Biotransformation of resveratrol: Synthesis of trans-dehydrodimers catalyzed by laccases from Myceliophtora thermophyla and from Trametes pubescens

Article abstract of DOI:10.1016/j.tet.2003.10.117

Laccases from different sources have been used, for the first time, for the selective oxidation of the stilbenic phytoalexin trans-resveratrol (3,5,4′-trihydroxystilbene, 1a) on a preparative scale. Specifically, the enzymes from Myceliophtora thermophyla

Full text of DOI:10.1016/j.tet.2003.10.117

Tetrahedron 60 (2004) 595–600
Biotransformation of resveratrol: synthesis of
trans-dehydrodimers catalyzed by laccases from
Myceliophtora thermophyla and from Trametes pubescens
Silvia Nicotra,a Maria Rita Cramarossa,b Adele Mucci,b Ugo Maria Pagnoni,b Sergio Rivaa
and Luca Fortib
,
*
aIstituto di Chimica del Riconoscimento Molecolare (ICRM), CNR, Via Mario Bianco 9, 20131 Milano, Italy
`
Dipartimento di Chimica, Universita di Modena e Reggio Emilia, Via Campi 183, 41100 Modena, Italy
b
Received 8 August 2003; revised 2 October 2003; accepted 24 October 2003
Abstract—Laccases from different sources have been used, for the first time, for the selective oxidation of the stilbenic phytoalexin trans-
resveratrol (3,5,40-trihydroxystilbene, 1a) on a preparative scale. Specifically, the enzymes from Myceliophtora thermophyla and from
Trametes pubescens gave the dehydrodimer 2a in 31 and 18% isolated yields, respectively. These results compare favorably with the reported
data for the chemically catalyzed dimerization of 1a (18% yields). The antioxidant properties of 2a have also been investigated.
q 2003 Elsevier Ltd. All rights reserved.
1. Introduction
exhibit a wide-array of biological activities, such as
antimicrobial, anti-HIV, anti-inflammatory;5 they are also
reported to be potentially important cancer chemoprotective
agents, being able to inhibit cellular events associated with
carcinogenesis.5,6 Due to their occurrence in various edible
vegetables and fruits, these compounds may represent
nutritionally important constituents.
trans-Resveratrol (3,5,40-trihydroxystilbene, 1a) is a
stilbenic phytoalexin produced by plants via a metabolic
sequence induced in response to biotic or abiotic stress
factors.1 It has been found in a multitude of dietary plants,
such as pines, legumes and grapevines: thus, relatively high
concentrations of this compound are present in grape juice
and in red wine.2 Resveratrol is one of the phenolic
compounds present in wine that could be responsible for the
decrease in coronary heart disease observed among wine
drinkers (French paradox).3 Growing evidence suggest that
resveratrol plays a role in the prevention of carcinogenesis.4
In addition to resveratrol, its oligomers, the so-called
‘viniferins’, have also been found in plants5 as a result of
infection or stress. Oligomeric stilbenes are reported to
Despite that, as many of these compounds are exclusively
obtained by extraction from natural sources, the studies of
their biological properties are limited by their very scarce
availability.
Few synthetic approaches to the oxidative coupling of
resveratrol have been reported. By treating 1a with 2,2-
diphenyl-1-picrylhydrazyl free radical (DPPH) a major
Keywords: Resveratrol; Laccase; Polyphenols; Biotransformation.
*
Corresponding author. Tel.: þ39-59-2055110; fax: þ39-59-373543; e-mail address: forti.luca@unimore.it
0040–4020/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2003.10.117

596
S. Nicotra et al. / Tetrahedron 60 (2004) 595–600
product was isolated in 18% yield and identified as trans-
resveratrol dehydrodimer 2a.7 On the other hand, the
treatment of 1a with FeCl3 failed to give 2a, affording the
isomeric dimer 1-viniferin (3) as the sole product.8
drawback of these biotransformations is the extensive
polymerization that may arise from the radical mechanism
of the oxidative process,23 producing a complex mixture of
poly-phenolic oligomers.
