On the reactions of two fungal laceases differing in their redox potential with lignin model compounds: products and their rate of formation

Article abstract of DOI:10.1021/jf901511k

Laceases (EC 1.10.3.2) are multicopper oxidases able to oxidize phenolic compounds such as lignin-related polyphenols. Since the discovery that so-called mediators effectively extend the family of lacease substrates, direct interactions between lignin-like materials and lacease have gained much less attention. In this work, the aim was to characterize oxidation products formed in direct laccase-catalyzed oxidation of different guaiacylic and syringylic lignin model compounds with two different laceases: a low redox potential Melanocarpus alborneces lacease and a high redox potential Trametes hirsuta lacease. By following the formation of different, mainly biphenylic (5-5) and benzylic oxidation products, it was found that although both of these enzymes generated practically the same pattern of products with particular types of syringyl and guaiacyl compounds, in some cases a clear difference in the rates of their formation was observed. The results also confirm further to the suggestions that syringylic compounds are able to act as mediators in their own oxidation reactions and also that in some instances acetylation of phenolic material may produce altered, unexpected structures.

Full text of DOI:10.1021/jf901511k

J. Agric. Food Chem. 2009, 57, 8357–8365 8357
DOI:10.1021/jf901511k
On the Reactions of Two Fungal Laccases Differing in Their
Redox Potential with Lignin Model Compounds: Products and
Their Rate of Formation
†
†
MAARIT
L
AHTINEN,*,† KRISTIINA
K
RUUS,‡ PETRI
H
EINONEN
,
AND
J
USSI
S
IPILA
†Laboratory of Organic Chemistry, Department of Chemistry, University of Helsinki, P.O. Box 55,
FIN-00014 HU, Finland, and ‡VTT, Technical Research Centre of Finland, P.O. Box 1000,
FIN-02044 VTT, Finland
Laccases (EC 1.10.3.2) are multicopper oxidases able to oxidize phenolic compounds such as
lignin-related polyphenols. Since the discovery that so-called mediators effectively extend the family
of laccase substrates, direct interactions between lignin-like materials and laccase have gained
much less attention. In this work, the aim was to characterize oxidation products formed in direct
laccase-catalyzed oxidation of different guaiacylic and syringylic lignin model compounds with two
different laccases: a low redox potential Melanocarpus albomyces laccase and a high redox
potential Trametes hirsuta laccase. By following the formation of different, mainly biphenylic (5-5)
and benzylic oxidation products, it was found that although both of these enzymes generated
practically the same pattern of products with particular types of syringyl and guaiacyl compounds, in
some cases a clear difference in the rates of their formation was observed. The results also confirm
further to the suggestions that syringylic compounds are able to act as mediators in their own
oxidation reactions and also that in some instances acetylation of phenolic material may produce
altered, unexpected structures.
KEYWORDS: Laccase; lignin; model compound; guaiacyl; syringyl; oxidation; product structure;
nuclear magnetic resonance spectroscopy; liquid chromatography-mass spectrometry; electrospray
ionization mass spectrometry; redox potential; Trametes hirsuta; Melanocarpus albomyces
INTRODUCTION
utilizing lignocellulosic material, laccase-synthetic mediator
reactions have gained much more attention compared to reac-
tions without such mediators. Obviously, laccase always reacts
directly with lignin to some extent. For example, laccases poly-
merize softwood lignin and oxidize lignin precursors to synthetic
lignin (7). Recently, there have also arisen questions about the
importance of so-called natural mediators (8). Therefore, a
thorough knowledge about the types of products formed and
reaction mechanisms involved in laccase-induced reactions with-
out synthetic mediators is of crucial importance in developing
new applications for laccase with lignin.
Reactions of laccase and lignin have been studied earlier with
some individual model compounds. These studies have mainly
focused on lignin model compounds containing a syringyl unit as
the phenolic moiety. Dimeric β-O-4 model compound syringyl-
glycol β-guaiacyl ether (7) was oxidized by Polyporus versicolor
laccase from benzylic position and also cleaved between CR and
phenyl ring to guaiacoxyacetaldehyde and 2,6-dimethoxy-p-ben-
zoquinone (9). Syringylglycerol β-guaiacyl ether (8) gave different
products in two different studies. Wariishi et al. (10) observed the
formation of guaiacoxy propionic acid and 2,6-dimethoxy-p-
benzoquinone (CR-aryl cleavage), syringaldehyde and guaiacoxy-
ethanol (CR-Cβ cleavage), and also guaiacol (O-Cβ cleavage)
by Coriolus versicolor laccase, when Kawai et al. (11) detected a
product of benzylic hydroxyl oxidation, 2,6-dimethoxy-p-hydro-
quinone and glyceraldehyde-2-guaiacyl ether (CR-aryl cleavage)
Laccases (EC 1.10.3.2) are multicopper oxidases able to cata-
lyze one-electron oxidation, with concomitant reduction of O2 to
H2O, of various substrates such as mono-, di-, and polyphenols,
aminophenols, diamines, and some inorganic compounds (1-3).
Lignin, an amorphous polyphenol accounting for approximately
30% of the organic carbon in the biosphere, is composed of three
different types of phenylpropane units and contains p-hydroxy-
phenyl, guaiacyl, and syringyl types as aromatic moieties (none,
one or two methoxyls ortho to the phenolic hydroxyl) (4, 5).
Softwood lignin contains mainly guaiacyl units, hardwood lignin
contains guaiacyl and syringyl units, and both lignins also have
low amounts of p-hydroxyphenyl units. Grass lignin contains
guaiacyl and syringyl units, and more p-hydroxyphenyl units than
wood lignin. The phenylpropane units are linked together to form
a seemingly randomly organized polymeric network, with several
types of linkages, the most abundant being the β-O-4 type
(45-60%) (5). Other types of linkages are β-5, β-β, 5-5, 5-5/β-
O-4 (dibenzodioxocin), 5-O-4, and β-1.
Laccase is able to oxidize directly only phenolic subunits of
lignin, but the substrate range can be widened to nonphenolic
units by the use of so-called mediators (6). Since the discovery of
laccase-mediator system and its potential in industrial processes
*Corresponding author (telephone þ358-9-191-50396; fax þ358-9-
191-50366; e-mail maarit.lahtinen@helsinki.fi).
© 2009 American Chemical Society
Published on Web 08/25/2009
pubs.acs.org/JAFC

8358 J. Agric. Food Chem., Vol. 57, No. 18, 2009
Lahtinen et al.
Figure 1. Lignin model compounds used in the study.
and guaiacol (O-Cβ cleavage) by the same C. versicolor laccase.
Oxidation of syringylic β-1 dimers by C. versicolor laccase yielded
products resulting from benzylic hydroxyl oxidation, CR-Cβ
cleavage, and alkyl-aryl cleavage (12, 13).
synthesized according to the previously reported methods. Synthesis of the
guaiacylic β-O-4 trimer used in LC-MS mass calibration will be published
elsewhere (19). The synthesized products were purified by crystallization,
with preparative HPLC or flash chromatography.
