Regioselectivity in lignin biosynthesis. The influence of dimerization and cross-coupling

Article abstract of DOI:10.1039/a907919j

We have studied the regioselectivity of oxidative phenol coupling in lignin formation using an oxidation system that distinguishes between dimerization reactions and cross-coupling reactions. We found that the regioselectivity of coupling was different in the two reactions. For instance, in coniferyl alcohol dimerization the formation of β-5 coupling product has a slight prevalence over the formation of β-O-4 product; in cross-coupling the β-0-4 mode is favoured in a ratio of ≈10:1. This ratio is higher than that found in isolated softwood lignins. The degree of cross-coupling was influenced only to a small extent by changes in the rates of conventional addition of coniferyl alcohol (Zulauf versus Zutropf conditions). We found that diffusion through a dialysis membrane did effectively suppress the dimerization of coniferyl alcohol. Of the different oxidants investigated, manganese triacetate in acetic acid yielded the highest proportion of cross-coupling product. The Royal Society of Chemistry 2000.

Full text of DOI:10.1039/a907919j

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Regioselectivity in lignin biosynthesis. The influence of
dimerization and cross-coupling
Kaisa Syrjänen and Gösta Brunow*
1
Department of Chemistry, Laboratory of Organic Chemistry, PO Box 55, FIN-00014
University of Helsinki, Finland
Received (in Cambridge, UK) 1st October 1999, Accepted 18th November 1999
We have studied the regioselectivity of oxidative phenol coupling in lignin formation using an oxidation system
that distinguishes between dimerization reactions and cross-coupling reactions. We found that the regioselectivity
of coupling was different in the two reactions. For instance, in coniferyl alcohol dimerization the formation of
β–5 coupling product has a slight prevalence over the formation of β–O-4 product; in cross-coupling the β–O-4
mode is favoured in a ratio of ≈10:1. This ratio is higher than that found in isolated softwood lignins. The degree
of cross-coupling was influenced only to a small extent by changes in the rates of conventional addition of coniferyl
alcohol (Zulauf versus Zutropf conditions). We found that diffusion through a dialysis membrane did effectively
suppress the dimerization of coniferyl alcohol. Of the different oxidants investigated, manganese triacetate in acetic
acid yielded the highest proportion of cross-coupling product.
and guaiacyl glycerol-β-guaiacyl ether 2 did not yield any cross-
Introduction
coupling products (Tables 1 and 2, Exp. 1). The coniferyl alco-
hol, with its lower oxidation potential, reacts by dimerization.
(Similarly, sinapyl alcohol dimerizes and does not couple with a
syringyl dimer.7) The abundance of arylglycerol-β-aryl ether
structures in natural guaiacyl lignins shows that cross-coupling
between coniferyl alcohol and guaiacyl groups in the polymer
does occur in spite of the oxidation-potential difference.
We have carried out oxidation experiments with coniferyl
alcohol 1 to find conditions under which cross-coupling
between phenols of unequal redox potentials can be achieved.
Different modes of addition of reagent, such as fast (Zulauf)
and slow (Zutropf), and different oxidants were tested. The
two competing reactions, cross-coupling and dimerization, are
shown in Scheme 1. To simplify the analysis of reaction prod-
ucts we replaced the β-ether model compound, which was used
in our previous experiments, with apocynol 3. We found that
the regioselectivity of coupling can be controlled by controlling
the rate of addition of monolignol.
