Laccase catalysed modification of lignin subunits and coupling to p-aminobenzoic acid

Article abstract of DOI:10.1016/j.molcatb.2013.07.014

Laccase catalysed oxidation of syringyl and guaiacyl subunits of lignin and their modification with an aromatic amine, p-aminobenzoic acid (PABA) were investigated. Laccase from Galerina sp. HC1 isolated earlier by us was used as the main catalyst, and Trametes versicolor laccase was used for comparison. Among the syringyl compounds, syringic acid and syringaldehyde were oxidised to 2,6-dimethoxy-1,4-benzoquinone, and in the presence of PABA yielded a cross-coupling imine product. The reaction with methyl syringol resulted in several products whose structures were determined. The possible oxidative coupling pathways were proposed for the formation of the identified products. Oxidation of syringol and the guaiacyl compounds resulted mainly in homooligomers by free radical mechanism, with a negligible tendency of reaction with the nucleophilic group of PABA. Similar treatment of Eucalyptus Kraft lignin, which is rich in syringyl moieties, showed the presence of identical products obtained with syringic acid and syringaldehyde.

Full text of DOI:10.1016/j.molcatb.2013.07.014

Journal of Molecular Catalysis B: Enzymatic 97 (2013) 45–53
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Laccase catalysed modification of lignin subunits and coupling to
p-aminobenzoic acid
Victor Ibrahim, Natalia Volkova, Sang-Hyun Pyo, Gashaw Mamo, Rajni Hatti-Kaul∗
Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history:
Laccase catalysed oxidation of syringyl and guaiacyl subunits of lignin and their modification with an
aromatic amine, p-aminobenzoic acid (PABA) were investigated. Laccase from Galerina sp. HC1 isolated
earlier by us was used as the main catalyst, and Trametes versicolor laccase was used for comparison.
Among the syringyl compounds, syringic acid and syringaldehyde were oxidised to 2,6-dimethoxy-1,4-
benzoquinone, and in the presence of PABA yielded a cross-coupling imine product. The reaction with
methyl syringol resulted in several products whose structures were determined. The possible oxidative
coupling pathways were proposed for the formation of the identified products. Oxidation of syringol and
the guaiacyl compounds resulted mainly in homooligomers by free radical mechanism, with a negligible
tendency of reaction with the nucleophilic group of PABA. Similar treatment of Eucalyptus Kraft lignin,
which is rich in syringyl moieties, showed the presence of identical products obtained with syringic acid
and syringaldehyde.
Received 13 March 2013
Received in revised form 22 July 2013
Accepted 23 July 2013
Available online xxx
Keywords:
Laccase
Syringyl subunits
Guaiacyl subunits
Oxidative coupling pathways
Structure elucidation
© 2013 Published by Elsevier B.V.
1. Introduction
The structure of lignin from Eucalyptus hard wood is known to be
rich in syringyl units, the S/G ratio varies between 3.2 and 6.4 in
Lignin is the second most abundant renewable polymer after
cellulose. Crude lignin is obtained in large quantities in the pulp
and paper industry, mainly as Kraft lignin and lignosulfonate. The
latter is used for several low value applications such as solid fuel,
dispersant, additive in particle board and wood– plastic composite
production, etc. The biopolymer has also been used as a starting
material for the production of some bulk chemicals, e.g. vanillin
and syringaldehyde [1]. One of the major reasons for the limited
value addition is the complexity of lignin in terms of composition
and structure, which complicates the transformation of the macro-
molecule to well defined products. Therefore, understanding of the
mechanisms of lignin biosynthesis, chemical or enzymatic modifi-
cation (e.g. oxidation), and reactivity towards different compounds
is often addressed through use of monomeric or dimeric model
compounds [2–4].
The composition of lignin varies depending on the botanical
source and the process of extraction. There are three monolignol
monomers methoxylated to different degrees: p-coumaryl alcohol,
coniferyl alcohol, and sinapyl alcohol, which are incorporated into
lignin in the form of phenylpropanoid subunits, p-hydroxyphenyl
(H), guaiacyl (G) and syringyl (S) linked in randomly organised
polymeric network. The S/G ratio is one of the important character-
istics in the selection of wood for the manufacturing of pulp [5,6].
native and 4.3 and 5.2 in Kraft lignin, as revealed by pyrolysis-gas
chromatography coupled to mass spectrometry (Py-GC–MS). The
main pyrolysis products identified in the Eucalyptus Kraft lignin
are guaiacol, syringol (or 2,6-dimethoxyphenol), 4-methylsyringol,
and syringaldehyde [7,8].