Enzymes. The M. albomyces laccase was overproduced in Tricho-
derma reesei and purified as described earlier (20). The T. hirsuta laccase
was produced in its native host and purified (21).
There are even fewer studies about oxidation of model com-
pounds containing guaiacyl units as the phenolic moiety (4, 5, 7).
These types of lignin substructures are, however, abundant in
softwoods. Vanillyl alcohol (2) has been oxidized by Trametes
versicolor laccase to 4,40-dihydroxy-3,30-dimethoxybenzophenone
and vanillin (1) (14). Oxidation of methyl to hydroxymethyl and
demethylation by T. versicolor laccase have also been observed
with diphenylmethane, R-5, and stilbene type models (15).
To understand more clearly the reactions of laccase and
lignocellulosic material, we investigated in this study how the
redox potential of the laccase and reaction time affect the product
pattern. Direct reactions between two types of laccases, one from
an ascomycete Melanocarpus albomyces (low redox potential
laccase) and another from a white rot fungus Trametes hirsuta
(high redox potential laccase), with eight different lignin model
compounds representative for softwood and hardwood lignins
(1-8, Figure 1) were elucidated. The formation of oxidation
products was analyzed as a function of time with high-perfor-
mance liquid chromatography (HPLC) and liquid chromatogra-
phy-mass spectrometry (LC-MS). By using these methods, the
order, by which the products were formed, and the formation
rates of the products were followed, as well as the differences
between low and high redox potential laccases. The structures of
the products, deduced on the basis of their mass values from LC-
MS, were further verified by fractionating the acetylated products
and analyzing them with nuclear magnetic resonance (NMR)
spectroscopy and high-resolution electrospray ionization mass
spectrometry (ESI-MS).
HPLC and LC-MS Analysis of Products as a Function of
Time. Analysis Equipment, Reagents, and Procedure. HPLC was
performed using reverse-phase columns (Agilent ZORBAX Eclipse XDB-
C8, 4.6 mm ꢀ 15 cm, 5 μm, or XDB-C18, 2.1 mm ꢀ 100 mm, 3.5 μm),
methanol-water as the eluent, and Agilent 1100 series HPLC. The C18
column was used for dimeric β-O-4 model compounds and the C8 column
for the other compounds. Gradient elution was applied starting from 50%
methanol (v/v). The flow rate was 0.1 mL min-1 at the gradient elution
phase (15 min) and 0.2 mL min-1 with 100% methanol.
LC-MS in negative-ion mode was acquired using the same HPLC
followed by Mariner ESI-TOF (PerkinElmer Biosystems). Ionization was
enhanced by spraying a 2.5% NH3 solution from a separate line with
syringe pump at 10 μL min-1 rate to the ionization chamber along with the
analyte. Mass calibration was done with the starting material present inthe
sample and guaiacylic β-O-4 trimer injected at the beginning of the
analysis.
Reaction Procedure for HPLC Analysis. Experiments were
carried out with both laccases, and 12.5 mM model compound solutions
were used. Solvent was dioxane/sodium succinate buffer (25 mM, pH 4.5)
2:8 with dimeric β-O-4 compounds and 1:9 with other compounds.
Dioxane was distilled over sodium before use. Enzyme dosages of 2 nkat
mL-1 for dehydrodivanillyl alcohol (4) and 1 nkat mL-1 for other
compounds were used. Laccase activity was determined using ABTS as
described by Niku-Paavola et al. (22). Enzyme was added to 10 mL of the
model compound solution. Samples (0.5 mL) were taken at 1, 5, 10, and
30 min and 1, 2, 4, and 24 h to vials containing 1-2 mg of NaN3, a known
inhibitor of laccase (23). A control sample (0 min) was taken before the
enzyme was added.
HPLC and LC-MS Samples. From each 0.5 mL sample two
samples were prepared for HPLC. Of the original sample, 0.4 mL was
diluted by adding 1.6 mL of water and 2.0 mL of methanol. The diluted
sample was divided into two 2 mL samples and to one was added 2 μL of
50 mg mL-1 guaiacol solution (guaiacol from Merck) to reveal any
material loss. All samples were analyzed with HPLC and some of them
with LC-MS to identify oxidation products. The mass spectral data are in
Table 1.
MATERIALS AND METHODS
General. All commercial reagents and solvents were used as received
unless otherwise mentioned. For preparative HPLC methanol-water was
used as the eluent (isocratic flow) with the following equipment: ISCO
2350 HPLC pump, Shimadzu SPD-6A UV spectrophotometric detector,
Shimadzu C-R6A Chromatopac data processor, reverse phase column
(Waters SymmetryPrep C18, 19 ꢀ 150 mm, 7 μm). Flash chromatography
purifications and fractionations were done with silica gel 60 (Merck) and
ethyl acetate-toluene as the eluent. For thin-layer chromatography silica
gel 60 F254 TLC aluminum sheets (Merck) were used.
NMR Analysis and High-Resolution ESI-MS of Acetylated
Oxidation Products. Analysis Equipment, Reagents, and Procedure.
A Varian Inova 500 MHz spectrometer was used for NMR measure-
ments, which were performed at 27 °C. Deuterated chloroform (CDCl3)
was used as a solvent and internal reference for all samples. High-
resolution mass spectra were acquired with Bruker Daltonics microTOF
ESI-TOF. Methanol/water (80:20) was used as a solvent, and the
concentration of all samples was 1 μg mL-1. Trifluoroacetic acid (0.1%)
was added to the sample to enhance ionization when necessary. For
internal mass calibration, Agilent ES Tuning Mix (G2421A; 1/50 dilution
Model Compounds and Enzymes. Model Compounds. Of the
used compounds vanillin was commercial (1, Merck). Other compounds
were prepared according to well-known methods. Vanillyl (2) and syringyl
(3) alcohols were reduced from the corresponding aldehydes with NaBH4
in ethanol. Dehydrodivanillyl alcohol (16) (4), guaiacylglycol β-guaiacyl
ether (17) (5), guaiacylglycerol β-guaiacyl ether (17) (6), syringylglycol
β-guaiacyl ether (9) (7), and syringylglycerol β-guaiacyl ether (18) (8) were

Article
J. Agric. Food Chem., Vol. 57, No. 18, 2009 8359
Table 1. Mass Spectral Data for Oxidation Products Studied with LC-MS
compound (m/z [M - 1]-)
-OCOCH3), 3.82 (6H, s, 3-OCH3, 5-OCH3), 3.83 (3H, s, 20-OCH3), 4.23
(1H, dd, 11.0 Hz, 3.9 Hz, β0H), 4.29 (1H, dd, 11.0 Hz, 7.8 Hz, βH), 6.12 (1H,
dd, 7.8 Hz, 3.9 Hz, RH), 6.67 (2H, s, 2-H, 6-H), 6.90-6.96 (4H, m, ArH);
13C NMR (125.69 MHz, CDCl3, 27 °C) δ 20.41 (arom -OCOCH3), 21.12
(aliph -OCOCH3), 55.94 (20-OCH3), 56.15 (3-OCH3, 6-OCH3), 72.23
(βC), 74.07 (RC), 103.67 (2-C, 6-C), 112.57 (30-C), 115.59, 120.94, 122.36
(40-60-C), 128.66 (4-C), 135.48 (1-C), 148.07 (10-C), 150.17 (20-C), 152.18 (5-
C, 3-C), 168.62 (arom -OCOCH3), 169.98 (aliph -OCOCH3); ESI-MS
(positive), m/z 422.1807 [M þ NH4]þ (C21H28NO8 requires 422.1809),
427.1350 [M þ Na]þ (C21H24NaO8 requires 427.1363), 443.1100 [M þ K]þ,
(C21H24KO8 requires 443.1103).