The final phases of the formation of lignin in the cell walls of
vascular plants involve oxidative coupling of p-hydroxy-
cinnamyl alcohol precursors (such as coniferyl alcohol) to
phenolic structures on the growing polymer chain. The coup-
ling is visualized as a more-or-less random process without
enzymic control of the regioselectivity of the process.1,2 The
validity of this concept has been tested many times by oxidation
of precursors with enzymes and with inorganic oxidants. The
dehydrogenation polymers (DHPs) have been found to contain
most of the structural units found in natural lignins; complete
identity between synthetic and natural lignins has, however, not
been achieved. The problems with reproducing lignin bio-
synthesis in vitro has recently led some investigators to chal-
lenge the whole concept of random polymerization.3 We feel
that the experimental evidence known so far still is compatible
with the original idea proposed by Freudenberg and Adler:1,4
that the polymerization itself is a purely chemical process and
that the enzymic control extends only to the rates of feeding of
the monomers into the reaction zone and the generation of the
phenoxyl radicals. This argument is supported by the results of
a recent study where it was established that lignins are not
optically active.5 A concept that has become increasingly
important in this process is that of cross-coupling, where the
monomer lignol radical, instead of dimerizing, reacts with
phenolic structures on the growing chain. When lignin form-
ation is reproduced in vitro, the main difficulty is to achieve
the necessary degree of cross-coupling whilst suppressing the
dimerization. In an effort to find practicable means of promot-
ing cross-coupling at the expense of dimerization, we have
studied model systems with p-hydroxycinnamyl precursors
(monolignols) and phenols representing structures of the poly-
mer chain. We found recently that bond formation in oxidative
phenol coupling is governed by a combination of factors such
as oxidation potential and radical reactivity.6,7 Phenols which
have similar oxidation potentials, such as coniferyl alcohol and
a syringyl β–O-4 dimer, yielded cross-coupling products in
good yield. It is more difficult to achieve cross-couplings that
are not favoured by similarity of oxidation potentials. In our
experiments with hydrogen peroxide and horseradish per-
oxidase, oxidation of equimolar mixtures of coniferyl alcohol 1
Results
In the oxidation experiments, we kept the pH lower than is
customary in work on lignin synthesis. We have shown that
lower pH favours the formation of dimers and suppresses
polymerization.8 Low pH also seems to yield a higher content
of β–O-4 structures and a low content of β–β coupling prod-
ucts.9 In each experiment we used equimolar amounts of
apocynol 3 and coniferyl alcohol 1. The products (1, 3, 5–7 and
9) were identified with the aid of authentic compounds. The
structure of compound 4 was determined by MS and NMR.
The dimeric β–O-4 compound 8 was identified by NMR using
published data.10 In the following discussion the coupling
products are classified as dimers when they are formed from two
identical phenols and cross-products when they are products of
coupling of two different phenols.
The product mixtures were analysed by HPLC and flash
chromatography. For quantitative analysis the products were
separated into three fractions A, B and C as shown in Fig. 1, I.
The fractions were weighed and the proportions of the indi-
vidual compounds were calculated from the integrals of signals
in the 1H NMR spectra. In the first fraction, A, there was only
J. Chem. Soc., Perkin Trans. 1, 2000, 183–187
This journal is © The Royal Society of Chemistry 2000
183

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Table 1 Reaction conditions for experiments 1–10. For details see Experimental section
Starting
materials
Exp.
Oxidant
Solvent
Zulauf
Zutropf
1
2
3
4
5
6
7
8
9
1, 2
1
H2O2–HRP
Acetone, 20% buffer (pH 3.5)
x
x
x
x
3
1, 3
1, 3
1, 3
1
1, 3
1, 3
1, 3
5.5 h
21 h
Mn(OAc)3
Glacial acetic acid
x
x
x
x
MnO2
FeCl3
Acetone 20%, buffer (pH 3.5)
Distilled water
10
Table 2 Yields of products in experiments 1–10
Monomer
Dimers (%)
recovery (%)
4 cross-
β–5
5 dimeric
β–5
7 cross-
β–O-4
8 dimeric
β–O-4
Total
yield
Exp.
1
2/3
6 5–5
9 β–β
Oligomers (%)
1
2
3
4
5
6
7
8
9
44
18
24
16
16
8
12
42
52
34
45
40.5
39
74
56
33
42
14
12
3a
8
14.5
12
15
14
33
10
36
50
45
31
31
34
3
3
15
10
9
22
9
15
10
5
10
9
14
8
11
50
26
5
8
8
6
2
1
6
9
4
3
11
1.5
1
2
23
28
44
1
1
2
18
5
5
10
4
18
a A tetrameric dioxepine (cf. ref. 11) was also formed (13%).
