Initial steps of the peroxidase-catalyzed polymerization of coniferyl alcohol and/or sinapyl aldehyde: Capillary zone electrophoresis study of pH effect

Article abstract of DOI:10.1016/S0031-9422(02)00573-3

Capillary zone electrophoresis has been used to monitor the first steps of the dehydrogenative polymerization of coniferyl alcohol, sinapyl aldehyde, or a mixture of both, catalyzed by the horseradish peroxidase (HRP)-H₂O₂ system. When coniferyl alcohol was the unique HRP substrate, three major dimers were observed (β-5, β-β, and β-O-4 interunit linkages) and their initial formation velocity as well as their relative abundance varied with pH. The β-O-4 interunit linkage was thus slightly favored at lower pH values. In contrast, sinapyl aldehyde turned out to be a very poor substrate for HRP except in basic conditions (pH 8). The major dimer observed was the β,β′-di-sinapyl aldehyde, a red-brown exhibiting compound which might partly participate in the red coloration usually observed in cinnamyl alcohol dehydrogenase-deficient angiosperms. Finally, when a mixture of coniferyl alcohol and sinapyl aldehyde was used, it looked as if sinapyl aldehyde became a very good substrate for HRP. Indeed, coniferyl alcohol turned out to serve as a redox mediator (i.e. shuttle oxidant ) for the sinapyl aldehyde incorporation in the lignin-like polymer. This means that in particular conditions the specificity of oxidative enzymes might not hinder the incorporation of poor substrates into the growing lignin polymer.

Full text of DOI:10.1016/S0031-9422(02)00573-3

Phytochemistry 62 (2003) 139–146
www.elsevier.com/locate/phytochem
Initial steps of the peroxidase-catalyzed polymerization
of coniferyl alcohol and/or sinapyl aldehyde:
capillary zone electrophoresis study of pH effect
David Fournanda, Bernard Cathalab, Catherine Lapierrea,*
aLaboratoire de Chimie Biologique, INRA/INA-PG, Institut National Agronomique, F-78850 Thiverval-Grignon, France
b
´
INRA/URCA, Equipe Paroi et Biomateriaux Fibreux, CRA 2 Esp R. Garros, F-51686 Reims Cedex, France
´
Received 26 July 2002; received in revised form 23 October 2002
Abstract
Capillary zone electrophoresis has been used to monitor the first steps of the dehydrogenative polymerization of coniferyl alco-
hol, sinapyl aldehyde, or a mixture of both, catalyzed by the horseradish peroxidase (HRP)–H2O2 system. When coniferyl alcohol
was the unique HRP substrate, three major dimers were observed (b-5, b-b, and b-O-4 interunit linkages) and their initial formation
velocity as well as their relative abundance varied with pH. The b-O-4 interunit linkage was thus slightly favored at lower pH
values. In contrast, sinapyl aldehyde turned out to be a very poor substrate for HRP except in basic conditions (pH 8). The major
dimer observed was the b,b0-di-sinapyl aldehyde, a red-brown exhibiting compound which might partly participate in the red col-
oration usually observed in cinnamyl alcohol dehydrogenase-deficient angiosperms. Finally, when a mixture of coniferyl alcohol
and sinapyl aldehyde was used, it looked as if sinapyl aldehyde became a very good substrate for HRP. Indeed, coniferyl alcohol
turned out to serve as a redox mediator (i.e. ‘‘shuttle oxidant’’) for the sinapyl aldehyde incorporation in the lignin-like polymer.
This means that in particular conditions the specificity of oxidative enzymes might not hinder the incorporation of poor substrates
into the growing lignin polymer.
# 2003 Elsevier Science Ltd. All rights reserved.
Keywords: Horseradish peroxidase; Coniferyl alcohol; Sinapyl aldehyde; Dehydrogenative polymerization; Dehydrodimers; Hydrogen peroxide;
Lignin; Lignification; Radical coupling reaction
1. Introduction
mutants and in genetically engineered variants of a
diverse range of plant species (Ralph et al., 2001a,b;
Pincon et al., 2001; Jouanin et al., 2000; Lapierre et al.,
2000; Sederoff et al., 1999; Piquemal et al., 1998;
Yahiaoui et al., 1998). These results highlight the need
for a better understanding of the factors controlling the
incorporation of phenolic compounds into lignins dur-
ing the enzyme-initiated oxidative coupling reactions.