m/z [M - 1]- for oxidation products
1 (151)
2 (153)
3 (-a)
4 (305)
5 (289)
6 (319)
7 (319)
8 (349)
301
151, 301, 303, 305, 423, 575
181
301, 303, 575, 577
577
637
317
347
Triacetate of 1-(4-hydroxy-3,5-dimethoxyphenyl)-2-(20-methoxy-
phenoxy)-1,3-propanediol (8): ESI-MS (positive), m/z 494.2008 [M þ
NH4]þ (C24H32NO10 requires 494.2021), 499.1563 [M þ Na]þ
(C24H28NaO10 requires 499.1575), 515.1304 [M þ K]þ (C24H28KO10
requires 515.1314).
a Syringyl alcohol was not ionized at the applied conditions.
in acetonitrile/water, 1:1) or sodium formiate (0.1% formic acid
and 2.5 mM NaOH in water/isopropanol, 1:1) was added to the
sample (1:1).
Tetraacetate of 3,30-bis[2-(2-methoxyphenoxy)-1-ethanol]-5,50-di-
methoxy-6,60-biphenyldiol (10): 1H NMR (499.82 MHz, CDCl3, 27 °C)
δ 2.07 (6H, br s, arom -OCOCH3), 2.12 (6H, br s, aliph -OCOCH3),
3.815, 3.817 (6H, s þ s, 2B-OCH3, 20B-OCH3), 3.87 (6H, br s,
5A-OCH3, 50A-OCH3), 4.22 (2H, dd, 11.0 Hz, 3.9 Hz, β2H, β20H),
4.30 (2H, dd, 11.0 Hz, 7.8 Hz, β1H, β10H), 6.12 (1H, dd, 7.8 Hz, 3.9 Hz,
R0H), 6.13 (1H, dd, 7.8 Hz, 3.9 Hz, RH), 6.88-6.96 (10H, m, ArH),
7.050, 7.046 (2H, d þ d, each 2.1 Hz, 4A-H, 40A-H); 13C NMR (125.69
MHz, CDCl3, 27 °C) δ 20.23 (6A-OCOCH3, 60A-OCOCH3), 21.01,
21.03 (R-OCOCH3, R0-OCOCH3), 55.89, 56.02 (5A-OCH3, 50A-
OCH3, 2B-OCH3, 20B-OCH3), 72.20, 72.24 (βC, β0C), 73.69, 73.75
(RC, R0C), 110.65, 110.68 (4A-C, 40A-C), 112.53 (3B-C, 30B-C), 120.26
or 120.92 (2A-C, 20A-C), 115.59, 120.26 or 120.92, 122.35 (4B-6B-C,
40B-60B-C), 131.13 (1A-C, 10A-C), 135.21, 135.24 (3A-C, 30A-C),
137.42, 137.45 (6A-C, 60A-C), 148.00 (1B-C, 10B-C), 150.11 (2B-C,
20B-C), 151.44, 151.45 (5A-C, 50A-C), 168.67 (6A-OCOCH3, 60A-
OCOCH3), 170.00 (R-OCOCH3, R0-OCOCH3); ESI-MS (positive),
Reaction Procedure for NMR Studies. Reaction was carried out
with that laccase which gave highest yield of all oxidation products at 24 h
so that they all could be identified. Thus, M. albomyces laccase was used
for compounds 2, 3, 6, 7, and 8 and T. hirsuta laccase for 1, 4, and 5.
Solvents and concentrations of the model compounds were the same as in
the procedure for HPLC analysis. The amount of the model compound
solution (50-150 mL) was dependent on the number of oxidation
products expected on the basis of HPLC and LC-MS analyses. Enzyme
dosage was 1 nkat mL-1 to final volume in all preparative oxidations.
After enzyme addition, solution was stirred for 24 h. Products were then
extracted twice with ethyl acetate. The organic phase was washed with
water and brine and dried with Na2SO4, and the solvent was evaporated.
In the case of vanillin (1), a considerable amount of solid material was
formed as could be expected when 9 is formed, and it was separated by
suction filtration after ethyl acetate was added. Solids were washed with
ethyl acetate and water, and the filtrate was then handled as in all other
cases. Yields of the mixtures (m-%) were as follows: 1, 0.25 g (86%); 2,
0.16 g (55%); 3, 0.28 g (81%); 4, 0.07 g (30%); 5, 0.29 g (100%); 6, 0.10 g
(50%); 7, 0.28 g (100%); 8, 0.26 g (85%). The mixture of the products was
then acetylated in pyridine/acetic anhydride (1:1) for 24 h at room
temperature. Yields after acetylation were as follows: 1, 0.36 g; 2, 0.21 g;
3, 0.34 g; 4, 0.09 g; 5, 0.39 g; 6, 0.17 g; 7, 0.30 g; and 8, 0.33 g.
m/z 764.2931 [M
þ
NH4]þ (C40H46NO14 requires 764.2913),
769.2452 [M þ Na]þ (C40H42NaO14 requires 769.2467), 785.2206
[M þ K]þ (C40H42KO14 requires 785.2206).
