamic acids (100 mmol) in ethanol
(500 cm3). Acetyl chloride (50 cm3) was added slowly
and the reaction monitored by tlc. After complete
disappearance of the acid, the solvent was removed in
vacuo and the crude product redissolved in ethyl
acetate and extracted with NaHCO (3% w/v). The
3
organic layer was then dried over anhydrous
Na SO , ültered and the solvent removed. Crys2 4
tallisation from ethyl acetate/petroleum ether (bp 40–
60¡C) gave the respective ethyl esters : Ethyl
4-hydroxycinnamate as white crystals, yield 68%,
NMR d (CD OD) 1.24 (3H, t, J 7.1, CH CH ),
H
3
2
3
4.16 (2H, q, J 7.1, CH CH ), 6.36 (1H, d, J 16,
2
3
C(8)H), 6.78 (2H, d, J 8.4, C(3 and 5)H), 7.53 (2H,
d, J 8.4, C(2 and 6)H), 7.56 (1H, d, J 16, C(7)H)
ppm. Ethyl 4-hydroxy-3-methoxycinnamate as white
crystals, yield 74%, NMR d (CD OD) 1.26 (3H, t,
H
3
J 7.1, CH CH ), 3.92 (3H, s, OCH ), 4.18 (2H, q, J
2
3
3
7.1, CH CH ), 6.39 (1H, d, J 16, C(8)H ), 6.87 (1H,
2
3
d, J 8.2, C(5)H), 7.14 (1H, dd, J 8.2 and 1.6,
C(6)H), 7.34, (1H, d, J 1.6, C(2)H), 7.58 (1H, d, J
16, C(8)H) ppm. Ethyl 3,5-dimethoxy-4-hydroxycinnamate as white crystals, yield 82%, NMR d
H
(CD OD) 1.28 (3H, t, J 7.1, CH CH ), 3.89 (6H, s,
3
2
3
OCH ), 4.19 (2H, q, J 7.1, CH CH ), 6.42 (1H, d, J
3
2
3
15.9, C(7)H), 7.02 (2H, s, C(2 and 6)H), 7.58 (1H, d,
J 15.9, C(8)H) ppm. ESR spectra (X-band) were
recorded as reported previously.
RESULTS
ESR spectra were recorded for the ethyl hydroxycinnamates using a continuous-ýow apparatus and
ammonium cerium(IV) nitrate as the one-electron
oxidant. The density of the unpaired electron at each
C atom was calculated directly from the hyperüne
coupling constants using the McConnell relationship. The positive spin density on the phenoxyl was
determined computationally by Austin Model 1 (Fig
1).12,13 The distribution of the unpaired electron
density increased on the phenolic oxygen and
decreased at C8 with the increasing extent of methoxylation of the aromatic ring. From this data, the
site at which coupling will occur was predicted. The
ability of substrates to reduce the peroxidase enzyme
intermediate, compound I, was shown by the signal
intensity at g \ 2.003 (Fig 2). The signal intensity was
lowest for ethyl 4-hydroxy-3,5-dimethoxycinnamate
indicating this compound to be the most easily oxidised. As the degree of substrate methoxylation
decreased, the signal intensity at g \ 2.003 became
stronger. This demonstrates the eþect of methJ Sci Food Agric 79 :408–410 (1999)
Figure 1. Structures repres enting the ethyl hydroxycinnamate
es ters s howing the dis tribution of the unpaired electron at the
carbon nuclei determined directly from the experimental ESR
s pectra via the McConnell relations hip. Theoretical values (%)
for the pos itive s pin dens ity res iding on the phenoxyl are given
in parenthes es .
oxylation on the ease of hydroxycinnamate oxidation
and therefore the extent of their participation in
coupling reactions.
DISCUSSION
It was predicted from the electron spin distribution
that the degree of methoxylation would direct the
type of linkage formed, with only the dimethoxylated
cinnamic acid demonstrating a preference for C–O
bond formation. Synthetic coupling of 4-hydroxy-3methoxycinnamic acid esteriüed to methyl-a-L-arabinofuranoside (a model for the most commonly
observed glycoside) resulted in 90% of products containing a C–C linkage.4 Since phenylpropanoids
appear to couple exclusively at C8, the incorporation
of the monomethoxylated cinnamate rather than the
dimethoxylated analogue as a cross-link to polysaccharide may be due to the additional strength
imparted by formation of a C–C linkage. The lower
capacity of the non-methoxylated compound to
reduce compound I would not facilitate the forma-
Figure 2. Low temperature ESR s pectra of peroxidas e, compound
I in the pres ence of cinnamate es ters : (A) ethyl
4-hydroxycinnamate, (B) ethyl 4-hydroxy-3-methoxycinnamate,
and (C) ethyl 3,5-dimethoxy-4-hydroxycinnamate. Spectral
width \ 3500 Gaus s .
409
WR Russell, MJ Burkitt, GJ Provan, A Chesson
tion of cross-links despite the preference for C–C
bond formation observed between non-methoxylated
dilignols. The comparative inability of ethyl 4hydroxycinnamate to reduce compound I, coupled
with the low unpaired spin density on the phenolic
oxygen, suggest that, when esteriüed, the nonmethoxylated compound will not readily participate
in further reactions. Despite the low reactivity of this
unit, the discovery of 4-hydroxycinnamic acid esteriüed to the primary alcohol of 3,5-dimethoxycinnamyl alcohol indicates that this ester is incorporated
into the cell wall.14 This demonstrates a requirement
for 4-hydroxycinnamate by the plant and it has been
suggested that its presence aids the coupling of 3,5dimethoxy-4-hydroxycinnamyl alcohol by a radical
transfer
mechanism.15
Although,
the nonmethoxylated cinnamate is oxidised to the phenoxyl
radical less easily than the substituted cinnamyl alcohols, its properties are such that it could facilitate
catalysis by some other mechanism and this requires
further study.
ACKNOWLEDGEMENTS
Financial support for this work was provided by The
Scottish Office, Agriculture, Environment and Fisheries Department.
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J Sci Food Agric 79 :408–410 (1999)
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