The in vitro dehydrogenative polymerization of
monolignols has been widely used in plant phenolic
research as a model to study lignification. However, the
chemical structure and molecular weight of dehydro-
genation polymers (DHP) are different from those of
native lignin. Among the different intermolecular link-
ages observed in vivo for a guaiacyl-type lignin (Fig. 1),
the b-O-4 interunit linkage is predominant and accounts
for about half of the total (Adler, 1977). In contrast, in
typical dehydrogenative polymerization performed in
Lignin is conventionally defined as a complex hydro-
phobic network of phenylpropanoid units derived from
the oxidative polymerization of one or more of three
types of hydroxycinnamyl alcohol precursors, thus giving
rise to p-hydroxyphenyl, guaiacyl and syringyl subunits
in lignin (Boudet et al., 1995; Whetten et al., 1998). These
alcohols can react with oxidative enzymes (peroxidases
and laccases) and then radically couple at several sites
with each other or with the growing oligomer to pro-
duce a complex polymer with a variety of intermolecular
linkages (Adler, 1977). Unexpected variation in lignin
subunit composition has been recently evidenced in
* Corresponding author. Tel.: +33-130-815-470; fax: +33-130-
815-373.
E-mail address: lapierre@grignon.inra.fr (C. Lapierre).
0031-9422/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(02)00573-3

140
D. Fournand et al. / Phytochemistry 62 (2003) 139–146
Fig. 1. Main interunit linkages in a guaiacyl-type lignin. R=H or lignin.
vitro with coniferyl alcohol as the unique monomer, the
main interunit linkages observed are the b-5, b-b and
b-O-4 linkages (Fig. 1) (Sarkanen, 1971). Despite this
discrepancy between natural and synthetic lignins, the in
vitro dehydrogenative polymerization remains a unique
tool to study the oxidative coupling reactions. However,
because of the time-consuming analytical HPLC or gas
chromatography techniques, the chemical structure of
DHP are conventionally determined after a given reac-
tion time, without any kinetic consideration. To over-
come this analytical limitation, we recently developed a
capillary zone electrophoresis technique for the rapid
determination of coniferyl alcohol oxidation products
(Fournand and Lapierre, 2001).
enzyme-initiated radical coupling reactions and (ii) to
elucidate the incorporation mode of sinapyl aldehyde,
an unusual monolignol, into lignins.
2. Results and discussion
2.1. Dehydrogenative dimerization of coniferyl alcohol
and further oligomerization
When coniferyl alcohol was used as the unique
monolignol substrate for the HRP–H2O2 system, very
high apparent turnover numbers were observed
(Table 1). Coniferyl alcohol thus turned out to be
rapidly oxidized by HRP. The highest turnover number
was observed at pH 6. Whatever the pH, three major
dehydrodimers were observed (Fig. 2, 4, 5, 6). They
were representative of b-5, b-O-4, and b-b interunit lin-
kages. Coupling modes producing 4-O-5 or 5-5 bonds
In this study, we have investigated, with a kinetic
point of view, the initial steps of the dehydrogenative
polymerization of coniferyl alcohol and/or sinapyl
aldehyde as a function of pH. Objectives of this investi-
gation were two-fold: (i) to test the effect of pH on the
Table 1
Apparent turnover numbers (k) of HRP towards different starting compounds as a function of pH. Initial concentration in the reaction medium was
0.67 mM for each compound
Starting compound
ka (sꢀ1
)
pH 4.5
pH 6
pH 8
Coniferyl alcohol
Sinapyl aldehyde
2936
89
4307
123
3465
660
Coniferyl alcohol/sinapyl aldehyde mixture
b-5 Dehydrodimer 4
b-O-4 Dehydrodimer 5
b-b Dehydrodimer 6
2182/2112
Nd
Nd
3053/3863
510
132
1628/2526
nd
nd
Nd
1197
nd
a
The HRP molecular mass used for calculation is 44,000 Da. Each k value results from 2 or 3 experiments. nd, Not determined. k is defined as
the number of substrate molecules that can be oxidized by one enzyme molecule per second.