Triacetate of 5-carboxaldehyde-50-hydroxymethyl-3,30-dimethoxy-2,20-
biphenyldiol (12): 1H NMR (499.82 MHz, CDCl3, 27 °C) δ 2.08 (3H, s, 20-
OCOCH3), 2.12 (3H, s, -CH2OCOCH3), 2.13 (3H, s, 2-OCOCH3), 3.88
(3H, s, 30-OCH3), 3.93 (3H, s, 3-OCH3), 5.10 (2H, s, -CH2-), 6.86 (1H, d,
1.8 Hz, 60-H), 7.00 (1H, d, 1.8 Hz, 40-H), 7.38 (1H, d, 1.7 Hz, 6-H), 7.51
(1H, d, 1.7 Hz, 4-H), 9.93 (1H, s, -COH); 13C NMR (125.69 MHz,
Fractionation of Oxidation Product Mixtures. Product mix-
tures were fractionated with flash chromatography or preparative HPLC
(2). Total yields (m-% to acetylated mixtures) of material recovered after
fractionation were as follows: 1, 0.31 g (86%); 2, 0.14 g (67%); 3, 0.05 g
(15%); 4, 0.08 g (89%); 5, 0.27 g (69%); 6, 0.13 g (76%); 7, 0.22 g (0.73%);
8, 0.25 g (76%). Fractionated oxidation products were analyzed with
NMR [1H, 13C, heteronuclear single quantum coherence (HSQC) and
heteronuclear multiple bond correlation (HMBC) experiments] and
high-resolution ESI-MS. Nearly all of the fractions contained only one
product.
CDCl3, 27 °C)
δ
20.04 (2-OCOCH3), 20.41 (20-OCOCH3), 21.03
(-CH2OCOCH3), 56.17 (30-OCH3), 56.33 (3-OCH3), 65.67 (-CH2-),
109.70 (4-C), 112.06 (40-C), 121.85 (60-C), 126.83 (6-C), 130.32 (1-C),
131.96 (10-C), 134.37, 134.39 (5-C, 5-C0), 137.35 (20-C), 142.74 (2-C),
151.37 (30-C), 152.21 (3-C), 167.76 (2-OCOCH3), 168.29 (20-OCOCH3),
170.54 (-CH2OCOCH3), 190.68 (-COH); ESI-MS (positive), m/z
448.1596 [M þ NH4]þ (C22H26NO9 requires 448.1602), 453.1160 [M þ
Na]þ (C22H22NaO9 requires 453.1156), 469.0902 [M þ K]þ (C22H22KO9
requires 469.0895).
Spectral Data for Selected Acetylated Oxidation Products. Tetra-
acetate of 5,50-bis(hydroxymethyl)-3,30-dimethoxy-2,20-biphenyldiol (4):
ESI-MS (positive), m/z 492.1856 [M þ NH4]þ (C24H30NO10 requires
492.1864), 497.1403 [M þ Na]þ (C24H26NaO10 requires 497.1418),
513.1145 [M þ K]þ (C24H26KO10 requires 513.1158).
Triacetate of 50-(500-carboxaldehyde-200-hydroxy-300-methoxyphenyl)-
6,9-bis(hydroxymethyl)-30,4,11-trimethoxydibenzo[d,f][1,3]dioxepin-2-spiro-40-
cyclohexa-20,50-dienone (13): 1H NMR (499.82 MHz, CDCl3, 27 °C) δ
2.14 (6H, s, aliph -OCOCH3), 2.21 (3H, s, arom -OCOCH3), 3.70
(3H, s, 30-OCH3), 3.90 (6H, s, 4-OCH3, 11-OCH3), 3.91 (3H, s, 300-
OCH3), 5.16 (4H, s, -CH2-), 5.90 (1H, d, 3.0 Hz, 20-H), 6.86 (1H, d,
3.0 Hz, 60-H), 7.02 (2H, d, 1.6 Hz, 5-H, 10-H), 7.16 (2H, d, 1.6 Hz, 7-H,
8-H), 7.45 (1H, d, 1.7 Hz, 600-H), 7.51 (1H, d, 1.7 Hz, 400-H), 9.92 (1H, s,
-CHO); 13C NMR (125.69 MHz, CDCl3, 27 °C) δ 20.50 (arom
-OCOCH3), 21.03 (two aliph -OCOCH3), 55.43 (30-OCH3), 56.05
(4-OCH3, 11-OCH3), 56.32 (300-OCH3), 66.01 (two -CH2-), 109.23 (2-
C = 10-C), 109.58 (20-C), 111.88 (400-C), 112.05 (5-C, 10-C), 120.30 (7-
C, 8-C), 125.66 (600-C), 129.82 (100-C), 134.06 (1O-C-C-C-C-3O-),
134.40 (6-C, 9-C), 134.54, 134.57 (50-C, 500-C), 138.97 (1O-C-,
3O-C), 142.18 (60-C), 143.44 (200-C), 150.67 (30-OCH3), 152.26 (300-
C), 153.04 (4-C, 11-C), 167.52 (arom -OCOCH3), 170.79 (two aliph
-OCOCH3), 178.03 (40;CdO), 190.55 (-CHO); ESI-MS (positive),
m/z 703.1987 [M þ H]þ (C37H35O14 requires 703.2021), 725.1819 [M þ
Diacetate of 1-(4-hydroxy-3-methoxyphenyl)-2-(20-methoxyphenoxy)-1-
1
ethanol (5): H NMR (499.82 MHz, CDCl3, 27 °C) δ 2.11 (3H, s, aliph
-OCOCH3), 2.31 (3H, s, arom -OCOCH3), 3.83 (3H, s, 20-OCH3), 3.85
(3H, s, 3-OCH3), 4.23 (1H, dd, 11.0 Hz, 3.9 Hz, β0H), 4.30 (1H, dd,
11.0 Hz, 8.0 Hz, βH), 6.15 (1H, dd, 8.0 Hz, 3.9 Hz, RH), 6.89-7.03 (6H, m,
ArH), 7.04 (1H, d, 1.6 Hz, 2-H); 13C NMR (125.69 MHz, CDCl3, 27 °C)
δ 20.62 (arom -OCOCH3), 21.12 (aliph -OCOCH3), 55.89, 56.00
(3-OCH3, 20-OCH3), 72.21 (βC), 73.76 (RC), 111.36 (2-C), 112.61 (30-C),
115.60 (arom C-H), 119.06 (6-C), 120.95, 122.36, 122.82 (each arom
C-H), 135.99 (1-C), 139.77 (4-C), 148.08 (10-C), 150.18 (20-C), 151.10
(3-C), 168.90 (arom -OCOCH3), 170.01 (aliph -OCOCH3); ESI-MS
(positive), m/z 392.1700 [M þ NH4]þ (C20H26NO7 requires 392.1704),
397.1238 [M þ Na]þ (C20H22NaO7 requires 397.1258), 413.0998 [M þ K]þ
(C20H22KO7 requires 413.0997).
Diacetate of 1-(4-hydroxy-3,5-dimethoxyphenyl)-2-(20-methoxyphenoxy)-
1
Na]þ (C37H34NaO14 requires 725.1841), 741.1566 [Mþ K]þ (C37H34
-
1-ethanol (7): In the same fraction with compound 18; H NMR (499.82
MHz, CDCl3, 27 °C) δ 2.12 (3H, s, aliph -OCOCH3), 2.32 (3H, s, arom
KO14 requires 741.1580).

8360 J. Agric. Food Chem., Vol. 57, No. 18, 2009
Lahtinen et al.