In vivo, a set of oxidative enzymes, including peroxidases and
laccases, catalyse the formation of monolignol radicals, which are
then assumed to undergo uncatalysed coupling to form the lignin
polymer [9–11]. In comparison to peroxidases, laccases have a
lower redox potential and can oxidise only phenolic structures with
low redox potential. The substrate spectrum of laccases is however
increased by the use of laccase mediator systems (LMSs) [12]. The
oxidation of Kraft lignin by laccase alone or by LMSs has long been
investigated for producing upgraded compounds [13–15]. Laccase
mediated modification of lignin has been used for the production
of safe adhesives in place of the phenol–formaldehyde based glues
[16,17], and for grafting of functional molecules to impart desired
functionalities [18]. Laccase treatment is also used for bleaching
of Kraft pulps as an eco-friendly alternative route to the classi-
cal chemical bleaching [19]. Laccases are now emerging as green
catalysts in synthetic chemistry and some interesting applications
such as synthesis of dyes, cosmetics and pharmaceutically active
compounds have been demonstrated [20–25].
The present study is a continuation of our recent work on the
reaction of laccase treated Eucalyptus Kraft lignin with soy protein
and chitosan, respectively to yield products with adhesive proper-
ties [17]. Although a number of studies have so far been reported
∗
Corresponding author. Tel.: +46 46 222 4840; fax: +46 46 222 4713.
E-mail address: rajni.hatti-kaul@biotek.lu.se (R. Hatti-Kaul).
1381-1177/$ – see front matter © 2013 Published by Elsevier B.V.
http://dx.doi.org/10.1016/j.molcatb.2013.07.014

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V. Ibrahim et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 45–53
Table 1
Chemical structures of the G-type and S-type compounds used in the study. Abbreviations are written in parentheses.
Lignin monomers
G-type
Compound number
R1
H
R2
H
General structure
Guaiacol (G)
1
R2
Vanillin (V)
Vanillic acid (VA)
Syringol (S)
Syringaldehyde (SD)
Syringic acid (SA)
4-Methyl syringol (MS)
2
3
4
5
6
7
H
H
OCH3
OCH3
OCH3
OCH3
CHO
COOH
H
CHO
COOH
CH3
R1
OCH3
S-type
OH
on laccase catalysed modification of different lignin model com-
pounds and their reaction with phenolic and aromatic compounds
[21,23–25], the minor differences in their molecular structures and
functional groups on the compounds result in varied reactivities,
reaction pathways and products formed. Herein, we investigate
laccase catalysed oxidation of different guaiacyl (G)- and syringyl
(S)-type subunits present in Kraft lignin and demonstrate the influ-
ence of a methoxy-substitution at ortho-position and also of the
functional groups on the homo- and cross-coupling reactions, and
the product profiles generated. The G-type compounds used were
guaiacol, vanillin and vanillic acid, while the S-type compounds
were syringol, 4-methyl syringol, syringaldehyde and syringic acid
(Table 1). p-Aminobenzoic acid was chosen as a model for aro-
matic amines that could represent on one hand toxic pollutants
in the environment and hence requiring detoxification, while on
the other provide possibilities of synthesising bioactive molecules.
Laccase from Galerina sp. HC1, isolated and characterised earlier by
Ibrahim et al. [26], was used for the study, and Trametes versicolor
laccase – a more well known enzyme – was used for comparison.
at 420 nm (Lambda Bio+ UV–vis spectrophotometer, PerkinElmer,
USA). The reaction mixture (1 mL) contained 20 mM sodium acetate
buffer pH 5, 1 mM ABTS, and a suitable amount of the enzyme sam-
ple. One unit of laccase activity was defined as the amount oxidising
1 ␮mol of ABTS per minute.