unchanged starting material, compounds 1 and 3, which are
easily identified from the spectra. HαЈ of apocynol gives a signal
at δ 5.90 (q) and HαЈ, HβЈ and HγЈ of coniferyl alcohol are at δ
4.75 (d), 6.27 (dt) and 6.64 (d), respectively. The second frac-
tion, B, consists of cross-β–5 (4), dimeric β–5 (5) and the 5–5
dimer from apocynol (6). HαЈ of compound 5 is at δ 6.64 (d),
but HαЈ of 4 and HαЈ of 6 overlap at δ 5.88 (m). Peak heights of
compounds 4 and 6 in the HPLC chromatogram I are roughly
1:2 and the yields are in this case estimated from the HPLC
curve taking into account the estimated absorbances at 280 nm
according to Pew and Connors.11 The third fraction, C, consists
of cross-coupled β–O-4 (7), dimeric β–O-4 (8) and β–β from
coniferyl alcohol (9). HαЈ of compound 8 is at δ 6.62 (d), HαЈ of
compound 7 is at δ 5.85 (m) and HβЈ of pinoresinol (9) is at
δ 3.14 (br s). The results of the quantitative estimations are
collected in Table 2.
have reported a β–O-4 cross-coupling product as a main prod-
uct in the oxidation of coniferyl alcohol with a guaiacyl model
compound, but no details were reported. In order to increase
the amount of cross-coupling by slow addition of oxidant and
coniferyl alcohol (Zutropf) the addition was extended over 5.5 h
and, further, to 21 h. This almost doubled the amount of cross-
coupling product and, furthermore, a small amount of cross-
coupled β–5 product 4 was identified for the first time. The
amount of 4 was only one-tenth that of 7, which means that
the regioselectivity of cross-coupling strongly favours β–O-4
structures over β–5 structures. A still slower addition of con-
iferyl alcohol was achieved by letting the coniferyl alcohol dif-
fuse through a dialysis membrane as suggested by Tanahashi
and Higuchi.14 In this case the coniferyl alcohol did not form a
dimer on oxidation; instead, it coupled with apocynol to give a
cross-coupled β–O-4 product 7 (Fig. 1, III). This again demon-
strates the strong tendency of monolignols to form β–O-4
products in cross-coupling.
Oxidation with H2O2–HRP
Coniferyl alcohol was first oxidized alone with 0.5 equivalents
of oxidant (Exp. 2). The β–5 (5), β–O-4 (8) (mixture of erythro
and threo isomers) and β–β (9) dimers were formed in 24, 16
and 12% yield, respectively, which is well in accord with earlier
results.1,2 Some coniferyl alcohol remained (36%) and 12% was
oxidized further to oligomeric products. When the oxidation
was repeated with addition of an equimolar amount of β–O-4
model compound 2 (Exp. 1), the result was very similar. Apart
from coupling products 5, 8 and 9, only dimeric products from
coniferyl alcohol were recovered together with unchanged 2.
Cross-coupling was then attempted with equimolar amounts
of coniferyl alcohol and apocynol. Again the main products
were the coniferyl alcohol dimers 5, 8 and 9 together with some
dimer 6 from apocynol. [Oxidation of apocynol 3 alone gave
the biphenyl 6 (34%) together with some of the corresponding
dioxepine.11,12] A small amount of cross-coupling product
(≈5%) was isolated and identified as the β–O-4 structure 7 by
comparison with an authentic sample. Erickson and Miksche13
Manganese triacetate oxidations
Manganese triacetate in glacial acetic acid has been used to
produce oligomeric lignols from coniferyl alcohol. In our
experiment, Mn(OAc)3 was added to an equimolar mixture of 1
and 3 all at once (Exp. 8). In the dimer fraction, 18% was β–O-4
cross-coupling product 7. The erythro:threo ratio was 65:35.
Coniferyl alcohol was also oxidized alone (Exp. 7) with
Mn(OAc)3 and the proportions of β–5, β–O-4 and β–β dimers
(22:50:2) are similar to those values observed by Landucci and
Ralph.15 The manganese triacetate–acetic acid system clearly
favours the formation of β–O-4 structures. The results of Exp. 8
are presented in Fig. 1, II.
MnO2 and FeCl3
The manganese dioxide and the ferric chloride oxidations (Exp.
9 and 10) gave similar results to the hydrogen peroxide–HRP
oxidations.