Diacetate of 9-carboxyaldehyde-50-(500-carboxyaldehyde-200-hydroxy-
300-methoxyphenyl)-6-hydroxymethyl-30,4,11-trimethoxydibenzo[d,f]-
[1,3]dioxepin-2-spiro-40-cyclohexa-20,50-dienone (14) or diacetate of
6-carboxyaldehyde-50-(500-carboxyaldehyde-200-hydroxy-300-methoxyphenyl)-
9-hydroxymethyl-30,4,11-trimethoxydibenzo[d,f][1,3]dioxepin-2-spiro-
40-cyclohexa-20,50-dienone (15): Numbering of atoms in NMR assignations
according to compound 14; 1H NMR (499.82 MHz, CDCl3, 27 °C) δ 2.15
(3H, s, -OCOCH3), 2.22 (3H, s, arom -OCOCH3), 3.71 (3H, s, 30-
OCH3), 3.92 (6H, br s, 300-OCH3, 4-OCH3), 3.97 (3H, s, 11-OCH3), 5.18
(2H, s, -CH2-), 5.87 (1H, d, 3.0 Hz, 20-H), 6.85 (1H, d, 3.0 Hz, 60-H), 7.05
(1H, d, 1.6 Hz, 5-H), 7.22 (1H, d, 1.6 Hz, 7-H), 7.44 (1H, d, 1.7 Hz, 600-H),
7.52 (1H, d, 1.7 Hz, 400-H), 7.55 (1H, d, 1.6 Hz, 10-H), 7.70 (1H, d, 1.6 Hz,
8-H), 9.93 (1H, s, 500-CHO), 10.05 (1H, s, 9-CHO); 13C NMR (125.69
MHz, CDCl3, 27 °C)
δ 20.48 (arom -OCOCH3), 21.04 (aliph
Figure 2. Oxidation of vanillin(1) and guaiacylic β-O-4 dimers (5 and 6) in
laccase-catalyzed reactions.
-OCOCH3), 55.49 (30-OCH3), 56.11, 56.27 (300-OCH3, 4-OCH3), 56.35
(11-OCH3), 65.89 (-CH2-), 109.14 (20-C), 110.02 (2-C = 10-C), 110.49
(10-C), 112.04 (400-C), 112.47 (5-C), 120.23 (7-C), 123.89 (8-C), 125.44 (600-
C), 129.66 (100-C), 133.25 (either of 1O-C-C-C-C-3O-), 134.58,
134.63, 134.67, 134.78, 134.87 (either of 1O-C-C-C-C-3O-, 50-C,
500-C, 6-C, 9-C), 138.99 (3O-C), 141.60 (60-C), 143.39 (200-C), 144.38
(1O-C-), 150.86 (30-C), 152.29 (300-C), 153.08 (4-C), 153.97 (11-C),
167.58 (arom -OCOCH3), 170.77 (aliph -OCOCH3), 177.91 (40-CdO),
190.50 (50-CHO), 190.84 (9-CHO); ESI-MS (positive), m/z 659.1759 [M þ
H]þ (C35H31O13 requires 659.1759), 681.1576 [M þ Na]þ (C35H30NaO13
requires 681.1579), 697.1340 [M þ K]þ (C35H30KO13 requires 697.1318).
Monoacetate of 1-(4-hydroxy-3,5-dimethoxyphenyl)-2-(20-methoxy-
phenoxy)-1-ethanone (18): In the same fraction with compound 7; 1H
NMR (499.82 MHz, CDCl3, 27 °C) δ 2.35 (3H, s, -OCOCH3), 3.87 (3H, s,
20-OCH3), 3.88 (6H, s, 3-OCH3, 5-OCH3), 5.27 (2H, s, βH), 6.86-6.98
(4H, m, ArH) 7.34 (2H, s, 2-H, 6-H); 13C NMR (125.69 MHz, CDCl3, 27
°C) δ 20.37 (-OCOCH3), 55.81 (20-OCH3), 56.31 (3-OCH3, 5-OCH3),
72.49 (βC), 105.21 (2-C, 6-C), 112.21 (30-C), 114.90, 120.86, 122.62 (40-60-
C), 132.54 (1-C), 133.37 (4-C), 147.32 (10-C), 149.75 (20-C), 152.38 (3-C, 5-
C), 168.06 (-OCOCH3), 193.78 (RC); ESI-MS (positive), m/z 361.1267 [M
þ H]þ, (C19H21O7 requires 361.1282), 383.1091 [M þ Na]þ (C19H20NaO7
requires 383.1101), 399.0823 [M þ K]þ (C19H20KO7 requires 399.0841).
Diacetate of 3-hydroxy-1-(4-hydroxy-3,5-dimethoxyphenyl)-2-(20-meth-
oxyphenoxy)-1-propanone (19): 1H NMR (499.82 MHz, CDCl3, 27 °C) δ
2.05 (3H, s, aliph -OCOCH3), 2.33 (3H, s, arom -OCOCH3), 3.75 (3H, s,
20-OCH3), 3.85 (6H, s, 2-OCH3, 6-OCH3), 4.47 (1H, dd, 12.1 Hz, 7.5 Hz,
γH), 4.72 (1H, dd, 12.1 Hz, 3.6 Hz, γ0H), 5.63 (1H, dd, 7.5 Hz, 3.6 Hz, βH),
6.83 (1H, td, 7.9 Hz, 1.5 Hz, 40-H or 50-H), 6.87 (1H, dd, 7.9 Hz, 1.5 Hz, 30-
H), 6.93 (1H, dd, 7.9 Hz, 1.5 Hz, 60-H), 7.00 (1H, td, 7.9 Hz, 1.5 Hz, 40-H or
50-H), 7.48 (2H, s, 2-H, 6-H); 13C NMR (125.69 MHz, CDCl3, 27 °C) δ
20.28 (arom -OCOCH3), 20.65 (aliph -OCOCH3), 55.63 (20-OCH3),
56.22 (3-OCH3, 5-OCH3), 64.37 (γC), 80.40 (βC), 105.76 (2-C, 6-C),
112.58 (30-C), 117.97 (60-C), 120.95, 123.44 (40-C, 50-C), 132.54 (1-C),
133.32 (4-C), 146.67 (10-C), 150.19 (20-C), 152.28 (3-C, 5-C), 167.93 (arom
-OCOCH3), 170.85 (aliph -OCOCH3), 194.24 (RC); ESI-MS (positive),
m/z 455.1284 [M þ Na]þ (C22H24NaO9 requires 455.1313), 471.1020 [M þ
K]þ (C22H24NaO9 requires 471.1052).