2.3. Oxidation and coupling reactions
For the oxidation reactions 5 mM of a model compound was
allowed to react with 0.4 U/mL laccase in 20 mM sodium acetate
buffer pH 5, at room temperature (∼22 ◦C) with mixing on rock-
ing table (Mixer 440, Swelab Instrument AB, Sweden). The reaction
was stopped by adding sodium azide to the final concentration of
40 ␮M. The coupling of equimolar amount of PABA (5 mM) to the
compounds was also investigated under similar conditions.
Oxidation of Kraft lignin (concentration 10 mg/mL) and coupling
with PABA (5 mM) was also studied as above.
2.4. Analyses
A high performance liquid chromatography (HPLC) system
(Jasco, Japan) consisting of two pumps (PU-980 Intelligent HPLC
Pumps), a degasser (DG-980-50 3-Line Degasser), a column oven
(Shimadzu CTO-10AC vp Column Oven) set at 35 ◦C, a UV detector
(UV-975 Intelligent UV-VIS Detector) and an autosampler (AS-950-
10 Intelligent Sampler) was used for the analysis of reactants and
products. Samples were diluted 4 times (or 12 times for the lignin
samples) in 40% (v/v) acetonitrile and injected (5 ␮L) into a reversed
phase C18 column (ACE 5 C18-AR, 250 × 4 mm, ACE®, Scotland)
equipped with a guard cartridge at the inlet. Elution was done
using a mobile phase made from solutions of 0.1% acetic acid (in
water) and 100% acetonitrile (ACN). Separation protocols employ-
ing different ACN gradients were developed for good resolution
of reaction components obtained with each substrate (Table 2).
Wavelengths that gave the highest absorbance were chosen after
spectrophotometric scanning of both the substrates and prod-
ucts. Many common products for different substrates were later
confirmed by using one HPLC protocol. It may be noted that the
2. Materials and methods
2.1. Chemicals
The chemicals used were procured from different sources: 2,2ꢀ-
azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium
salt (ABTS), polyethylene glycol (PEG) 8000, guaiacol (G), vanillin
(V), syringaldehyde (SD), deuterated chloroform (CDCl3), deu-
terium oxide (D2O) and ethyl acetate were from Sigma–Aldrich®
(USA), syringol (2,6-dimethoxyphenol) (S) from Acros Organics
(USA), vanillic acid (VA) and syringic acid (SA) from Alfa Aesar®
(Germany), 4-methyl syringol (MS) from SAFCTM (USA), para-
aminobenzoic acid (PABA), PEG 4000 from Fluka (Switzerland) and
sodium azide from BDH Chemicals (England), polyethylene glycol
monomethyl ether (mPEG 5000) from Shearwater polymers (USA),
series of polystyrene molecular mass standards PS 10000, 30000,
and 150000 from Fluka (Germany), silica gel 60 (40–63 ␮m),
HPLC grade acetonitrile (LiChrosolv®) and acetic acid from Merck
(Germany), chloroform (HiPerSolv®) from BDH Prolabo® (EC), and
deuterated dimethylsulfoxide ((CD3)2S O)) from Amar Chemicals
(Switzerland). Eucalyptus Kraft lignin was kindly provided by
Innventia AB (Sweden). All the reagents used were of analytical
grade.
Table 2
HPLC protocols used to follow the laccase catalysed modification of guaiacyl and
syringyl compounds.
Substrate (abbreviation)
Flow rate
(mL min−1
Detection
wavelength (nm)
Elution (%
of ACN)
)
Guaiacol (G)
Vanillin (V)
1
1
229
290
290
269
290
290
273
36–40
40
2.2. Enzyme preparation and assay
Vanillic acid (VA)
Syringol (S)
Syringaldehyde (SD)
Syringic acid (SA)
4-methyl syringol (MS)
0.5
0.5
0.5
0.5
0.8
20–40
20–40
20–40
28–40
28–40
Galerina sp. HC1 laccase was produced and partially purified as
described earlier [26], while T. versicolor laccase was purchased
from Fluka BioChemika (Switzerland) and used as such. The lac-
case activity was determined by following the oxidation of ABTS

V. Ibrahim et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 45–53
47
substrates and their respective oxidation products would have dif-
ferent molar extinction coefficients, and so the peak areas in HPLC
chromatogram would not give actual product yields. However, for
convenience an estimation of the amounts of the formed products
is based on HPLC peak areas.