Different studies have shown that resveratrol is transformed
in vivo (for instance by incubating 1a with Botrytis cinerea
cultures) into various oxidized products (dimers), such as
2a.9,10 On the other hand, to the best of our knowledge, there
are only four reports on the in vitro biocatalyzed oxidation
of 1a: the first two describe a horseradish peroxidase–
hydrogen peroxide promoted dimerization of 1a to 2a,11 the
third reports on the oxidative degradation of 1a by action of
a soybean lipoxygenase (products structures not identi-
fied),12 while the fourth describes the performances of a
purified laccase isolated from a B. cinerea strain.13 The
latter reaction was run on analytical scale, monitored by
HPLC and the formed product was identified as 1-viniferin 3
simply by comparison with the HPLC elution time of an
authentic sample.
At variance, this has not been the case with trans-
resveratrol. Following an optimized protocol for the use of
laccases in organic solvents,24 resveratrol (a compound that
is poorly soluble in aqueous buffer solutions) has been
dissolved in n-BuOH (pre-saturated with 25 mM phosphate
buffer, pH 6.5) and submitted to the action of the laccase
from Myceliophtora thermophyla supported on glass beads.
The suspension has been shaken at 45 8C for 4 days and the
formation of a more polar product has been observed by
TLC. Similarly to resveratrol, this compound exhibits an
intense purple color under UV irradiation (254 nm) on TLC
plates: this information indicates that the new molecule
maintains the extended conjugation of the stilbene system.
The product has been isolated by flash chromatography in
31% yield and identified as the trans-dehydrodimer 2a by
EI-MS and extensive mono- and bi-dimensional NMR
analysis.
As a part of our general interest in biocatalysis14 and, more
specifically, in enzyme-catalyzed carbon–carbon bond
formation,15 we have recently started an investigation on
the synthetic potential of laccases. In this paper we report on
the preparative-scale oxidation of trans-resveratrol and of
some of its derivatives by action of two laccases, isolated
from Mycelyopthora thermophyla and from Trametes
pubescens, respectively.
The EI-MS shows a molecular ion peak at m/z 454,
corresponding to the structure of a dehydrodimer of
resveratrol (m/z calcd for C28H22O6 is 454), while 1H
and 13C NMR data, reported in Table 1, suggest the
substituted dihydrobenzofurane structure 2a, in accordance
with the values reported in literature.10b To unambiguously
assign the relative stereochemistry of the stereogenic
centers of C-7 and C-8, a detailed NMR study has been
performed.
2. Results and discussion
Initial inspection of the 1H NMR spectrum of 2a in DMSO-
d6 has shown the presence of three different types of
aromatic OH’s at 9.14, 9.17 and 9.48 ppm (integral: 2H, 2H
and 1H, respectively) suggesting a penta-phenolic structure.
Additionally, the 1H NMR spectrum exhibits signals due to
one 4-hydroxybenzene moiety (multiplet at 6.85 ppm, AA0
system, 2H, and 7.24 ppm, BB0 system, 2H), to two 3,5-
dihydroxybenzene moieties (doublet at 6.20 ppm, 2H;
triplet at 6.26 ppm, 1H; triplet at 6.29 ppm, 1H; doublet at
6.54 ppm, 2H), to two trans olefinic protons (doublets at
6.90 and 7.06 ppm), and to two aliphatic protons (doublets
at 4.47 and 5.45 ppm).