(R-OCOCH3), 56.12 (20-OCH3), 56.16 (3-OCH3, 5-OCH3), 101.06 (2-C, 6-
C), 112.91 (30-C), 118.88 (60-C), 121.01, 124.78 (40-C, 50-C), 128.65 (4-C),
131.63 (1-C), 133.81 (RC), 134.07 (βC), 146.37 (10-C), 150.14 (20-C), 152.27
(3-C, 5-C), 168.24 (R-OCOCH3), 168.65 (arom -OCOCH3); ESI-MS
(positive), m/z 420.1652 [M þ NH4]þ (C21H26NO8 requires 420.1653),
425.1202 [M þ Na]þ (C21H22NaO8 requires 425.1207), 441.0948 [M þ K]þ
(C21H22KO8 requires 441.0946).
Monoacetate of 1-(4-hydroxy-3,5-dimethoxyphenyl)-2-(20-methoxy-
1
phenoxy)-2-propen-1-one (22): H NMR (499.82 MHz, CDCl3, 27 °C) δ
2.35 (3H, s, -OCOCH3), 3.85 (3H, s, 20-OCH3), 3.88 (6H, s, 2-OCH3, 6-
OCH3), 4.73 (1H, d, 2.5 Hz, γH), 5.29 (1H, d, 2.5 Hz, γ0H), 6.95 (1H, td, 7.9
Hz, 1.3 Hz, 50-H), 6.98 (1H, dd, 7.9 Hz, 1.3 Hz, 30-H), 7.06 (1H, dd, 7.9 Hz,
1.6 Hz, 60-H), 7.16 (1H, td, 7.9 Hz, 1.6 Hz, 40-H), 7.40 (2H, s, 2-H, 6-H); 13C
NMR (125.69 MHz, CDCl3, 27 °C) δ 20.43 (-OCOCH3), 55.77 (20-
OCH3), 56.28 (3-OCH3, 5-OCH3), 100.55 (γC), 106.98 (2-C, 6-C), 112.83
(30-C), 121.29 (50-C), 121.63 (60-C), 125.87 (40-C), 132.76 (4-C), 133.96 (1-
C), 143.12 (10-C), 150.85 (20-C), 151.91 (3-C, 5-C), 157.66 (βC), 168.14
(-OCOCH3), 189.32 (RC); ESI-MS (positive), m/z 762.2765 [2M þ NH4]þ
(C40H44NO14 requires 762.2756), 767.2327 [2M þ Na]þ (C40H40NaO14
requires 767.2310), 783.2101 [2M þ K]þ (C40H40KO14 requires 783.2050).
RESULTS
Eight different lignin model compounds were used to study
reactions of laccases with lignin (Figure 1): vanillin (1), vanillyl
alcohol (2), syringyl alcohol (3), dehydrodivanillyl alcohol (4),
guaiacylglycol β-guaiacyl ether (5), guaiacylglycerol β-guaiacyl
ether (6), syringylglycol β-guaiacyl ether (7), and syringylglycerol
β-guaiacyl ether (8). Two different laccases were used: Melano-
carpus albomyces and Trametes hirsuta laccases. The T1 copper
redox potentials are 470 mV for M. albomyces laccase (20) and
780 mV for T. hirsuta laccase (24).
Structures of Oxidation Products. The oxidation products were
first deduced on the basis of the data from LC-MS and later
confirmed with NMR and high-resolution ESI-MS. The same
oxidation products were obtained with both laccases and, thus,
the fractionation and detailed analysis were performed with only
one of the laccases.
With vanillin (1) and guaiacylic β-O-4 dimers 5 and 6, only one
type of oxidation product, a 5-5 coupling product, was observed
(Figure2). These kinds ofproducts were highlyexpectedastypical
and well-known oxidation products in guaiacylic lignin model
compound oxidations.
Vanillyl alcohol (2) gave the most complicated distribution of
oxidation products (Figure 3). Three different kinds of 5-5
coupling products were formed: one formed straightforward
from two radicals of vanillyl alcohol (4), and the other two had
one (12) or two (9) benzylic hydroxyl groups oxidized. Vanillin
(1), a product formed when the benzylic hydroxyl of vanillyl
alcohol is oxidized, was also formed. The structures of trimeric
and tetrameric (based on mass values in MS) oxidation products
remained unfortunately unresolved, because they were in the
same fraction in nearly equimolar amounts.
Triacetate of 10,15-dihydro-1,3,6,8,11,13-hexamethoxy-2,7,12-triol-
5H-tribenzo[a,d,g]cyclononene (20): 1H NMR (499.82 MHz, CDCl3,
27 °C) δ 2.31 (9H, s, -OCOCH3), 3.76 (9H, s, 3-OCH3, 8-OCH3, 13-
OCH3), 3.88 (9H, s, 1-OCH3, 6-OCH3, 11-OCH3), 4.05 (3H, d, 13.8 Hz,
50-H, 100-H, 150-H), 4.45 (3H, d, 13.8 Hz, 5-H, 10-H, 15-H), 7.20 (3H, s, 4-
H, 9-H, 14-H); 13C NMR (125.69 MHz, CDCl3, 27 °C) δ 20.61
(-OCOCH3), 30.10 (-CH2-), 56.06 (3-OCH3, 8-OCH3, 13-OCH3),
60.85 (1-OCH3, 6-OCH3, 11-OCH3), 110.53 (4-C, 9-C, 14-C), 131.50 (2-
C, 7-C, 12-C), 125.21, 138.65 (CdC in cyclononatriene ring), 150.24 (3-C,
8-C, 13-C), 150.61 (1-C, 6-C, 11-C), 168.74 (-OCOCH3); ESI-MS
(positive), m/z 642.2554 [M þ NH4]þ (C33H40NO12 requires 642.2545),
647.2097 [M þ Na]þ (C33H36NaO12 requires 647.2099), 663.1843 [M þ
K]þ (C33H36KO12 requires 663.1838).
Diacetate of 1-(4-hydroxy-3,5-dimethoxyphenyl)-2-(20-methoxyphen-
oxy)-1-ethen-1-ol (21): 1H NMR (499.82 MHz, CDCl3, 27 °C) δ 2.32
(3H, s, R-OCOCH3), 2.33 (3H, s, arom -OCOCH3), 3.82 (6H, s, 3-OCH3,
5-OCH3), 3.88 (3H, s, 20-OCH3), 6.61 (2H, s, 2-H, 6-H), 6.82 (1H, s, βH),
6.93 (1H, td, 7.9 Hz, 1.3 Hz, 50-H), 6.96 (1H, dd, 7.9 Hz, 1.3 Hz, 30-H), 7.10
(1H, td, 7.9 Hz, 1.6 Hz, 40-H), 7.14 (1H, dd, 7.9 Hz, 1.6 Hz, 60-H); 13C
NMR (125.69 MHz, CDCl3, 27 °C) δ 20.41 (arom -OCOCH3), 20.61

Article
Dehydrodivanillyl alcohol (4, Figure 4) gave the same benzylic
J. Agric. Food Chem., Vol. 57, No. 18, 2009 8361
The only oxidation products observed for syringylic β-O-4 dimers
7 and 8 were 18 and 19 (Figure 6), resulting from benzylic
hydroxyl oxidation.