Liquid chromatography coupled to mass spectrometry (LC–MS)
was used to determine the molecular mass of the products. Similar
chromatographic conditions were used as for the HPLC analysis.
The QSTARpulsar-i-Q-TOF tandem mass spectrometer (PE Sciex,
Canada) was employed. The turbospray source was set to positive
ion mode with a needle voltage +4900 V. The quadrupole system
was set to scan m/z 120–1200 in TOF-MS mode and 50–700 for
MS/MS with 1 second per scanning cycle.
Gel permeation chromatography (GPC) with chloroform as
mobile phase was used for the determination of the average molar
mass of guaiacol and syringol oxidation products. PEG 4000 and
8000, mPEG 5000, and PS 10000, 30000 and 150000 (5 mg/mL)
were used as molecular mass standards. Standards and samples
were dissolved in chloroform and injected (2 ␮L) into a GPC col-
umn (K-805, 5 ␮m, 300 mm × 8 mm, Shodex, Japan) at a flow rate of
0.5 mL min−1. A PerkinElmer Series 200 HPLC system was used with
dual detection by UV/vis detector (785A, Perkin Elmer, USA) and
Evaporative Light Scattering Detector (Alltech® 3300 ELSD, Grace
Davison Discovery Sciences, USA).
much lower (6–16 h) resulting primarily in water insoluble prod-
ucts. As an example, a comparison of V and SD (both bearing
aldehyde groups at the R2 site) oxidation is shown in Fig. 1. The
high rate of oxidation of S-compounds is attributed to the higher
number of methoxy groups (Table 1), which decreases their redox
potential and increases the electron density at the phenoxy group
allowing them to be easily oxidised by laccase [27,28]. In case of the
G-compounds, the generation of free radicals, initiated by deproto-
nation of the phenolic hydroxyl groups on the molecules, favours
their coupling with each other thus resulting in the formation of
homo-molecular polymers [24]. The products obtained after longer
reaction times with laccase are dimers, oligomers or polymers; in
an earlier report a range of oligomers (dimer to pentamer) were
obtained on treatment of vanillic acid by a laccase from the fungus
Rhizoctonia praticola [29].
Among the S-compounds, syringol behaved similar to the G-
compounds on oxidation by laccase and was totally converted to
an insoluble purple coloured product, with a molecular mass of
38.2 kDa according to GPC analysis. Treatment of syringol with a
laccase from Pycnoporus coccineus in an organic medium reported
earlier has also shown the formation of a homopolymer [30]. This
preference to undergo homo-oligomerization is directly associ-
ated with the absence of substituents on the R2 position (see
Table 1) of syringol which provides an additional site for radi-
cal formation. Unlike syringol, the oxidation products of SD and
SA were soluble, while those of MS were partially insoluble, and
all reaction mixtures were yellowish-brown in colour. The HPLC
analyses done according to Table 2 revealed one major product
peak (10) with retention times of 11.2 min, 8.3 min and 5.2 min,
in case of SD, SA and MS, respectively (Fig. 2a–c); the reten-
tion time of the product was similar when analysed using the
same HPLC protocol. The product (10) was confirmed to be 2,6-
dimethoxy-1,4-benzoquinone (2,6-DMBQ) with a molecular mass
of 168.04 g mol−1 (cal. 168.04 g mol−1) by NMR and mass spectro-
metric analyses (Table 3 and Supplementary Fig. S1). The HPLC–MS
analyses of minor oxidation products revealed masses that cor-
responded to dimeric compounds of SD and SA with m/z of 319
(cal. 318.11 g mol−1 (11)) and 335 (cal. 334.10 g mol−1 (12)), respec-
tively (Supplementary Figs. S2 and S3). The peaks separated by
18 Da appear in low m/z regions of the mass spectrum from
[M+NH4] adducts. The oxidation pattern remained unchanged dur-
ing 24 h reaction time. A HPLC pattern comparable to that of SD and
SA was obtained for MS, with product (13) eluting as a broad peak
at RT13.5 min (Fig. 2c).