Laccases are oxidoreductases (the so-called ‘blue oxi-
dases’), widely distributed in fungi and in some bacteria and
higher plants and able to catalyze the oxidation of various
phenolic compounds at the expense of molecular oxygen.16
The active site of these enzymes consists of a metallic
cluster containing four copper atoms, all of them being
involved in the redox process via a radical cyclic
mechanism.17 The oxidation of a specific phenolic deriva-
tive depends on the redox potential difference between the
target compound and the so called ‘T1’-copper. Laccases
show an exceptional substrate versatility and therefore are
potentially suitable biocatalysts for the mild oxidation of
organic compounds. So far, the main limitation to their use
has been their scarce availability. However, mainly to
satisfy the demand for new ‘green’ processes by the textile
and pulp and paper industries, some of these enzymes have
recently been cloned and overexpressed and are becoming
commercially available.18
1
The assignments have been made on the basis of H, 13C-
inverse detected single-quantum (HSQC)25 and multiple-
bond (HMBC)26 correlation experiments. The long-range
correlation between H-2,6 at 7.24 ppm and C-7 at
94.72 ppm, and between H-10,14 at 6.20 ppm and C-8 at
58.52 ppm allows to establish that the A ring is bonded to
C-7 whereas the B ring is bonded to C-8. The low, but
detectable, observed correlation between H-7 at 5.45 and
C-40 at 161.3 ppm confirms the presence of the fused
oxygenated five-member ring and is in favor of a dihedral
angle H-7–C-7–O–C-40 close to 908.
Literature reports on the synthetic applications of laccases
can be divided into two groups. In the first one, these
enzymes are used to oxidize a suitable chemical compound
(i.e., Tempo), which in turn acts as a ‘mediator’ for the
oxidation of the synthetic target;19 one example is the
laccase-mediated production of aldehydes from the corre-
sponding alcohols.20
The value of 3J(H-7,H-8) measured from 1H NMR spectrum
(7.97 Hz) suggests a predominant conformation with the
two H-7 and H-8 protons in a pseudo trans-axial arrange-
ment, whereas the 3J(H-70,H-80) is 16.33 Hz, as expected for
a double bond with a trans configuration. The distinction
The second group of reports describes the direct oxidation
of phenolic derivatives, such as estradiol21 and penicillin
X,22 to give dimers and higher oligomers. The main

S. Nicotra et al. / Tetrahedron 60 (2004) 595–600
Table 1. H and 13C NMR data for compounds 2a and 2b
H (mult., J Hz)
597
1
d
dC
2aa,b
2bc
2ab
2bc
C-1
133.28
129.23
116.85
159.10
129.47
127.89
115.29
157.65
H-2,6
H-3,5
7.24 (BB0 multiplet)
6.85 (AA0 multiplet)
7.20 (BB0 multiplet)
6.76 (AA0 multiplet)
C-2,6
C-3,5
C-4
H-7
H-8
5.45 (d, 7.96)
4.47 (d, 7.96)
5.54 (d, 8.13)
4.76 (d, 8.13)
C-7
C-8
C-9
94.72
58.52
145.91
92.08
54.80
143.80
H-10,14
H-12
6.20 (d, 2.16)
6.29 (t, 2.16)
6.92 (d, 2.10)
6.95 (t, 2.10)
C-10,14
C-11,13
108.11
160.44
118.74
151.08
C-12
C-10
103.08
132.43
114.79
130.08
H-20
H-30
7.43 (dd, 8.26, 1.63)
6.87 (d, 8.26)
7.47 (dd, 8.45, 1.36)
6.94 (d, 8.45)
C-20
C-30
C-40
C-50
129.29
110.81
161.30
132.81
128.24
109.67
159.14
130.76
H-60
H-70
H-80
7.26 (broad s)
7.06 (d, 16.33)
6.90 (d, 16.33)
7.24 (broad s)
7.26 (d, 16.35)
7.02 (d, 16.35)
C-60
C-70
C-80
C-90
124.62
129.80
127.92
141.46
123.07
130.24
124.07
139.79
H-100,140
6.54 (d, 2.10)
6.26 (t, 2.10)
7.21 (d, 2.10)
C-100,140
C-110,130
106.42
160.22
116.73
151.02
H-120
11,13-CH3
110,130-CH3
6.82 (t, 2.10)
2.22
2.26
C-120
103.43
114.29
20.74
20.74
168.80
168.90
11,13-CH3
110,130-CH3
11,13-CvO
110,130-CvO
OH
9.53
a
Single broad signal due to hydroxy protons was found at 8.20.
