The early oxidative biodegradation steps of residual kraft lignin models with laccase

Article abstract of DOI:10.1016/S0968-0896(98)00173-4

A number of model compounds resembling the fundamental bonding patterns of residual kraft lignin, including a series of stilbenes, were incubated with laccase from Trametes versicolor in the presence and absence of delignification 'mediators' ABTS and HBT. The condensed kraft lignin model compounds seem to undergo initial degradation by laccase mainly via benzylic oxidation, demethylation and hydroxylation reactions. Phenolic 5-5', diphenylmethane and α-5 lignin models were found to be degraded mainly via side-chain oxidation reactions. Among the models studied, a phenolic stilbene was found to be the most reactive, yielding several products showing side-chain oxidation/transposition, demethoxylation and hydroxylation reactions. Non-phenolic 5-5', diphenylmethane and stilbene model compounds were found unreactive even in the presence of the laccase-mediator system. Copyright (C) 1997 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(98)00173-4

BIOORGANIC &
MEDICINAL
CHEMISTRY
Bioorganic & Medicinal Chemistry 6 (1998) 2161±2169
The Early Oxidative Biodegradation Steps of Residual Kraft
Lignin Models with Laccase
Claudia Crestinia and Dimitris S. Argyropoulosb*
aAgrobiology & Agrochemistry Department, Tuscia University, Via San Camillo De Lellis s.n.c., 01100 Viterbo, Italy
bPaprican and Department of Chemistry, Pulp and Paper Research Centre, McGill University, 3420 University Street, Montreal,
H3A 2A7 Canada
Received 9 March 1998; accepted 20 July 1998
AbstractÐA number of model compounds resembling the fundamental bonding patterns of residual kraft lignin,
including a series of stilbenes, were incubated with laccase from Trametes versicolor in the presence and absence of
deligni®cation `mediators' ABTS and HBT. The condensed kraft lignin model compounds seem to undergo initial
degradation by laccase mainly via benzylic oxidation, demethylation and hydroxylation reactions. Phenolic 5-50,
diphenylmethane and a-5 lignin models were found to be degraded mainly via side-chain oxidation reactions. Among
the models studied, a phenolic stilbene was found to be the most reactive, yielding several products showing side-chain
oxidation/transposition, demethoxylation and hydroxylation reactions. Non-phenolic 5-50, diphenylmethane and stil-
bene model compounds were found unreactive even in the presence of the laccase-mediator system. # 1998 Elsevier
Science Ltd. All rights reserved.
Introduction
order to elucidate the deligni®cation mechanism cata-
lyzed by laccase, several studies have been carried out
on both phenolic and non-phenolic monomeric, aryl-
glycerol b-O-4 and b-1 ether lignin model com-
pounds.3±5 The use of radical mediators has become of
interest since the early report that non-phenolic lignin
model compounds, unreactive towards laccase alone,
could be oxidized in the presence of laccase and ABTS,3
and that kraft pulp could be deligni®ed. Since then, 1-
hydroxybenzotriazole was found to mediate pulp
deligni®cation in the presence of laccase.6 It is also likely
that the relatively small radical mediators can act as
di€usible lignin oxidizing agents since they can access
inner lignin structures in the cell wall as opposed to the
relatively large enzyme.7
Residual lignin from kraft pulping processes is a three
dimensional polymer linked mainly by condensed (i.e.,
carbon±carbon) bonds and some ether linkages between
dihydrophenyl propionic, diphenylmethane and stilbene
units, most of which are not readily hydrolyzable. In
view of the renewed interest in alternative bleaching
processes, the use of enzymes as mild and environmen-
tally benign bleaching agents is being considered by the
pulp and paper industry.1 Among these laccase (benze-
nediol: oxygen oxidoreductase; E.C. 1.10.3.10), a multi-
copper oxidase which performs one-electron oxidations
of a variety of aromatic substrates, holds a pre-eminent
position. Laccase alone, however, has little e€ect on
pulps, but when used in the presence of mediators such
as 2,20 -azinobis-(3-ethyl-benzthiazoline-6-sulfonate)
(ABTS) or 1-hydroxybenzotriazole (HBT) laccase has
been shown to e€ectively demethylate kraft pulps.2 In
Kraft lignin contains signi®cant amounts of condensed
structures such as 5-50, a-5, diphenylmethane, and stil-
bene units.8±13 To date there are no literature studies
dealing with the behaviour of laccase toward such lignin
moieties, despite the fact that their presence is very sig-
ni®cant within the kraft lignin backbone. The aim of
this work is to elucidate the reactivity of laccase toward
condensed lignin model compounds in the presence or
Key words: laccase; oxidation; lignin models.