2.5. Isolation of reaction products for structure determination
The products obtained in the laccase catalysed reactions with
various substrates were isolated from the reaction mixture by dif-
ferent procedures. The main oxidation product of syringic acid was
extracted from 12 mL reaction volume with 24 mL ethyl acetate
followed by rotary evaporation (Heidolph, UK), while the product
obtained from the reaction with PABA was used directly for analysis
after freeze-drying.
The major oxidation product of syringaldehyde was identified
directly by LC–MS analysis of the reaction mixture without any
extraction. The product of methyl syringol was isolated through
extraction by two volumes of ethyl acetate from a 40 mL reaction
volume followed by evaporation of the solvent under nitrogen flow.
The reactions of syringaldehyde and 4-methyl syringol with PABA
were run in 200 mL volume, and the resulting reaction mixture
was centrifuged (25 min at 2800 × g). Separation (or purification) of
the products from the supernatant and insoluble fraction was per-
formed using a silica gel column (2.5 cm diameter × 80 cm length)
and a solvent mixture of chloroform:methanol:water (65:31:4,
v/v/v) based on preliminary screening trials on TLC plates coated by
the same type of silica gel. Five millilitre fractions were collected
and analysed by reversed phase HPLC using the conditions stated
in Table 2. The fractions containing the same product were pooled
together and dried by rotary evaporation.
Based on the HPLC peak areas, it is seen that the formation of
putative oligomeric products upon laccase catalysed oxidation of
the S-type substrates is in the order: S (100%) > MS ((13), 37%) > SD
((12), 32%) > SA ((11), 15%) (Fig. 2). It is clear that the presence
and the nature of a substituent group at the para position (R2 in
Table 1) with respect to the hydroxyl group on the phenolic ring,
has a drastic influence on minimising the tendency of the phenolic
free radicals to couple with each other and instead to be further
oxidised enzymatically to form quinonoid derivatives. SA gives the
highest yield of the quinone product (10), while SD oxidation is
slightly slower, as it would first undergo oxidation to SA, and gives
higher amount of oligomer.
The purified compounds were dissolved in 750 ␮L of deuter-
ated solvent(s) and analysed by 1H NMR, 13C NMR, correlation
spectroscopy (COSY) and heteronuclear multiple bond correlation
(HMBC) using a 400 MHz NMR system (UltraShield Plus 400, Bruker,
Germany). The purity of the compounds was based on HPLC deter-
mination and ranged between 80 and 98%.
3.2. Laccase catalysed oxidative coupling of S- and G-type
compounds with PABA
3. Results and discussion
3.1. Laccase catalysed oxidation of S- and G-type compounds
The oxidation of phenolic/aromatic substrates by laccase, fol-
lowed by heteromolecular coupling can be a promising possibility
for the synthesis of new compounds [18]. PABA was used as a model
for aromatic amines for reaction with G- and S-compounds in the
presence of laccase. Incubation of PABA alone with laccase resulted
in no reaction as reported earlier even for dichloroaniline [31].
Oxidation of the S- and G-type compounds catalysed by lac-
case, and monitoring the reaction by HPLC analysis showed the
S-compounds (S, SD, SA, and MS) to be completely oxidised within
1 h while the oxidation rates of G-compounds (G, V, and VA) were

48
V. Ibrahim et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 45–53
100
80
60
40
20
0
100
(a)
(b)
80
60
40
20
0
0
3
6
9
12
15
18
21
24
0
0.5
1
1.5
2
2.5
3
Reaction time (h)
Reactiontime (h)
Fig. 1. Time course for the laccase catalysed oxidation of (a) vanillin (V) and (b) syringaldehyde (SD). Symbols indicate (a): (ꢀ) vanillin (2) with HPLC retention time (RT) of
3.4 min, (᭹) oxidation product (8) (RT 4.9), (ꢁ) oxidation product (9) (RT 5.3), and (b): (᭹) syringaldehyde (5) (RT 14.1), (ꢀ) oxidation product (10) (RT 11.2), (ꢁ) oxidation
product (11) (RT 23.2). The HPLC analyses were performed according to Table 2 and Section 2.4; in case of vanillin isocratic elution with 40% acetonitrile at a flow rate of
1 mL min−1 was employed while a gradient of 20–40% acetonitrile at a flow rate of 0.5 mL min−1 was used for separation in case of syringaldehyde. The compounds were
detected by absorbance at 290 nm.