Aceton-d6
DMSO-d6.
b
c
between the ethylenic protons has been made through the
analysis of the H,C long-range correlation found in the
HMBC spectrum: the signal of H-70 at 7.06 ppm shows a
correlation with the carbon signal at 124.62 ppm, directly
bonded to H-60 (i.e., C-60), whereas the signal due to H-80 at
6.90 ppm is correlated to the carbon signal0at 106.42 ppm,
directly bonded to H-100, 140 (i.e., C-100,14 ).
stereoisomer (21418). In this way the isolated product has
been unambiguously identified as the compound 2a. The
same product could be isolated, albeit in a lower yield
(18%), when the bio-catalytic oxidation of 1a was
performed using a laccase from Trametes pubescens in a
biphasic system acetate buffer—AcOEt.24
The structure of 2a indicates that the dimerization reaction
proceeds exclusively through the phenoxy radical derived
from the 40-OH of 1a. As further evidence, it has been found
that the 40-O-methyl resveratrol derivative 1c is unreactive
under the same reaction conditions, whereas the 3,5-
diacetylated derivative 1b is oxidized to give the expected
trans-dehydrodimer 2b, whose structure has been assigned
The relative stereochemistry of C-7 and C-8 carbons has
been proposed on the basis of the analysis of the NOESY27
NMR spectrum (400 ms mixing time) and of the computer-
aided energy-minimized stereostructure obtained.
The NOESY spectrum clearly shows the absence of NOE
in-phase cross-peaks between H-7 and H-8, and the
presence of positive in-phase cross peaks between H-7
and H-10,14 (of comparable intensity with respect to those
between H-7 and H-2,6) and between H-8 and H-2,6 (of
comparable intensity with respect to those between H-8 and
H-10,14), thus confirming that H-7 and H-8 are in a
prevalent trans-axial arrangement. The remaining NOE
observed (i.e., between H-10,14 and H-60) is in accordance
with the conclusions drawn from the H,C correlation
experiments. According to the modified Karplus-equation
reported by Haasnoot,28 the 3J(H-7,H-8) value found for the
compound 2a (7.96 Hz) would result in a dihedral angle of
about 21408, which is in good accordance with that found
from the computer-aided calculation for the trans-dia-
1
by H and 13C NMR analysis using HMBC, NOESY and
ROESY experiments (Table 1).
The antioxidant activity of 2a has been determined by the
DPPH reduction method,29 by plotting, as indicated in the
Section 4, the remaining percentage of DPPH as a function
of the molar ratios of 2a over DPPH. The resveratrol dimer
reacts with DPPH, reaching a steady state after about 3 h.
An EC50 of 5.2^0.4 (mmol 2a/mmol DPPH) has been
determined, which is slightly greater than that observed for
resveratrol [4.1^0.3 (mmol/mmol DPPH)] and comparable
with that of its biologically active analogue pterostilbene
[5.0^0.4 (mmol/mmol DPPH)].4c

598
S. Nicotra et al. / Tetrahedron 60 (2004) 595–600
3. Conclusions
mixed and left to dry at room temperature with occasional
mixing for nearly 2 days.
In this paper we have shown the usefulness of isolated
laccases for the synthesis of the resveratrol dimer 2a in good
yields and under very mild reaction conditions (atmospheric
air, enzyme, solvent). To our knowledge, this is the first
report on the preparative-scale oxidative dimerization of 1a
catalyzed by this group of oxidative enzymes. Due to the
wide-array of biological activity exhibited by resveratrol
oligomers, the compounds synthesized may serve as lead for
the development of new drugs and as nutraceuticals.