Products Resulting from Acetylation of Syringylic Compounds.
In the case of syringyl compounds a few products were observed
only after acetylation. These were formed from product mixtures
of syringyl alcohol (3) and of β-O-4 dimers 7 and 8.
The product from acetylation of oxidized syringyl alcohol (3)
was cyclotrisyringylene (20) (Figure 7). This product was also
formed in small amounts when 3 was acetylated without oxida-
tion by laccase. It is also highly probable that syringyl alcohol was
hydroxyl oxidized products 9 and 12 observed with vanillyl
alcohol (2). In addition, dibenzodioxepin-type structures, with
one or two oxidized benzylic hydroxyls, were observed (13-15).
It was impossible to determine whether 14 or 15 or both were
present in the product mixture. The tetramer found in the
oxidation of vanillyl alcohol (2) is probably like structure 14 or
15, because it has the same mass value and dehydrodivanillyl
alcohol is an oxidation product of vanillyl alcohol (2).
Oxidation products detected for syringyl alcohol (3, Figure 5)
were syringaldehyde (16) and 2,6-dimethoxy-p-benzoquinone
(17). The latter of these was observed by absorption spectrum
from HPLC, and the structure was confirmed with NMR,
because it was not ionized at the applied LC-MS conditions.
Figure 5. Oxidation products of syringyl alcohol (3) in laccase-catalyzed
reactions.
Figure 3. Oxidation products of vanillyl alcohol (2) in laccase-catalyzed
reactions.
Figure 6. Oxidation products of dimeric β-O-4 compounds containing
phenolic syringyl unit (7 and 8) in laccase-catalyzed reactions.
Figure 4. Oxidation products of dehydrodivanillyl alcohol (4) in laccase-catalyzed reactions.

8362 J. Agric. Food Chem., Vol. 57, No. 18, 2009
Lahtinen et al.
even further polymerized during acetylation because of the low
yield of material recovered after fractionation. It was actually
clearly seen that dark material remained above the silica gel and
did not dissolve during fractionation.
Products 21 and 22 were most probably formed from oxidation
products 18 and 19 and can be explained by the acidic proton next
to a carbonyl group formed in oxidation and the presence of basic
pyridine (Figure 8).
Oxidation Product Amounts Recovered from Fractionation.
Yields of oxidation products and starting material recovered
from oxidations after fractionation to the original starting
material are shown in Table 2. In cases when there was more
than one product in a fraction, amounts were estimated on the
1
basis of H NMR integrals (structures were first assured from
HSQC and HMBC spectra).
Formation of Oxidation Products as a Function of Time. For-
mationofproducts wasanalyzed asa function oftimewithHPLC
and LC-MS analyses. Analyses were performed with both lac-
cases to reveal possible differences between the enzymes.
First detection times of oxidation products for M. albomyces
laccase are in Table 3, and those for T. hirsuta laccase are in
Table 4.
Figure 7. Product detected after acetylation of laccase-oxidized syringyl
alcohol (3).
DISCUSSION
On the basis of our results, the main reactions of lignin model
compounds oxidized by laccase are formation of 5-5 dimers and
oxidation of a benzylic hydroxyl group. Only one product was
formed by these reactions with vanillin (1) and β-O-4 dimers 5-8.
From these compounds, the guaiacylic model compounds gave
5-5 coupling products and syringylic models were oxidized from
the benzylic position.
From syringyl alcohol (3), two oxidation products were formed:
the benzylic hydroxyl oxidation product syringaldehyde (16) and
2,6-dimethoxy-p-benzoquinone (17). The latter has been previously
shown to be formed from syringaldehyde through 2,6-dimethoxy-
p-hydroquinone (25), which we did not, however, detect.
Vanillyl alcohol (2) and dehydrodivanillyl alcohol (4) gave very
similar products, which is well understood by the fact that 4 is the
main product from oxidation of 2. Actually, one of the reasons
why 4 was also taken to the study was, that it revealed how some
products of 2 were formed. Identified products of vanillyl alcohol
(2) were, besides dehydrodivanillyl alcohol (4), dehydrodivanillin
(9), biphenylic compound of vanillin and vanillyl alcohol (12),
and vanillin (1). Formation of 9 and 12 from vanillyl alcohol (2)
could in principle take place in two ways: (1) two molecules of 2
are coupled to form 4, which is then further oxidized from
benzylic positions or (2) 2 is first oxidized to form 1, followed
by 5-5 coupling. The first reaction is probably dominating, because
vanillin (1) and dehydrodivanillin (9) were formed after 12 and
dehydrodivanillyl alcohol (4) gave also products 9 and 12.
The other products that were formed from dehydrodivanillyl
alcohol (4) were of dibenzodioxepin type (13-15), which had one
Figure 8. Tentative mechanism for products formed from oxidation pro-
ducts 18 and 19 as a result of acetylation.
Table 2. Yield of Material Recovered from Oxidations (Reaction Time = 24 h) after Fractionation
model
yield (%)
product
yield (%)
product
yield (%)
product
yield (%)
product
yield (%)
total yield (%)
1
2
3
4
5
6
7
8
35.1
0.7
9
43.5
2.7
78.6
<24.5b
12.6
1
4
<13.9a
2.1
9
4.7
3.0
8.6
12
2.5
2.9
c
7.6
16
9
7.5
3.4
17
12
20
13
3.1
14,15
25.6
26.7
8.0
10
11
18
19
18.7
3.6
35.9
29.5
12.0
39.5
65.6
30.4
47.5
21
22
5.7d
3.1d
62.6
a Low amount of unidentified material was present; yield calculated from the total weight of the fraction. b The true yield might be slightly higher, because structures of trimeric
and tetrameric compounds could not be resolved (yield of their fraction was 7% in m-%). c Starting material was not recovered, probably because it reacted during acetylation.
d A product resulting from a reaction of the oxidation product during acetylation.

Article
J. Agric. Food Chem., Vol. 57, No. 18, 2009 8363
or two oxidized benzylic hydroxyls. The formation of dibenzo-
dioxepins as lignin dehydrogenation products is a known reac-
tion (26,27), but as far as we know, oxidation of their side chains
as well has not beenreported before. As was mentioned before, we
could not confirm whether the structure with two benzylic
hydroxyls oxidized was 14 or 15. The “parent” dibenzodioxepin,
in which no benzylic hydroxyls are oxidized, was absent from the
product mixture. This is understandable in a way that benzylic
hydroxyl oxidation of dehydrodivanillyl alcohol (4) starts well
before any dibenzodioxepins were detected and the benzylic
oxidation may also be the most preferred reaction.