184
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Discussion
It has long been presumed that, in the preparation of synthetic
lignin, a slow rate of addition of monomer gives a more ‘lignin-
like’ product.16 Experimental results supporting this idea were
found by Lai and Sarkanen.17 They found that slow addition of
monomer produced a synthetic lignin with a larger proportion
of β–O-4 structures. This was interpreted as the result of
‘end-wise’ polymerization which constitutes cross-coupling of
the monomer with phenols on the polymer chain. This effect
has so far only been observed in polymerization experiments
under conditions that are notoriously difficult to reproduce in a
satisfactory manner. The present experiments reveal the actual
coupling step and make it possible to determine the regio-
selectivity of the cross-coupling reaction without interference
from other reactions as in the formation of oligomeric DHPs.
Using H2O2–HRP and fast addition of coniferyl alcohol (the
Zulauf method) we obtained 5% cross-coupling product. With
slow addition (Zutropf) the yield of cross-coupled β–O-4 dimer
rose to ≈10% and a small amount (≈1%) of cross-β–5 dimer was
detected. Diffusion through a dialysis membrane14 proved to
yield a slow enough rate of addition. In a very slow reaction,
coniferyl alcohol formed only cross-coupling product. This
demonstrates that it is possible to regulate the degree of cross-
coupling by controlling the rate of addition of monolignol.
The oxidation potential of the oxidant may also influence the
rate of cross-coupling. In experiments with Mn(OAc)3–acetic
acid, Landucci and Ralph15 have reported that oxidation of
coniferyl alcohol with Mn(OAc)3 gives mainly β–O-4 dimers.
In our experiments a large amount (50%) of β–O-4 dimer
from coniferyl alcohol was formed but also a remarkable
amount (18%) of cross-β–O-4 dimer, and only 1% of cross-β–5
dimer (Exp. 8). In this case both the oxidant and the solvent
polarity were different from those in the HRP oxidations.
Solvent polarity has been shown to influence the regioselectivity
of coupling.8,18
In conclusion, it is evident from these studies that the ratio of
cross-coupling to dimerization of monolignols to a large extent
determines the outcome of lignin biosynthesis. This in turn is
determined mainly by relative oxidation potentials of mono-
lignols and the rates of addition of the monolignols. The role of
the oxidant still remains to be determined.
Experimental
General
All mps were measured on an Electrothermal (digital melting
point) apparatus in open capillary tubes and are uncorrected.
The buffered aqueous solution was obtained using citric acid
(0.01 M)–phosphate (0.02 M) buffer (pH 3.5). Horseradish per-
oxidase (EC 1.11.1.7) was from Serva, activity 450 U mgϪ1. 30%
aq. hydrogen peroxide (Merck) was diluted to give a 3%
solution (≈0.8 mol dmϪ3) before use. Silica gel for column
chromatography was Merck Kieselgel 60 (230–400 mesh). TLC
was performed on silica gel plates (Merck Kieselgel 60 F254).
Spots were made visible with UV light. In the dialysis experi-
ment regenerated cellulose tubular membrane (Cellu Sep T2,
nominal MWCO 6000–8000) was used. 1H NMR and 13C
NMR spectra were recorded at 200 MHz with a Varian Gemini
instrument. Deuteriochloroform was used as solvent. Mass
spectra were recorded on a JEOL JMS-SX102 instrument.
HPLC was performed using a Waters 600 pump, LiChrospher
Si 60 (5 µm) columns (0.4 × 25 cm and 1 × 25 cm) and Waters
996 UV spectrophotometric detector with detection at 280 nm.
Hexane–ethyl acetate (11:9) was used as eluent. The injection
volume was 20 or 500 mm3. Evaporations were conducted
under reduced pressure at a temperature less than 40 ЊC. Prod-
ucts were acetylated with dry acetic anhydride and pyridine
(1:1) overnight at room temperature.19 THF was purified by
distillation over sodium.
Scheme 1 Dimerization and cross-coupling products.