Table 3
1H and 13C NMR chemical shifts for the oxidation product of syringic acid by Galerina sp. laccase.
Atom number
1
Assignment of chemical shifts ((CD3)2S O)
Structure
1H NMR
13C NMR
5.97 (sa, 2H)
187.58
2
3
4
5
107.52
157.73
175.01
56.93
3.75 (s, 6H)
a
Singlet.
Incubation in the presence of G-molecules led to almost negligi-
ble cross-coupling reactions; V did not react at all while extremely
low consumption of PABA was observed during reaction with G
and VA giving 5 minor products in agreement with earlier reports
involving reaction of chloroaniline with G using R. praticola and T.
versicolor laccase [32] and of PABA with VA catalysed by R. praticola
laccase [33].
2,6-DMBQ was isolated after the oxidation reaction and then incu-
bated with PABA, or if the latter was added to the reaction mixture
after oxidation of SA and the enzyme inactivated by addition of
sodium azide. Hence, the presence of active laccase seemed to be
Table 4
HPLC peak area percentage of products obtained during oxidative coupling (over
3 h) of syringaldehyde (SD), syringic acid (SA) and 4-methyl syringol (MS), respec-
tively, with p-aminobenzoic acid (PABA) catalysed by Galerina sp. laccase (GLac) or
T. versicolor laccase (TLac). The HPLC protocols for the different substrates listed in
Table 2 were used.
Even syringol (S) did not exhibit any coupling with PABA and
lower consumption of S was noted in comparison to the system
without the amine. Inclusion of PABA in the reaction mixture with
SD, SA and MS, respectively, showed significant reduction in the
major oxidation product (10) and disappearance of the oligomers
(11, 12 and 13), while a new major product and other minor ones
appeared as revealed by HPLC (Fig. 2aꢀ–cꢀ and Table 4). Mikolasch
and Schauer have earlier distinguished homo-molecular coupling
products as well as hetero-molecular hybrid dimers during laccase
catalysed reaction between substrates with different structures
[34]. In our experiments, the reaction was most efficient with SA,
and PABA was completely consumed within 3 h. In case of SD, more
product peaks appeared on the chromatogram at the expense of
the amount of the major product peak, while more 2,6-DMBQ and
even PABA was left unconsumed at the end of the reaction. Reac-
tion of PABA with MS led to the formation of several products in
varying amounts; TLac displayed significantly higher reaction effi-
ciency than GLac. The difference in catalytic efficiency of two other
laccases from M. albomyces and T. hirsuta has earlier been attributed
to two factors: (i) the difference in redox-potential between T1 cop-
per of the respective laccase and the oxidised substrate, and (ii) the
specificity towards that substrate (Kcat/Km) [28].
Substrates
Peak retention time in
Peak area (%)
min (compound number)
GLac
TLac
SD + PABA
6.4 (PABA)
7.1
11.2 (10)
20.6 (14)
22.1
1.6
5.8
21.4
30.6
2.4
8
9.8
5.7
29.3
22.9
2.1
23.4
3.9
24.0 (15)
28.7
25
0
11.8
11.5
SA + PABA
MS + PABA
8.3 (10)
13.5
14.4 (14)
18.4
4.7
5.2
83.4
6.1
6.9
0.2
86.7
5.8
3.6
4.0 (PABA)
4.3
6.1
8.5
11.0 (17)
13.0 (15)
16.5 (18)
19.5
0.8
57.2
0
14.3
1.5
10.1
4.9
4.1
0.4
11.5
5.9
14.7
0
3
When PABA was replaced with para-hydroxybenzoic acid, no
coupling with SA was noted even at a lower reaction pH (pH 3),
which highlights the importance of the stronger nucleophilicity of
the amino group for coupling. Moreover, no product was detected if
0.6
15.8
0
8

V. Ibrahim et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 45–53
49
Fig. 2. HPLC chromatograms of the reaction mixtures obtained on laccase catalysed oxidation of (a) syringaldehyde, (b) syringic acid and (c) 4-methyl syringol, and their
coupling to PABA (aꢀ–cꢀ). The HPLC protocols for the different substrates listed in Table 2 were used. The different curves correspond to pure substrate (- - -), substrate oxidised
by GLac (—) and TLac (· · ·· · ·). The reaction time was 3 h.
essential for the coupling to occur. These observations suggest that
the aromatic amine reacts directly with the decarboxylation inter-
mediate generated by the enzymatic reaction prior to the formation
of the stable quinone product (10). The latter is presumably formed
by the addition of water molecule to the intermediate oxidation
product instead of the aromatic amine.