Furthermore, the bio-oxidation of resveratrol, affording
dimeric product(s), is interesting not only from a chemical
point of view but it has an utmost importance, for example,
for the wine-making industry. The use of enzyme
preparations in the wine-making industry to improve the
quality of wine is well established:30 for example, the color
stabilization by removal of excess phenols using laccase has
been proposed. Immobilization of enzymes for their use in
wine making is another interesting possibility.
4.2.1. Oxidation of resveratrol catalyzed by M. thermo-
phyla laccase. Resveratrol (100 mg) was dissolved in 6 ml
of n-butanol saturated with 25 mM phosphate buffer, pH
6.5. 200 mg (120 U) of M. thermophyla laccase supported
onto glass beads was added and the suspension was shaken
at 45 8C for 4 days. The enzyme and a little amount of a
brown precipitate were separated by filtration, the solvent
was evaporated and the crude residue was purified by
flash chromatography (eluent: petrol 40–60 8C/ethyl
acetate/methanol¼6:4:0.5) to give 31 mg of 2a (31%
1
yield). Pale yellow amorphous solid. H NMR (acetone-
d6, TMS): 4.47 (d, J¼8.0 Hz, 1H, H-8), 5.45 (d, J¼8.0 Hz,
1H, H-7), 6.20 (d, J¼2.2 Hz, 2H, H-10,14), 6.26 (t,
J¼2.1 Hz, 1H, H-120), 6.29 (t, J¼2.2 Hz, 01H, H-12), 6.54
(d, J¼2.1 Hz, 2H, H-100,140), 6.85 (AA multiplet, 2H,
H-3,5), 6.87 (d, J¼8.3 Hz, 1H, H-30), 6.90 (d, J¼16.3 Hz,
1H, H-80), 7.06 (d, J¼16.3 Hz, 1H, H-70),0 7.24 (BB0
multiplet, 2H, H-2,6), 7.26 (broad s, 1H, H-6 ), 7.43 (dd,
J¼8.3, 1.6 Hz, 1H, H-20). 13C NMR (acetone-d6, TMS):
58.52, 94.72, 103.08, 103.43, 106.42, 108.11, 110.81,
116.85, 124.62, 127.92, 129.23, 129.29, 129.80, 132.43,
132.81, 133.28, 141.46, 145.91, 159.10, 160.22, 160.44,
161.30.
As regards the antioxidant activity of 2a, this dimer is able
to scavenge DPPH radical with an EC50 value comparable
with that observed for resveratrol and its analogues,
disclosing a preliminary positive information on the
bioactivity of 2a. Being easily scaled up, this simple and
selective biotransformation can afford the dimeric pro-
duct(s) in sufficient amounts to allow the evaluation of the
biological activity of compound 2a and of other resveratrol
derivatives (i.e., 2b): in particular, the protection against
lipid peroxidation, the effects on cell proliferation and the
ability to inhibit replicative DNA polymerase are under
investigation in our laboratory and will be reported in due
course.
MS (EI, 70 eV): m/z (%)¼454 (39, Mþ); 228 (100); 179
(93); 123 (82).
Anal. calcd for C28H22O6: C, 74.00%; H, 4.88%; O,
21.12%. Found C, 74.06; H, 4.93.
4.2.2. Oxidation of resveratrol catalyzed by T. pubescens
laccase. Resveratrol (100 mg) was dissolved in 4 ml of ethyl
acetate, while the laccase (110 U) was dissolved in 4 ml of
20 mM acetate buffer, pH 4.5. The biphasic system was
shaken at room temperature for 24 h. The organic solvent
was evaporated and the crude residue was purified by
flash chromatography (eluent: petrol 40–60 8C/ethyl
acetate/methanol¼6:4:0.5) to give 18 mg of 2a (18% yield).
4.2.3. Attempted oxidation of 40-0O-methyl-resveratrol
(1c) by M. thermophyla laccase. 4 -O-Methyl-resveratrol
(1c, 3 mg) was dissolved in 0.5 ml of n-butanol saturated
with 25 mM phosphate buffer, pH 6.5. 50 mg (30 U) of
M. thermophyla laccase supported onto glass beads were
added and the system was shaken at 45 8C. No reaction was
observed after 48 h.