In all experiments there was an oligomeric fraction. This
material was similar to DHPs. From 13C NMR spectra it could
be seen that in HRP–H2O2 oxidations there were more β–O-4
than β–5 linkages in Zutropf experiments, while in Zulauf
experiments equal amounts of β–5 and β–O-4 linkages were
found. The oligomeric fraction in Mn(OAc)3 oxidations
consisted mainly of β–O-4 structures with some β–5 and β–β
structures present.
J. Chem. Soc., Perkin Trans. 1, 2000, 183–187
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Fig. 1 HPLC chromatograms: I, H2O2–HRP oxidation, Zutropf. II, Mn(OAc)3 oxidation, Zulauf. III, H2O2–HRP oxidation, addition through
dialysis membrane. (The erythro and threo forms of 7 and 8 are marked in chromatogram II.)
(3 H, s, OCH3), 3.90 (1 H, m, HβЈ), 4.28–4.56 (2 H, m, HγЈ), 5.57
Materials
(1 H, d, J 6.6 Hz, HαЈ), 5.87 (1 H, q, HαЈ), 6.86–7.06 (5 H, ArH);
m/z 472 (Mϩ, 1%), 412 (45), 352 (15), 310 (100), 295 (27), 278
(14), 195 (7) and 175 (8) (Found: Mϩ, 472.1726. C25H28O9
requires M, 472.1733).
Compounds 5 and 9 were isolated from the oxidation mixture
of coniferyl alcohol (Exp. 2) and identified by comparison of
retention time and NMR spectra with those of authentic
compounds.23,24 Compound 6 was prepared from acetovan-
illone according to published procedures.25,26 The β–O-4
cross-coupling product 7 was synthesized for HPLC identifi-
cation. The synthesis is based on the procedure developed by
Nakatsubo and co-workers, substituting carbonyl-protected
acetovanillone for guaiacol.27–31 Mp 62–65 ЊC; m/z 364 (Mϩ,
Coniferyl alcohol 1 was prepared from vanillin (commercial
grade, Fluka). A Knoevenagel reaction with vanillin and
malonic acid20 followed by esterification with ethanol–sulfuric
acid gave ethyl ferulate. Reduction of ethyl ferulate to coniferyl
alcohol was done with DIBAL-H as described by Quideau and
Ralph.21 Apocynol 3 was prepared from acetovanillone (com-
mercial grade, Aldrich) as described by Bailey and Dence.22
Compound 4 has not been reported previously. A sample was
isolated from an oxidation experiment (Exp. 5) and identified
by 1H NMR, MS and HRMS. Spectral data for the triacetate of
compound 4: δH 1.57 (3 H, CH3), 2.09 (3 H, COCH3), 2.12 (3 H,
s, COCH3), 2.35 (3 H, s, COCH3), 3.86 (3 H, s, OCH3), 3.96
186
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5%), 346 (19), 328 (8), 316 (12), 298 (24), 269 (7), 239 (34), 194
(88), 176 (100), 150 (95); HRMS (Found: Mϩ, 364.1510.
C19H24O7 requires M, 364.1522). Compound 7 has been
synthesized earlier by a different route and the NMR spectrum
has been published by Landucci and Ralph.32
mmol) in 5 cm3 of acetone, were added in ten portions to the
solution over 30 min. The mixture was then stirred for an
additional 30 min and extracted with ethyl acetate.
Dialysis experiment
In experiment 3 some tetrameric dioxepine from apocynol
(cf. ref. 11) was isolated. Spectral data for the dioxepine tetra-
acetate: δH 1.56 and 1.65 (together 9 H, 2 d, J 6.6 Hz, 3 × CH3),
2.10, 2.15, 2.16 and 2.21 (each 3 H, s, COCH3), 3.73, 3.88, 3.92
and 3.94 (each 3 H, 4 s, 4 × OCH3), 5.85–6.25 (4 H, m, HαЈ and
quinone H), 6.81–7.17 (7 H, ArH and quinone H); δC 21.2 (Ar-
COCH3), 22.1 and 23.0 (COCH3 and CH3), 56.1 (OCH3), 56.7
(OCH3), 109.9–153.5 (arom.), 168.8 (COCH3), 170.8 (COCH3)
Apocynol (0.17 g, 1.0 mmol) dissolved in acetone (1 cm3), and
HRP (20 mg) in buffer (10 cm3), were put into the dialysis tube.