D. Fournand et al. / Phytochemistry 62 (2003) 139–146
141
Fig. 2. Chemical structure of coniferyl alcohol, sinapyl aldehyde and their main oxidation products. Coniferyl alcohol 1, sinapyl aldehyde 2,
syringaldehyde 3, b-5 dehydrodimer 4, b-O-4 dehydrodimer 5, b-b dehydrodimer 6, b-b0-di-sinapyl aldehyde 7.
were insignificant, if any. This was in accordance with
previous results (Sarkanen, 1971; Syrjanen and Brunow,
1998) showing that coupling modes producing a cova-
lent bond at the b-carbon of at least one of the partici-
pating radicals were the dominant ones, probably
because of a higher reactivity of b-radicals. Relative
amount of the three major compounds was also in
accordance with literature (Sarkanen, 1971). In all cases,
the b-5 dehydrodimer 4 accounted for about half of the
total, the b-O-4 dehydrodimer 5 and b-b dehydrodimer 6
being formed approximatively in equal amount. Some
slight differences were nevertheless observed as a func-
tion of pH. As shown in Fig. 3, the relative initial for-
mation rate of the b-O-4 dehydrodimer 5 was slightly
improved at pH 4.5 (28% instead of 25 and 23% at pH
6 and 8, respectively). As described by Sipila and Bru-
now (1991), the formation of the b-O-4 dehydrodimer
differs from the other two dimers in that it requires the
addition of water to an intermediate quinone methide.
This water addition proceeds more rapidly at acidic pH
(Sipila and Brunow, 1991). The unidentified products
observed in higher amounts at pH 6 and 8 might corre-
spond to quinone methides. These products could be
observed by capillary zone electrophoresis (CZE) in
large amount during the first five minutes of an oxi-
dative dimerization performed in a concentrated med-
ium containing acetone (data not shown), and their
abundance was correlated to a yellow coloration of the
reaction medium at the early stage of the oxidation.
Quinone methides are known to give such a coloration
in solution (Brunow et al., 1989). Moreover, Brunow
and co-workers also described a higher stability of qui-
none methides in alcaline conditions.
The decrease of dehydrodimer concentration as a
function of time (Fig. 3, A1–A3) suggests that they are
involved in oligomerization reactions. It was never-
theless possible to stop the reaction at a stage where
more than 80% of initial coniferyl alcohol appeared in a
dimerized form. As for coniferyl alcohol dimerization,
the lowest dimer oligomerization was observed at pH
4.5. According to Fig. 3 (A1–A3), it seems that b-b and
b-5 dehydrodimers undergo oligomerization more
rapidly than b-O-4 dehydrodimer. Considering the
maximum amount of each compound at the beginning of
oligomerization, these results suggest that b-b dehydro-
dimer 6 is more reactive towards the HRP–H2O2 sys-
tem. To confirm these results, different oxidation
reactions were performed using standard dehydrodimers
4, 5 and 6 as substrate for the HRP–H2O2 system. At
pH 6, the initial turnover number of b-b dehydrodimer

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D. Fournand et al. / Phytochemistry 62 (2003) 139–146
Fig. 3. Formation of dehydrodimers with the HRP–H2O2 system as a function of pH. Substrate: (A), coniferyl alcohol; (B), sinapyl aldehyde; (C),
equimolar mixture of coniferyl alcohol and sinapyl aldehyde. (1), pH 4.5; (2), pH 6; (3), pH 8. (*), Coniferyl alcohol; (*), sinapyl aldehyde; (&),
b-5 dehydrodimer 4; (^), b-O-4 dehydrodimer 5; (&), b-b dehydrodimer 6; (ꢁ), unidentified products; (- - -), sum of oxidation products; (^),
b-b0-di-sinapyl aldehyde 7; (+), syringaldehyde 3; (~), sum of b-5, b-O-4, and b-b dehydrodimers (4, 5, 6); (~), sum of syringaldehyde and b-b0-di-
sinapyl aldehyde (3, 7).