*Corresponding author. Tel: 39 761 357 234; fax: 39 761 357
242; e-mail: crestini@unitus.it
0968-0896/98/$ - see front matter # 1998 Elsevier Science Ltd. All rights reserved
PII: S0968-0896(98)00173-4

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C. Crestini, D. S. Argyropoulos/Bioorg. Med. Chem. 6 (1998) 2161±2169
absence of ABTS and HBT. A series of model com-
pounds resembling the fundamental bonding patterns
of residual kraft lignin were thus incubated with lac-
case from Trametes versicolor. In order to elucidate
the initial steps in the oxidation degradation products,
the reactions were conducted under conditions of lim-
ited oxygen supply and low enzyme activity. This
experimental design allowed for the early metabolites
of the reaction to be detected, characterized and
quanti®ed. Our e€orts were also supplemented with
experiments where oxygen was bubbled in the reactor
accompanied by extensive degradation of the examined
models.
The presence of radical mediators did not signi®cantly
a€ect the degree of conversion and product distribution
(Table 2, Entry 1). Compounds 2, 3, and 4 are all pro-
ducts of side-chain oxidation of the benzyl group to
hydroxymethyl, aldehyde and double oxidation to di-
aldehyde, respectively; the major component being, under
all experimental conditions examined, the benzyl alco-
hol derivative 2. Along with side-chain oxidation pro-
ducts, compound 5,
a metabolite produced upon
demethylation of one of the methyl-aryl ether moieties,
was also identi®ed. In the presence of higher enzyme
activity another metabolite with a high molecular
weight was detected, and tentatively assigned as being a
compound possibly emerging via a radical coupling
pathway.4,14,15 Table 2 (Entry 1) shows the quantitative
amounts of the metabolites obtained.
Results
Biodegradation of 5-50 condensed models dihydrocreosol
1 and dihydromethyl veratrole 6
When the fully methylated model 6 was incubated under
the same experimental conditions, no reaction occurred,
with the exception that traces of the dialdehyde 7 were
detected in the presence of ABTS when high enzyme
levels were used (Scheme 1). When the reaction was
performed in the presence of bubbled oxygen, the extent
of degradation was found to be 61.7% for model com-
pound 1 in the presence of laccase alone, while the non-
phenolic compound 6 was essentially unreacted even in
the presence of ABTS or HBT.
Compound 1 was incubated with laccase both in the
presence and in the absence of the radical mediators
ABTS and HBT to give compounds 2, 3, 4, and 5 in
variable yields (Scheme 1). When the enzyme load was
0.5 U/mL the extent of degradation ranged from 9.4 to
10.3% (Table 1). In the presence of a lower enzyme load
the conversion of 1 was 4.6±6.5%.
Scheme 1. Laccase biodegradation products of 5-50 condensed models 1 and 6. 2 is the major product recovered.