Coniferyl alcohol (0.18 g, 1.0 mmol) as a solution in acetone
(1 ml), and H2O2 (0.5 mmol), were added to the buffer solution
(250 ml) and poured into the conical flask. The tube was then
placed into the solution and stirred with a magnetic stirrer for
17 h. The contents of the tube and the outer buffer solution
were extracted separately with ethyl acetate, and the organic
layers were dried (Na2SO4) and evaporated.
and 178.9 (C᎐O); m/z 788 (Mϩ, 3%), 728 (1), 668 (9), 608 (12),
᎐
568 (9), 548 (15), 504 (12), 488 (32), 462 (17), 446 (46), 341 (46),
298 (68), 281 (100) (Found: Mϩ, 788.2667. C42H44O15 requires
M, 788.2680).
References
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Lignin, Springer-Verlag, Heidelberg, 1968, vol. 2.
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All oxidation experiments were done under an argon atmos-
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hexane 9:11).
6 G. Brunow, I. Kilpeläinen, J. Sipilä, K. Syrjänen, P. Karhunen,
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Oxidations with H2O2–HRP
Zulauf method—general procedure.—Apocynol (0.28 g, 1.66
mmol) and coniferyl alcohol (0.30 g, 1.66 mmol) were dis-
solved in acetone (25 cm3) and horseradish peroxidase (10 mg;
450 U mgϪ1) in buffer solution (100 cm3) was then added. H2O2
(0.83 mmol), diluted to 5 cm3 with buffer, was added to the
solution over 30 min. The mixture was stirred for an addi-
tional 30 min and then extracted with ethyl acetate.
9 N. Terashima and R. H. Atalla, in 8th ISWPC, ed. anon., Gummerus
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16 K. Freudenberg, Angew. Chem., 1956, 68, 508.
Zutropf method.—Apocynol (0.28 g, 1.66 mmol) and horse-
radish peroxidase (10 mg; 450 U mgϪ1) were dissolved in acet-
one (1 cm3)–buffer (4 cm3). H2O2 (0.83 mmol) diluted to 15 cm3
with buffer, and coniferyl alcohol (0.30 g, 1.66 mmol), dissolved
in acetone (6 cm3)–buffer (9 cm3), were added gradually to the
solution via syringe pump over 5.5 h (Exp. 5). The mixture
was then stirred for an additional 30 min and extracted with
ethyl acetate. In Exp. 6, addition was done over 21 h with
peristaltic pumps following the procedure described by Kirk
and Brunow.33
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Apocynol (0.28 g, 1.66 mmol) and coniferyl alcohol (0.30 g,
1.66 mmol) were dissolved in glacial acetic acid (30 cm3), and
solid Mn(OAc)3 (0.67 g, 2.5 mmol) was added to the solution.
The mixture was stirred for 30 min, poured into water, and
extracted with ethyl acetate. The ethyl acetate layer was also
washed several times with freshly prepared 10% aq. NaHCO3.
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Oxidation with MnO2
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Apocynol (0.17 g, 1.0 mmol) and coniferyl alcohol (0.18 g, 1.0
mmol) were dissolved in acetone (15 cm3)–buffer (60 cm3) and
the mixture was added to dry MnO2 34 (0.43 g, 5.0 mmol). The
mixture was stirred for 15 min, then was filtered through a bed
of Celite and extracted with ethyl acetate.
31 R. Sterzycki, Synthesis, 1979, 724.
32 L. L. Landucci and S. Ralph, J. Wood Chem. Technol., 1997, 17, 361.
33 T. K. Kirk and G. Brunow, Methods Enzymol., 1988, 161, 65.
34 G. Cahiez and M. Alami, Encyclopedia of Reagents for Organic
Synthesis, ed. L. A. Paquette, Wiley, Chichester, 1995, vol. 5, p. 3230.
Oxidation with FeCl3
Apocynol (0.17 g, 1.0 mmol) was dissolved in acetone (10 cm3)–
distilled water (55 cm3). A solution of FeCl3 (0.16 g, 1.0 mmol)
in 5 cm3 of water, and a solution of coniferyl alcohol (0.18 g, 1.0
Paper a907919j
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