Table 5 presents the NMR data (chemical shifts assignments
as well as short and long range homo- and hetero-nuclear cou-
pling) and structure elucidation of the major products obtained in
the laccase catalysed reactions with SD and SA, and some prod-
ucts from MS with PABA, and Scheme 1 proposes the reaction
pathways involved leading to the formation of these products. Cou-
pling between SA (6) and PABA occurs most likely by a nucleophilic
attack of the latter’s amino group onto the decarboxylated interme-
diate generated by the action of laccase. Earlier reports suggested
however that the attack is made on the carbonyl group of the
quinonic oxidation products, as reflected in the reaction between
SA or VA with aniline [33,35]. Tatsumi et al. [33] reported on the for-
mation of a Michael’s type adduct in the reaction between aniline
and protocatechuic acid which lacks an ortho-methoxy group.
The reaction with SD (5) also led to the formation of com-
pound (14) and an additional compound (15), which appears to

Table 5
1H and 13C chemical shifts assignments for the main products formed (in Fig. 2) during laccase catalysed reaction of S-type compounds with PABA for 3 h. Supporting information from COSY and HMBC NMR experiments is also
provided and corresponding structures elucidated.
Compound
number
Atom number
Assignment of chemical shifts ((CD3)2S O)
Structure
1H
13C
COSY
HMBC
14
COOH
12.72 (ba, 1H)
167.47
1
2
3
4
5
6
127.20
130.94
120.78
154.34
157.55
111.62 98.83
7.98 (db, Jc4.72 Hz, 2H)
6.98 (d, J4.72 Hz, 2H)
2, 4, -COOH
1, 3
6.52 (d, J2.16 Hz, 1H),
5.97 (d, J2.16 Hz, 1H)
6, 8
7
7
8
9
155.71, 154.96
176.09
56.56, 56.27
3.82 (s, 3H), 3.59 (s, 3H)
15
COOH
169.39
132.30
130.53
120.70
153.33
157.97
112.49
94.88
1
2
3
4
7.93 (d, J8.4 Hz, 2H)
6.87 (d, J8.4 Hz, 2H)
3
2
4
1, (3)
5
6
6.51 (s, 1H)
6.23 (s, 1H)
(6ꢀ)
(6)
8
8
6ꢀ
7
154.35
140.11
178.01
56.37
7ꢀ
8
9
3.86 (s, 3H)
(6)
7
10
11
12
13
NH
COOH
141.87
120.59
130.34
130.93
7.17 (d, J8.4 Hz, 2H)
7.77 (d, J8.4 Hz, 2H)
12
11
13, (11)
10
8.69 (s, 1H)
163.40

Table 5 (Continued)
Compound
number
Atom number
Assignment of chemical shifts ((CD3)2S O)
1H
Structure
13C
COSY
HMBC
17
COOH
1
168.06
117.87
2
3
4
5
6
7
8
9
7.65 (d, J8.8 Hz, 2H)
6.61 (d, J8.8 Hz, 2H)
131.44
111.64
152.86
46.78
129.66
105.46
148.41
134.89
56.42
3
2
2, 4, -COOH
1, 3
4.20 (d, J6.0 Hz, 2H)
6.63 (s, 2H)
NH , (3)
4, 7
5, 7, 9
8.23 (b, 1H, low intensity) OH)
3.73 (s, 6H)
10
NH
8
6.86 (td, J5.6 Hz, 1H)
5
18e
COOH
167.86
1
2
3
4
5
6
7
8
117.93
131.34
111.83
152.45
54.56
128,42
104.62
148,43
134.74
56.34
7.69 (d, J8.8 Hz, 2H)
6.75 (d, J9.2 Hz, 2H)
3
2
2, 4, COOH
1
4.60 (s, 4H)
6.44 (s, 4H)
(7)
(5)
4, 5, 6, 7, (9)
5, 7, 9
9
10
OH
3.66 (s, 12H)
8.23 (b, low intensity)
8
a
Broad.