C. Crestini, D. S. Argyropoulos/Bioorg. Med. Chem. 6 (1998) 2161±2169
2163
Table 1. Extent of biodegradation of Kraft lignin models after
treatment with laccase
Table 2. Product pro®les for the biodegradation of condensed
models after treatement with laccase
Reaction Conditionsa
Reaction Conditionsa
Compound
A
B
C
D
E
F
Entry
1
Products
A
B
C
D
E
(%) of conversion of the model compoundsb
Yield (%)b
1
6.5
Ð
4.6
Ð
9.4
Ð
10.3
TRACE
40.4
9.8
Ð
61.7
Ð
2
1.8 1.3
0.6 0.3
6.0
0.0
0.1
0.2
5.6
0.1
0.2
0.1
2.8
1.4
0.0
0.0
14.4
0.8
0.6
14.2
0.4
0.5
Ð
6
3
8
14.9
Ð
8.6 41.8
Ð
36.5 100
Ð Ð
38.8 100
4
Ð
0.1
12
13
Ð
Ð
29.5
5
0.3 0.2
8.0 6.1
0.3 0.1
0.4 0.2
5.0 3.6
1.4 1.4
0.4 0.2
9.7 12.8 32.9
2
3
9
10
29.3
0.7
0.7
20.8
0.6
0.8
0.6
5.1
31.9
0.8
0.5
22.0
0.6
0.8
0.7
3.4
a A: Laccase 0.05 U/ml; B: Laccase 0.05 U/mL, ABTS 1 mM; C:
Laccase 0.5 U/mL; D: Laccase 0.5 U/mL, ABTS 1 mM; E:
Laccase 0.5 /mL, HBT 1 m; F: Laccase 0.5 U/mL, bubbled
oxygen. Model compounds 6 and 12 were also treated with
laccase and ABTS or HBT 1 mM. In both cases (experiments
carried out in triplicate), the non-phenolic models were found
to be completely unreactive.
11
14
15
16
17
18+19c
Ð Ð
1.8 0.7
2.3
b Measured by quantitative GC analyses.
Entry 1: Product pro®les for the biodegradation of 5-50 con-
densed model 1; Entry 2: product pro®les for the biodegrada-
tion of diphenylmethane model 8; Entry 3: Product pro®les for
the biodegradation of a-5 condensed model 13.=
Biodegradation of model compounds (3,30-dihydroxy-
4,40-dimethoxy-6,60-dimethyl) diphenylmethane 8 and
(3,304,40-tetramethoxy-6,60-dimethyl) diphenylmethane
12
a
A: Laccase 0.05 U/mL; B: Laccase 0.05 U/mL, ABTS 1 mM;
C: Laccase 0.5 U/mL; D: Laccase 0.5 U/mL, ABTS 1 mM; E:
Laccase 0.5 U/mL, HBT 1 mM.
b
Evaluated by quantitative GC analyses.
Due to the partial overlaping of the CG peaks of these iso-
mers, they were integrated together.
c
Compound 8 was treated with laccase from Trametes
versicolor to give 9, 10, and 11 in variable yields (Scheme
2). The extent of conversion ranged from 41.8±36.5 % in
incubations performed with 0.5 U/mL and 14.9±8.6% in
the presence of 0.05 U/mL of laccase (Table 1).
the presence of bubbled oxygen, the phenolic model
compound 2 was completely degraded, while the non-
phenolic model 12 was found completely inert, even in
the presence of ABTS or HBT (Table 1).
The presence of radical mediators did not signi®cantly
a€ect the reaction pathway and the extent of degrada-
tion. In this case two products witnessing side-chain
oxidations, 9 and 10 arising from the oxidation at the
benzylic position to hydroxymethyl and aldehyde,
respectively, were detected. A demethylated product, 11,
was also identi®ed (Scheme 2). In the presence of higher
amounts of laccase two more metabolites were detected,
both of high molecular weight. In any case, the major
metabolite detected was compound 9, that is, the pro-
duct of side-chain oxidation to benzyl alcohol (Table 2,
Entry 2). The lack of further oxidation products to
carboxylic derivatives was expected. In fact the laccase
catalyzed oxidation of aromatic methyl and hydroxy-
methyl groups with oxygen usually is very selective and
stops to benzaldehyde step. In the presence of excess of
oxygen, autooxidation processes may yield to carboxylic
derivatives.
Biodegradation of the ꢀ-5 model compound (2,40-
dihydroxy-3,30-dimethoxy-5-methyl)-diphenylmethane 13
The extent of biodegradation of the a-5 model com-
pound 13 with laccase ranged from 29.5±38.7% in the
presence of 0.5 U/mL of enzyme to 9.6±20.4% in the
presence of 0.05 U/mL of enzyme (Table 1). The main
metabolite detected, 14, is a product of side-chain oxi-
dation to benzyl alcohol. Besides 14, two products, 15
and 16, of oxidation to aldehyde and carboxylic moi-
eties respectively were identi®ed, albeit in low amounts
(Scheme 3). The formation of 16 can be rationalized by
autooxidation of 14 or 18 in the presence of oxygen. A
product indicating side-chain oxidation in the benzylic
position, compound 17, was also identi®ed. Besides side-
chain oxidation, two demethylation metabolites, 18, 19,
were detected (Table 2, Entry 3). High molecular weight
metabolites were not detected. In general the radical
mediators ABTS and HBT had no e€ect on the extent
of degradation and the distribution of metabolites.