Interestingly, we were not able to find any degradation
products with syringylic β-O-4 dimers 7 and 8 as others have
done, and as was mentioned before, the two previous studies for
syringylglycerol β-guaiacyl ether (8) gave two sets of
products (9-11). Also, the products that we found for vanillyl
alcohol (2) were different from those that others found, except for
vanillin (1) (14). The reasons for different results are not very
clear, but a few that might affectthe outcomeare different enzyme
activities, reaction temperatures, substrate concentrations, etc.
The differences are not most probably due to different laccases,
because we observed the same products with both laccases used.
On the other hand, Wariishi et al. and Kawai et al. had different
products in the case of syringylglycerol β-guaiacyl ether using the
same laccase from C. versicolor (10, 11).
Compared to other reaction conditions, the greatest difference
seems to be in the amounts of the used enzyme activities. These
are, however, difficult to compare because of the different activity
determination methods. For example, Kawai et al. (13) used an
activity of 2400-4800 nkat mL-1 (activity determined with
syringaldazine) and Crestini et al. (14) used 10 U mL-1 [equal
to 167 nkat mL-1, activity measured with ABTS, 2,20-azinobis(3-
ethylbenzthiazoline-6-sulfonate)]. In our study, the activity used
was 1-2 nkat mL-1 (activity determined with ABTS). Thus, it
seems that the most important factor, when reaction product
distributions are concerned, is the amount of the used activity.
The amount is probably proportional to the concentration of
radicals formed, which most likely has an impact to the course of
the reaction.
Table 3. First Detection Times of Oxidation Products in Reaction Catalyzed
by M. albomyces Laccase
model 1 min 5 min 10 min 30 min 1 h 2 h 4 h
24 h
The products that resulted from acetylation were observed
only in the case of syringyl compounds. This is also an indication
of their higher reactivity in general compared to guaiacyl com-
pounds and can be seen also in practice, for example, in model
compound synthesis. Compound 22 was also detected by Kawai
et al. (11) with GC-MS after acetylation, but they thought it was
formed during GC analysis. Compounds similar to cyclotrisyr-
ingylene (20), for example, cyclotriveratrylene and its derivatives,
1
2
3
4a
5
6
7
8
9
4
12 1, 9, trimer, tetramer
17
13 9, 14, 15
16
12
10
18
11
19
are usually synthesized in acidic conditions [for example, Veriot
´
et al. (28)], but are apparently also formed in basic conditions.
Because there might be quite complicated reactions, as in the case
of syringyl alcohol (3) that can oligomerize or polymerize, the
high reactivity of syringylic compounds should be taken into
account when they are modified prior to analysis.
a Enzyme dosage twice as high (based on activity) as in other experiments.
Table 4. First Detection Times of Oxidation Products in Reactions Catalyzed
by T. hirsuta Laccase
model 1 min 5 min 10 min 30 min 1 h 2 h 4 h
24 h
By examining the total material yields at different stages, after
laccase oxidation and after fractionation of acetylated products,
it can be seen that the greatest yield loss for syringyl alcohol (3)
occurred during fractionation. It shows also that polymerization
occurred indeed during acetylation and not at the oxidation stage.
With vanillyl alcohol (2), dehydrodivanillyl alcohol (4), and
guaiacylglycerol β-guaiacyl ether (6), greatest yield loss occurred
during enzymatic oxidation, or most probably at the isolation
stage, indicating that they were polymerized already during
laccase oxidation.
1
2
3
4a
5
6
7
8
9
4
12
1
9, trimer, tetramer
17
9, 14, 15
16
12
13
19
10
18
11
a Enzyme dosage twice as high (based on activity) as in other experiments.
Figure 9. Previously suggested mechanisms for benzylic hydroxyl oxidation: upper mechanism taken from Kirk et al. (9) and lower from Kawai et al. (13).

8364 J. Agric. Food Chem., Vol. 57, No. 18, 2009
Lahtinen et al.
laccase with these compounds are coupling to biphenylic
(5-5) products and benzylic hydroxyl oxidation. If there is a
choice between reactions in the same system, as is the case
with guaiacylic compounds, formation of biphenylic comp-
ounds dominates and this reaction also occurs at a faster
rate. Also, high and low redox potential laccases, T. hirsuta and
M. albomyces, form in practice the same reaction products, but
there are some clear differences in the formation rates. Interest-
ingly, T. hirsuta laccase forms products more quickly from
guaiacylic models, whereas M. albomyces laccase seems to be
slightly faster with syringylic models. This may be a sign of
difference in substrate specificity between the laccases, support-
ing our earlier findings (31). On the basis of comparison to
other studies, it seems that the product distributions and the
reaction routes are dependent on the dosages of the used enzyme
activities.
Figure 10. Mechanism suggestion for benzylic hydroxyl oxidation cata-
lyzed by laccase.
As was mentioned earlier, coupling to 5-5 dimers seems to be
faster compared to benzylic hydroxyl oxidation. The other
possible products, dibenzodioxepins and p-benzoquinone, are
formed even later. Results also show that for guaiacylic com-
pounds benzylic hydroxyl oxidation is not preferred. Guaiacylic
β-O-4 dimers were not oxidized to ketones at all, and side-chain
oxidation of dehydrodivanillyl alcohol (4) took place more
rapidly than that of vanillyl alcohol (2). This result is also
consistent with a previous finding that in the early reaction stages
laccase polymerizes phenolic lignin model compounds even when
there is a mediator present, and a mediator then degrades the
formed polymer (29).
NOTE ADDED AFTER ASAP PUBLICATION
After this paper was published ASAP August 25, 2009, a
correction was made to 13C NMR data for compound 20; the
corrected version was reposted August 26, 2009.
LITERATURE CITED
(1) Baldrian, P. Fungal laccases-occurrence and properties. FEMS
Microbiol. Rev. 2006, 30, 215–242.
(2) Solomon, E. I.; Sundaram, U. M.; Machokin, T. E. Multicopper
oxidases and oxygenases. Chem. Rev. 1996, 96, 2563–2605.
(3) Thurston, C. F. The structure and function of fungal laccases.
Microbiology 1994, 140, 19–26.
There were also some differences in the reaction rates between
the two laccases used. T. hirsuta laccase formed more rapidly
dehydrodivanillin (9) from vanillin (1) and products that were
formed after primary products from vanillyl alcohol (2) and
dehydrodivanillyl alcohol (4). Tetrameric 11 from guaiacylgly-
cerol β-guaiacyl ether (6) was also formed more rapidly by
T. hirsuta laccase. M. albomyces laccase was faster in forming
19 from syringylglycol β-guaiacyl ether (8).
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As was mentioned above, the product distributions in different
studies seem to correlate with the amount of enzyme activity used.
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activity used is high, it becomes more probable that two radicals
couple in a way that degradation products are formed.
In conclusion, on the basis of studies performed with different
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Received May 8, 2009. Accepted July 27, 2009. The research was
supported by the international Ph.D. program in Pulp and Paper
Science and Technology in Finland (PaPSaT).