6 was effectively two-fold and ten-fold higher than that
of b-5 dehydrodimer 4 and b-O-4 dehydrodimer 5,
respectively (Table 1). This was probably due to the two
phenolic sites present in compound 6, which increased
the possibilities of phenoxy radical formation. The
turnover number of b-O-4 dehydrodimer 5 was surpris-
ingly low and did not correspond to the relative
decrease of dimer concentration during oligomerization
(Fig. 3, A2). In spite of its lower reactivity towards the
HRP-driven oxidation, the b-O-4 dehydrodimer 5 might
be oxidized by phenoxy radicals from other dehydro-
dimers, thus acting as ‘‘shuttle oxidants’’.
turnover numbers were about 30–40-fold lower than
those obtained with coniferyl alcohol, except in slightly
alcaline conditions (Fig. 3, B1–B3, and Table 1). At pH
8, the apparent turnover number surprisingly increased
and was only 6-fold lower than that obtained with con-
iferyl alcohol. Sinapyl aldehyde thus turned out to be a
poor HRP substrate in acidic and neutral conditions.
These results highlighted a strict relationship between
the HRP substrate specificity and the pH of the reaction
medium. Candeias and co-workers recently showed that
substrate specificity of peroxidases combined two
aspects. The first one is a Michaelis-Menten kinetic
control, which is related to dissociation constants of the
enzyme-substrate complexes. The second one is a
thermodynamic driving force, which corresponds to the
reorganization energies of electron-transfer within those
complexes and which is related to the difference of
2.2. Dehydrogenative dimerization of sinapyl aldehyde
When sinapyl aldehyde was used as the unique mono-
mer substrate for the HRP–H2O2 system, the apparent

D. Fournand et al. / Phytochemistry 62 (2003) 139–146
143
oxidation potentials between the peroxidase and the
substrates (Candeias et al., 1997). Both the dissociation
constants of the enzyme-substrate complexes and the
thermodynamic driving force are dependent on pH and
could explain the higher turnover number observed at
pH 8 with sinapyl aldehyde as substrate.
Russell et al. (2000) recently showed that the coupling
rate of sinapyl aldehyde was lower than that of coniferyl
aldehyde (monomethoxylated aldehyde) in spite of a
higher oxidation rate (phenoxy radical formation).
These authors proposed that sinapyl aldehyde formed a
more stable radical responsible for the decreased rate of
coupling in the presence of the HRP–H2O2 system
(Russell et al., 2000). In addition, we could also suggest
that pH had an influence on the stability of the sinapyl
aldehyde radical which could undergo dimerization
more rapidly at high pH values.
After radical coupling, the b,b0-di-sinapyl aldehyde
(Fig. 2, 7) was the main dehydrodimer observed at pH 6
and pH 8 (about 80% of total oxidation products;
Fig. 3, B2–B3). This compound exhibited a brown-red
coloration. Such interunit linkages might thus partly
participate in the red coloration usually observed in
cinnamyl alcohol dehydrogenase (CAD) deficient
angiosperms (Halpin et al., 1998; Yahiaoui et al., 1998;
Baucher et al., 1999). In contrast, this dehydrodimer
represented only about 20% of total oxidation products
at pH 4.5, the main dehydrodimer being a still uni-
dentified product (Fig. 3, B1).
HRP–H2O2 system, a wide range of oxidation com-
pounds was formed. The apparent turnover numbers
determined for each of the starting phenolic substrates
revealed unexpected results (Table 1). Coniferyl alcohol
still remained a very good substrate for HRP since
the apparent turnover numbers were higher than 1500
sꢀ1 whatever the pH. On the other hand, whereas
sinapyl aldehyde was previously shown to be a poor
substrate for HRP when used as the unique starting
material, it appeared as a very good substrate (apparent
turnover numbers higher than 2000 sꢀ1) when added in
combination with coniferyl alcohol (Fig. 3, C1–C3). In
other words, coniferyl alcohol promoted the oxidation
of sinapyl aldehyde. Two hypotheses could be proposed
to account for this observation: (i), coniferyl alcohol
may act as a redox mediator (i.e., ‘‘shuttle’’ oxidant) for
the sinapyl aldehyde oxidation (Fig. 4A), as previously
described for the isoeugenyl acetate or 4-methoxy-
mandelic acid oxidation by lignin peroxidase in the
presence of veratryl alcohol (ten Have et al., 1999; Tien
and Ma, 1997); (ii), coniferyl alcohol could efficiently
reduce compound II and thereby promote the sinapyl
aldehyde oxidation by closing the catalytic cycle
(Fig. 4B), as previously described for the p-anisyl alco-
hol oxidation by lignin peroxidase in the presence of
veratryl alcohol (Koduri and Tien, 1994).