When the etheri®ed diphenylmethane model 12 was
incubated with the enzyme no reaction was detected
under any of the experimental conditions used in this
e€ort (Scheme 2). When the reaction was carried out in

2164
C. Crestini, D. S. Argyropoulos/Bioorg. Med. Chem. 6 (1998) 2161±2169
Scheme 2. Laccase biodegradation products of diphenylmethane models 8 and 12. 9 is the major product recovered.
Scheme 3. Laccase biodegradation products of a-5 model compound 13. 14 is the major product recovered.
When the reaction was performed in the presence of
bubbled oxygen, the model compound 13 was com-
pletely degraded.
lignin.8 Stilbene units are known to be fairly oxygen
sensitive,16,17 and under our reaction conditions they
reacted even in the absence of laccase. It is noteworthy
that the nature of the products obtained from laccase
incubation and direct oxygen degradation are the same.
In the absence of enzyme the degradation reached 24%
of the starting material, while in the presence of 0.5 U/
mL of laccase the extent of degradation ranged from
77.6 to 90.0% (Table 3).
Biodegradation of 4,40-dihydroxy-3,30-dimethoxystilbene
20 and 3,30,4,40-tetramethoxystilbene 25
Stilbenoids, which are formed during pulping, constitute
a signi®cative structural feature of the residual kraft

C. Crestini, D. S. Argyropoulos/Bioorg. Med. Chem. 6 (1998) 2161±2169
2165
Table 3. Extent of begradation of Stilbene models
Table 4. Product pro®les for the degradation of Stilbene
models by laccase
Reaction Conditionsa
Reaction Conditionsa
Compound
A
B
C
D
Product
A
B
C
D
Conversion of the model compounds
20 and 25 (%)b
Yield (%)b
20
25
24.0
9.0
77.6
9.3
80.6
9.1
90.0
9.3
21
22
23
24
26
27
28
3.4
3.4
17.0
13.3
26.8
28.0
1.0
21.6
22.4
7.0
8.1
32.5
12.6
17.1
27.0
a
A: Blank; B: Laccase 0.5 U/mL; C: Laccase 0.5 U/mL, ABTS
1 mM; D: Laccase 0.5 U/mL, HBT 1 mM.
26.8
1.6
1.6
0.9
1.6
0.9
1.6
0.9
b Evaluated by quantitative GC analyses.
0.9
TRACE
TRACE
TRACE
TRACE
From the incubation of 4,40-dihydroxy-3,30-dimethoxy-
stilbene 20, four main metabolites were detected in
comparable amounts (Scheme 4). Two of them, com-
pounds 21 and 23 were products of ole®nic oxidation
and rearrangement,17 while 22 and 24 were character-
ized as products of aromatic hydroxylation and deme-
thoxylation, respectively (Scheme 4). In Table 4 the
amounts of the detected metabolites obtained both in
the presence and absence of radical mediators are
reported. For these models ABTS and HBT showed an
activating e€ect on the oxidation of the stilbene moiety
compared to laccase alone.
a
A: Blank; B: Laccase 0.5 U/mL; C: Laccase 0.5 U/mL, ABTS
1 mM; D: Laccase 0.5 U/mL, HBT 1 mM.
b
Evaluated by quantitative GC analyses.
carried out in the presence of ABTS and HBT, no dif-
ferences in the reactivity of the system were apparent,
the only metabolites detected being the hydroxylation
product 27 and traces of 28, a product of ole®nic oxi-
dation and rearrangement.17
Discussion
The non-phenolic stilbene model 25 showed a somewhat
reduced reactivity toward laccase as witnessed by the
fact that its oxidation products were obtained in
amounts roughly equivalent to those obtained in the
absence of enzyme (Table 3). When the reaction was
Laccases are oxygen-dependent enzymes that reduce
oxygen to water. The reaction mechanism of laccase on
lignin model compounds has been widely studied to
understand the mechanisms of the biological degradation
Scheme 4. Laccase biodegradation products of stilbene models 20 and 25.