Doublet.
Coupling constant.
Triplet.
b
c
d
e
This compound was dissolved in a mixture of deuterated solvents CDCl3:(CD3)2S O:D2O (25:75:15).

52
V. Ibrahim et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 45–53
O
OH
(PABA)
(a)
O
R
O
H2N
OH
H3C
CH3
O
O
N
O
Laccase, O2
OH
H3C
CH3
R = H
R = OH
O
O
(5)
(6)
(14)
(b)
O
OH
O
OH
R´
PABA
PABA
O
H3C
CH3
O
O
N
O
N
O
OH
Laccase, O2
Laccase, O2
OH
H3C
CH3
H3C
O
O
O
R´ = COH
R´ = CH3
N
(5)
(7)
H
(14)
(15)
CH3
(c)
O
OH
O
OH
CH3
H3C
O
O
CH3
OH
O
(16)
.
PABA
Laccase, O2
H3C
CH3
H N
CH2
O
O
N
Laccase, O2
H2
C
CH2
.
H3
C
CH3
CH2
O
O
H3C
CH3
(7)
H3C
CH3
O
O
O
O
HO
OH
H3
CH3
C
O
O
OH
O H
(17)
(18)
(16)
Scheme 1. Oxidative coupling of syringaldehyde (5), syringic acid (6), and 4-methyl syringol (7) with p-aminobenzoic acid (PABA) catalysed by laccase.
be formed by addition of a second molecule of PABA to (14) via
demethoxylation and nucleophilic attack (Scheme 1b). In case of
MS (7), several products were formed. The compounds (15), (17)
and (18) were identified to be among the main products formed
(Scheme 1c). Compound (18) would most likely be formed by addi-
tion of one PABA to the oxidation product (16) of MS (with an
m/z = 167 (cal. 166.06) as detected by mass spectrometry), while
coupling of another (16) to (17) results in the formation of the
heterotrimer (18).
Among the four products (14, 15, 17 and 18), only (17) has been
produced earlier by chemical synthesis using hazardous solvents
and high temperature, and has been reported to possess activ-
ity as a hypolipidemic drug [36]. The other three compounds are
completely new structures; related structures with a C–N linkage
produced by laccase catalysed reaction between aromatic or cyclic
amines and substituted phenolic compounds have been shown to
display antibiotic activity [37–39].
Preliminary experiments were also conducted with Eucalyp-
tus Kraft lignin in place of the S-/G-compounds. Treatment with
laccase showed the presence of SA, SD and 2,6-DMBQ in the sol-
uble fraction of the reaction mixture, indicating that a fraction of
the Kraft lignin was degraded to its constitutive monomers. The
reaction with PABA resulted in 4 products, with the major prod-
uct being compound (14). Higher conversion of PABA was obtained
with TLac, probably due to its higher redox potential as mentioned
above.
4. Conclusion
The study shows that the difference in the number of methoxy
groups in guaiacyl- and syringyl subunits of lignin results in
significant difference in their reactivity on treatment with lac-
case. The G-type compounds with one methoxy group are prone
to homo-oligomerization due to coupling of phenolic radicals,
while one extra methoxy group in S-type compounds makes
them (except syringol) more prone to rapid oxidation with
laccase and – reaction with other aromatic molecules with a
nucleophilic moiety. Furthermore, the substitution on the para
position of the phenolic hydroxyl on the S-compounds led
to the formation of diverse hetero-oligomeric products. Three
new products (14, 15 and 17) were identified, which would
be interesting to test for bio-activity. Laccase catalysed synthe-
sis of product (15), identified as a hypolipidemic agent could
provide a green alternative to the conventional chemical synthe-
sis.
Acknowledgments
The Swedish Foundation for Strategic Environmental Research
(MISTRA) is thanked for financing the project. Dr Martin Hedström’s
expert help with the mass spectrometry analyses is highly appre-
ciated.

V. Ibrahim et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 45–53
53
Appendix A. Supplementary data
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Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.molcatb.
2013.07.014.
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