If coniferyl alcohol only served to close the cataly-
tic cycle, then at the most 1 mol sinapaldehyde
would be expected to be oxidized per mol of con-
iferyl alcohol consumed. As shown in Fig. 3 (C2–C3)
and Table 1, we observed that sinapyl aldehyde was
oxidized more rapidly than coniferyl alcohol at pH 6
and 8, which supports the first redox mediation
hypothesis. The apparent turnover numbers reported in
Table 1 thus have to be considered as total oxidation
rates and not as actual turnover numbers. This oxida-
tion rate was the highest at pH 6, as it was observed
when coniferyl alcohol was the unique substrate, but
more than 60% of this activity still remained at pH 4.5
and 8.
Syringaldehyde observed at pH 6 and 8 probably
resulted from the heterolytic cleavage of the Ca–Cb
bond in sinapaldehyde, as previously reported for the
veratryl alcohol-mediated oxidation of isoeugenyl ace-
tate by lignin peroxidase (ten Have et al., 1999).
2.3. Dehydrogenative dimerization of a coniferyl alcohol/
sinapyl aldehyde mixture
When an equimolar mixture of coniferyl alcohol and
sinapyl aldehyde was submitted to oxidation by the
Fig. 4. Proposed mechanism for the coniferyl alcohol-mediated oxidation of sinapyl aldehyde (A) and for the catalytic cycle completion allowed by
coniferyl alcohol (B). CA, coniferyl alcohol; Sa, sinapyl aldehyde.

144
D. Fournand et al. / Phytochemistry 62 (2003) 139–146
could be oxidized at a rate higher than a more reactive
one (i.e., coniferyl alcohol) provided that the oxidation
potentials of the two species enable the redox mediation
phenomenon.
3. Experimental
3.1. Chemicals
Sinapyl aldehyde and syringaldehyde were purchased
from Aldrich Chemical Co (Milwaukee, WI). Coniferyl
alcohol was synthesized according to the method of
Ludley and Ralph (1996). Horseradish peroxidase type
II (200 purpurogallin units per mg solid) was purchased
from Sigma Chemical Co (St. Louis, MO). The internal
standard 3,4,5-trimethoxybenzoic acid was obtained
from Fluka (Buchs, Switzerland) and hydrogen peroxide
(35 wt.% solution) from Acros (New Jersey). Reaction
buffers were prepared from sodium dihydrogen phos-
phate (NaH2PO4, 2H2O) and di-sodium hydrogen
phosphate (Na2HPO4, 2H2O) (analytical reagents; Pro-
labo, France).
Fig. 5. Electropherogram of a standard mixture. 1, Coniferyl alcohol;
2, sinapyl aldehyde; 3, syringaldehyde; 4, b-5 dehydrodimer; 5, b-O-4
dehydrodimer; 6, b-b dehydrodimer; 7, b,b0-di-sinapyl aldehyde; IS,
internal standard; EOF, electroosmotic flow. See Fig. 2 for compound
structures.
Many other oxidation products were observed in
addition to the different homodimers previously formed
from each of the phenolic compounds as unique sub-
strate. For figure clarity (Fig. 3, C1–C3), the previously
identified products were grouped in two classes. Class I
was composed of b-5, b-b, and b-O-4 dehydrodimers
resulting from coniferyl alcohol oxidation and class II
was composed of b,b0-di-sinapyl aldehyde and syring-
aldehyde. Most of the other non identified oxidation
products probably resulted from cross-coupling reac-
tions. As shown in Fig. 3 (C1–C3), the ratio between
class I and class II compounds was strictly dependent
on pH. At pH 4.5, the class II compounds could not be
detected whereas they were prominent at pH 8, which
confirmed the highest reactivity of sinapyl aldehyde in
alcaline conditions.
These results highlighted the importance of pH on
the radical coupling modes of a mixture of mono-
lignols. Determination of the actual pH value within
plant cell walls is very difficult, but the notion does
exist in so far as enzymes need water to be active and
monolignols have to diffuse from cytosol to wall. This
pH value is probably not constant since cell wall com-
position (pectin, protein and lignin content) varies in
time and space. Thus, pH might be a physico-chemical
parameter which participates in the spatio-temporal
variability of lignins, in addition with the different
expression levels of genes encoding enzymes involved in
the lignification pathway.