2166
C. Crestini, D. S. Argyropoulos/Bioorg. Med. Chem. 6 (1998) 2161±2169
of wood and evaluate the possible use of this oxidative
enzyme in deligni®cation processes.4,14,15 The phenoxy
radical intermediates formed during this process can
further disproportionate and consequently initiate lignin
degradation.4,14,15
suggests that during the laccase deligni®cation of kraft
pulp the process of degradation of stilbenes could play
an important role.
The condensed kraft lignin model compounds seem to
initially be degraded by laccase, mainly via side chain
oxidation, demethylation and hydroxylation reactions.
These transformations can be considered as aids to
deligni®cation since they increase the hydrophilic nature
of the lignin moieties. Most signi®cantly, however, such
reactions introduce activated sites on lignin structures.
Oxidative coupling reactions that inhibit deligni®cation
were found to occur only in minor amounts under the
acidic conditions examined, and are therefore con-
sidered irrelevant.
The main early metabolites detected were, for all the 5-
50 diphenylmethane and a-5 models, products of oxida-
tion at the benzylic positions. The oxidation of methyl
groups on aromatic rings to benzyl alcohols or benzal-
dehydes is usually accomplished in organic synthesis
using transition metal oxidants, but it is rather dicult
to stop the reaction at the aldehyde stage, since the oxi-
dation products are more easily oxidized than the start-
ing materials. The occurrence of this selective oxidation
on benzylic groups to aldehyde on non-phenolic systems
in the presence of the laccase-ABTS system, has recently
been reported.18
The inertness of the non-phenolic 5-50, diphenylmethane
and stilbene models toward laccase is in accord with
data previously reported for methoxy and dimethoxy
benzenes.21,22 However, their inertness in the presence
of radical mediators was unexpected. In fact, it has been
reported that the radical mediator ABTS is able to bring
about the oxidation of non-phenolic systems character-
ized by a redox potential higher than laccase such as
veratryl alcohol or non-phenolic b-O-4 model com-
pounds.3 In contrast, recent ®ndings have also shown
that the reactivity of benzylic alcohols in the presence of
the laccase-mediator system depends on the substitution
pattern on the aromatic ring.18,23 In particular, 2,6-dis-
ubstituted benzylic alcohols were found to be unreactive
with the laccase-mediator system.
Besides the products of side-chain oxidation, deme-
thoxylation and perhaps oxidative coupling products
were also detected. Such reactivity patterns have already
been observed for laccase oxidations of lignins and lig-
nin model compounds.2,15,19 This behaviour is analo-
gous to the overall reactivity found between hydroxy
radicals and lignin model compounds. This analogy is
reasonable since the main intermediates involved in such
reactions are believed to be phenoxy radicals.20
The phenolic diphenylmethane and a-5 structures, 8 and
13, respectively, were found to be remarkably more
reactive than the 5-50 dicreosol model compound 1, that
was degraded by the laccase treatments only in low
amounts. The diphenylmethane model 8, which showed
the highest reactivity, also showed the highest amount
of high molecular weight metabolites, probably arising
from radical coupling reactions.4,14,15 The strongly
enhanced reactivity of 5-50, diphenylmethane and a-5
models in the presence of bubbled oxygen (which caused
their near complete degradation), constitutes experi-
mental evidence supporting our contention that the
other experiments were performed under limited oxygen
supply. This experimental design e€ectively allowed the
isolation and characterization of the early metabolites
occurring during the laccase and laccase-mediator oxi-
dative processes.
Most of the work on laccase and laccase/mediator sys-
tems has been carried out in aqueous bu€ers. In princi-
ple one could argue that the presence of dioxane in our
experiments could a€ect the functionality of laccase and
the laccase-mediator reaction rate and/or redox poten-
tial. However there are several literature examples of the
use of laccases in the presence of considerable amounts
of organic solvents.18,23±25
In an e€ort to address this concern, the activity of lac-
case was assayed under these conditions. The presence
of 50% dioxane reduced the enzyme oxidation rate by
about 70±80%, but the enzyme was still functional. In
fact assays of enzymatic activity of laccase kept 24 h in
50% dioxane solution showed residual 80% activity.
With respect to the redox potential of the laccase-
mediator system in the presence of dioxane, laccase-
mediator treatments in mixed solvents have been
reported to occur successfully under analogous reac-
tion conditions.18,23,24
The degradation pathway of the phenolic stilbene model
20, besides showing demethylation and hydroxylation
reactions, was shown to yield a product of benzylic
oxidation, 21, and a product arising from complex oxi-
dation-transposition reactions, 23. These transforma-
tions are characteristic of stilbenes in the presence of
hydroxy radicals.4,14 Once again this similarity can be
due to the presence of common phenoxy radical
intermediates. The high reactivity of the stilbene unit
According to our data, the radical mediators ABTS and
HBT do not play an important role in the degradation
of non-phenolic 5-50, diphenylmethane and stilbene

C. Crestini, D. S. Argyropoulos/Bioorg. Med. Chem. 6 (1998) 2161±2169
2167
models. It has been recently reported that laccase, in the
presence of either ABTS or HBT can e€ectively
delignify kraft pulps.5 On the basis of our data one may
suggest that the role of such mediators, in kraft pulps, is
to act as di€usible lignin oxidizing agents of phenolic
systems, since these relatively small compounds can
access the inner lignin structure with greater facility
than the relatively large enzyme.
123.53 (CH), 132.59 (C) 132.81 (C), 144.60 (C ), 152.30
(C ). Mass Spectrometry data are reported in Table 5.
(3,304,40-Tetramethoxy-6,60-dimethyl) diphenylmethane
1
12. H NMR (CDCl3) d 2.24 (s, 6H, CH3), 3.76 (s, 6H,
OCH3), 3.85 (s, 6H, OCH3), 3.96 (s, 2H, CH2), 6.52±
6.60 (m, 4H, CH); 13C NMR (CDCl3) d 21.30 (CH3),
29.07 (CH2), 55.62 (OCH3), 60.37 (OCH3), 111.21 (CH),
122.87 (CH), 133.29 (C) 134.46 (C), 144.88 (C ), 152.66
(C). Mass Spectrometry data are reported in Table 5.
Experimental
Oxidation of residual kraft lignin model compounds. Due
to the low solubility of kraft lignin model compounds in
water, their oxidation was performed in a 1/1 (v/v)
dioxane:water solvent mixture. The substrate (2 mM)
was dissolved in dioxane:acetate bu€er pH 5 0.05 M
(10 mL, 1/1 v/v), in the presence or absence of either
ABTS or HBT (1 mM), and treated with puri®ed laccase
from Trametes versicolor supplied by Dr. R. Bourbon-
nais of PAPRICAN with either 0.05 U/mL or 0.5 U/mL
at 40 ꢀC. The reaction mixtures were kept in open vials
under vigorous stirring in order to ensure constant oxy-
gen saturation of the solutions throughout the experi-
ment.5 The reactions were conducted in the dark in
order to avoid possible photochemical oxidation.
1H and 13C NMR spectra were recorded on a Varian
XL 300 spectrometer. Mass Spectroscopy (MS) was
performed with Hewlett-Packard 5971 mass-selective
detector on a Hewlett-Packard 5890 gas chromatograph.
All solvents were ACS reagent grade and were redis-
tilled and dried according to standard procedures.
Chromatographic puri®cations were performed on
columns packed with Merck silica gel 60, 230±400 mesh
for ¯ash technique. Thin-layer chromatography was
carried out using Merck platten Kieselgel 60 F254.
Characterization of metabolites. Gas chromatography
and gas chromatography-mass spectrometry of the
reaction products were performed using a DB1 column
(30 mÂ0.25 mm and 0.25 mm ®lm thickness), and an
isothermal temperature pro®le of 100 ꢀC for the ®rst two
min, followed by a 20 ꢀC/min temperature gradient to
300 C and ®nally an isothermal period at 300 ꢀC for
10 min. The injector temperature was 280 ꢀC. Chroma-
tography grade helium was used as the carrier gas. The
fragmentation patterns were compared to those of
authentic samples.17,26±31
After 24 h the reactions were halted by heating the mix-
tures in boiling water. A suitable amount of benzoic
acid as internal standard (2 mM), dissolved in dioxane,
was then added to the reaction mixtures. The solvent
was evaporated under reduced pressure, the residues
were solubilized in ethyl acetate and pyridine. The solu-
tions were dryed over anhydrous MgSO4 and ®ltered.