3.2. Preparation of coniferyl alcohol homodehydrodimer
models
Coniferyl alcohol homodehydrodimers were purified
by preparative chromatography (from 1 g of coniferyl
alcohol), using Et2O/CH2Cl2/MeOH (20/5/1, v/v/v) as
an eluent, from a HRP-catalyzed oxidative dimerization
of coniferyl alcohol. The reaction was performed at pH
4.5 without internal standard in a 77-fold concentrated
medium [containing 18.5% (v/v) acetone] compared to
the standard oxidation reaction. 13C NMR spectra were
recorded on a Bruker AC250 spectrometer.
b-5 Dehydrodimer (Fig. 2, 4) (yield 43%): 13C NMR
(62.9 MHz, acetone-d6), ꢀ 54.68 (Ab), 56.19 (AOCH3),
56.29 (BOCH3), 63.37 (Bg), 64.53 (Ag), 88.49 (Aa),
110.39 (A2), 111.53 (B2), 115.65 (A5), 116.02 (B6),
119.53 (A6), 128.24 (Bb), 130.32 (B5), 130.53 (Ba),
131.85 (B1), 134.27 (A1), 145.10 (B3), 147.22 (A4),
148.33 (A3), 148.86 (B4).
Racemic b-O-4 dehydrodimer (Fig. 2, 5) (yield 7%):
13C NMR (62.9 MHz, acetone-d6), ꢀ 56.18 (AOCH3),
56.24 (BOCH3), 61.74/61.81 (Ag, erythro/threo), 63.24
(Bg), 73.70/73.74 (Aa, erythro/threo), 86.47/88.18 (Ab,
erythro/threo), 110.72/110.86 (B2, threo/erythro), 111.33
(A2), 115.13/115.21 (A5, erythro/threo), 119.05/119.34
(B5, erythro/threo), 120.21/120.30 (B6, erythro/threo),
120.40/120.48 (A6, erythro/threo), 129.48/129.52 (Bb,
erythro/threo), 129.87 (Ba), 132.71/132.82 (B1, erythro/
threo), 133.73/134.16 (A1, threo/erythro), 146.59/146.77
(A4, erythro/threo), 147.93/147.99 (A3, erythro/threo),
148.46/149.05 (B4, erythro/threo), 151.56/151.74 (B3,
threo/erythro).
Moreover, the incorporation rate of monolignols into
the growing polymer is often related to the reactivity
of these monolignols towards oxidative enzymes (per-
oxidases and laccases). This study revealed that a
poorly reactive phenolic species (i.e., sinapyl aldehyde)

D. Fournand et al. / Phytochemistry 62 (2003) 139–146
145
b-b Dehydrodimer (Fig. 2, 6) (yield 9%): 13C NMR
(62.9 MHz, acetone-d6), ꢀ 55.15 (Cb), 56.17 (OCH3),
72.14 (Cg), 86.57 (Ca), 110.50 (C2), 115.50 (C5), 119.56
(C6), 134.06 (C1), 146.77 (C4), 148.26 (C3).
4, 5 (threo form), 1, 6, 2, IS, 3, and 7, as shown in
Fig. 5.
Acknowledgements
3.3. Preparation of sinapyl aldehyde homodehydrodimer
models
We are grateful to the Institut National de la
Recherche Agronomique (INRA, France) for the spe-
cial ANS grant received for this study.
b,b0-Di-sinapyl aldehyde was purified by TLC (from
0.58 g of sinapyl aldehyde) from a HRP-catalyzed oxi-
dative dimerization of sinapyl adehyde. The reaction
was performed at pH 8 without internal standard in a
77-fold concentrated medium (containing 18.5% (v/v)
acetone) compared to the standard oxidation reaction.
b,b0- Di -sinapyl aldehyde (Fig. 2, 7) (yield 22%): 13C
NMR (62.9 MHz, acetone-d6), ꢀ 56.63 (OCH3), 109.19
(C2,C6), 125.95 (Cb), 134.15 (C1), 140.56 (Ca), 149.09
(C3,C5), 155.20 (C4), 194.52 (Cg).
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