The organic solvent was evaporated under reduced
pressure. In order to analyze the reaction products the
residues were dissolved in 1 mL of pyridine and sub-
mitted to silylation by addition of N,O-bis(tri-
methylsilyl)-acetamide. After 30 min the mixtures were
subjected to gas chromatography (GC) and gas chro-
matography-mass spectrometric (GC-MS) analyses.
Kraft lignin model compounds. The model compounds 1,
8, 13, 20, and 25 were synthesized by known meth-
ods.26,27 Non-phenolic models dihydromethyl veratrole
6 and (3,304,40-tetramethoxy-6,60-dimethyl) diphenyl-
methane 12 were synthesized by methylating the corre-
sponding phenols
1
and
8
using dimethylsulfate
Enzyme assays. Laccase activity was determined by oxi-
dation of ABTS.32 The assay mixture contained 0.5 mM
ABTS, 0.1 M sodium acetate, pH 5.0, and a suitable
amount of enzyme. Oxidation of ABTS was fol-
(Me2SO4) under alkaline conditions. Phenols 1 and 8
(0.1 mol) were dissolved in 10 mL of NaOH 30% (aq
solution) under magnetic stirring, and Me2SO4
(0.2 mole) were added at 60 ꢀC during 1 h. After 2 h the
reaction mixtures were cooled, neutralized with ammo-
nium chloride, and extracted with ethyl acetate. The
organic solvent was evaporated under reduced pressure,
and the crude products were crystallized from hexane-
ethyl acetate to give 6 and 12 in quantitative yield.
lowed by absorbance increase at 420 nm (e420
=
3.6Â104 M 1cm 1). Enzyme activity was expressed in
units (U=mmol of ABTS oxidized per minute).4,7 The
determination of the laccase activity was also performed
in a dioxane/Na-acetate bu€er 1/1 (v/v) solvent system.
Oxidation of kraft lignin model compounds in the
presence of bubbled oxygen. The model substrate
(2 mM) was dissolved in dioxane:acetate bu€er pH 5
0.05 M (10 mL, 1/1 v/v) and treated with laccase 0.5 U/
mL at 40ꢀC in the presence or absence of either ABTS
1
Dihydromethyl veratrole 6. H NMR (CDCl3) d 2.33 (s,
6H, CH3), 3.64 (s, 6H, OCH3), 3.88 (s, 6H, OCH3),
6.64±6.74 (m, 4H, CH); 13C NMR (CDCl3) d 21.26
(CH3), 55.79 (OCH3), 60.64 (OCH3), 112.58 (CH),

2168
C. Crestini, D. S. Argyropoulos/Bioorg. Med. Chem. 6 (1998) 2161±2169
Table 5. Mass spectrometric data
Product Derivativea MS (m/z) data (%)
1
Ð
274 (M, 100), 241 (24), 227 (21), 199 (19)
1
-Si (CH3)3 418 (M, 38), 403 (M-15, 18), 388 (M-30, 26), 287 (14), 179 (11), 73 (100)
-Si (CH3)3 506 (M, 28), 329 (100), 147 (8), 73 (87)
-Si (CH3)3 432 (M, 21), 417 (M-15, 26), 301 (8), 73 (100)
2
3
4
-Si (CH3)3 446 (M, 12), 431 (M-15, 18), 357 (4), 315 (8), 73 (100)
-Si (CH3)3 476 (M, 16), 373 (7), 358 (10), 147 (14), 73 (100)
5
6
Ð
Ð
Ð
302 (M, 100), 287 (M-15, 16), 272 (M-30, 21) 256 (93), 241 (25), 213 (23), 128 (28)
330 (M, 100), 315 (M-15, 12), 300 (M-30, 18), 284 (96), 269 (27), 241 (22)
288 (M, 84), 255 (21), 241 (8), 151 (33), 138 (100), 123 (7)
7
8
8
-Si (CH3)3 432 (M, 56), 417 (M-15, 12), 402 (M-30, 24), 73 (100)
-Si (CH3)3 520 (M, 9) 505 (M-15, 5), 417 (7), 343 (22), 73 (100)
-Si (CH3)3 446 (M, 28), 431 (M-15, 62), 357 (6), 269 (8), 179 (8), 73 (100)
-Si (CH3)3 490 (M, 13), 431(8), 342 (66), 269 (6), 193 (8), 117 (12), 73 (100)
9
10
11
12
13
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
Ð
Ð
316 (M, 100), 301 (M-15, 11), 269 (23), 255 (21), 165 (26), 151 (99), 135 (78)
274 (M, 95), 241 (12), 151 (28), 150 (100), 149 (22), 124 (18), 121 (13)
-Si (CH3)3 419 (M+1, 26), 418 (M, 100), 403 (M-15, 26), 388 (M-30, 91), 358 (23), 343 (18), 179 (20), 73 (81)
-Si (CH3)3 506 (M, 14), 491 (M-15, 4), 476 (M-30, 6), 387 (8), 297 (9), 209 (11), 147 (12), 73 (100)
-Si (CH3)3 432 (M, 22), 414 (M-15, 13), 402 (M-30, 16), 193 (16), 73 (100)
-Si (CH3)3 520 (M, 18), 505 (M-15, 11), 430 (7), 417 (11), 343 (28), 193 (11), 147 (13), 73 (100)
-Si (CH3)3 432 (M, 24), 417 (M-15, 22), 372 (8), 314 (11), 223 (14), 179 (21), 121 (16), 73 (100)
-Si (CH3)3 476 (M, 22), 446 (M-30, 9), 417 (14), 387 (19), 343 (12), 314 (13), 179 (17), 73 (100)
-Si (CH3)3 476 (M, 26), 446 (M-30, 13), 417 (18), 387 (12), 343 (9), 314 (15), 179 (19), 73 (100)
-Si (CH3)3 417 (M+1, 31), 416 (M, 100), 401 (M-15, 4), 386 (M-30, 28), 356 (19), 281 (5), 253 (14), 178 (11), 73 (96)
-Si (CH3)3 432 (M, 6), 417 (M-15, 4), 223 (M-209, 100), 209 (M-223, 12), 179 (21), 165 (9), 73 (32)
-Si (CH3)3 504 (M, 71), 489 (M-15, 6), 474 (M-30, 11), 444 (M-60, 4), 342 (8), 73 (100)
-Si (CH3)3 418 (M, 63), 403 (M-15, 28), 388 (M-30, 25), 373 (M-45, 12), 358 (M-60, 26), 223 (58), 193 (62), 165 (60), 73 (100)
-Si (CH3)3 475 (M+1, 16), 474 (M, 46), 444 (M-30, 8), 356 (18), 253 (6), 73 (100)
Ð
Ð
301 (M+1, 21), 300 (M, 100), 285 (M-15, 39), 225 (16), 210 (14), 210 (9), 181 (14), 152 (15), 139 (14), 128 (16)
316 (M, 5), 302 (36), 271 (24), 227 (12), 207 (16), 165 (100), 137 (22)
-Si (CH3)3 389 (M+1, 11), 388 (M, 69), 373 (M-15, 6), 358 (M-30, 5), 345 (12), 315 (14), 287 (18), 73 (100)
302 (32), 271 (M-31, 24), 227 (14), 207 (16), 165 (100) 137 (19)
Ð
a-: underivatized; -Si (CH3)3: trimethylsilylated with N,O-bis(trimethylsilyl)-acetamide.
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reaction mixtures. All reactions were conducted in the
dark in order to avoid photochemical oxidation. After
4 h the reactions were halted by heating the mixtures in
boiling water.
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Acknowledgements
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The authors would like to acknowledge Mr. R. Bour-
bonnais for supplying the puri®ed enzyme used in this
e€ort, and Dr. F. Archibald for providing constructive
criticism during the writing of this paper. This work was
supported by the Paprican Maintaining Member Com-
panies, and forms a part of a research program under
the auspice of the Protein Engineering Network Centre
of Excellence (PENCE).
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