4. Experimental
4.1. Materials and methods
Laccase from Myceliophtora thermophyla was from Novo-
zymes A/S, Denmark, while the laccase from Trametes
pubescens was provided by Professor Haltrich.31 Com-
pounds 1b32 and 1c4c were synthesized following the
procedures reported in the literature. TLC: precoated silica
gel 60 F254 plates (Merck). Flash chromatography: silica gel
60 (70–230 mesh, Merck). Mass spectra were acquired with
a combined HPLC particle beam MS-engine. NMR spectra
were recorded in aceton-d6 or DMSO-d6 solutions at
400 MHz and the chemical shift reported are in ppm
relative to tetramethylsilane as external standard. Energy
minimized stereostructure of 2a was obtained by MMþ
calculation using HyperChem 5.0 molecular modeling
program. Antiradical activity by DPPH: spectrophotometric
data were acquired using a UV/VIS spectrometer.
4.2.4. Oxidation of 3,5-di-O-acetyl-resveratrol (1b) by M.
thermophyla laccase. 3,5-Di-O-acetyl-resveratrol (1b,
35 mg) was dissolved in 8 ml of n-butanol saturated with
25 mM phosphate buffer, pH 6.5. 150 mg (90 U) of the
M. thermophyla laccase supported onto glass beads was
added and the system was shaken at 45 8C for 4 days. The
enzyme and a little amount of a brown precipitate were
separated by filtration, the solvent was evaporated and the
crude residue was purified by flash chromatography (eluent:
petrol 40–60 8C/ethyl acetate/methanol¼75:25:5) to give
9 mg of 2b (26% yield). Pale yellow oil. 1H NMR (acetone-
d6): 2.22 (s, 3H, CH3), 2.26 (s, 3H, CH3), 4.76 (d, J¼8.1 Hz,
1H, H-8), 5.54 (d, J¼8.1 Hz, 1H, H-7), 6.76 (AA0 multiplet,
4.2. Adsorption of the laccase from M. thermophyla on
glass beads
M. thermophyla laccase was dialyzed against 25 mM
phosphate buffer pH 6.5 and lyophilized. The enzyme was
dissolved in the same buffer (60 mg/ml) and the solution
was dropped onto glass beads (1 ml/3 g). The slurry was

S. Nicotra et al. / Tetrahedron 60 (2004) 595–600
599
2H, H-3,5), 6.82 (t, J¼2.1 Hz, 1H, H-120), 6.92 (d,
J¼2.1 Hz, 2H, H-10,14), 6.94 (d, J¼8.5 Hz, 1H, H-30),
6.95 (t, J¼2.1 Hz, 1H, H-12), 7.02 (d, J¼16.4 Hz, 1H,
H-80), 7.20 (BB0 multiplet, 2H, H-2,6), 7.21 (d, J¼2.1 Hz,
2H, H-100,140), 7.24 (broad s, 1H, H-60), 7.26 (d, J¼16.4 Hz,
1H, H-70), 7.47 (dd, J¼8.5, 1.4 Hz, 1H, H-20), 9.53 (s, 1H,
OH). 13C NMR (acetone-d6): 20.74 (2C), 54.80, 92.08,
109.67, 114.29, 114.79, 115.29, 116.73, 118.74, 123.07,
124.07, 127.89, 128.24, 129.47, 130.08, 130.24, 130.76,
139.79, 143.80, 151.02, 151.08, 157.65, 159.14, 168.80,
168.90.
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Passilly-Degrace, P.; Berlot, J.-P. Int. J. Mol. Med. 2002, 10,
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25.70%. Found C, 69.47; H, 4.91.
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as a free radical, following the method described by
Berset,29 using different concentrations of 2a (0.069,
0.137, 0.296, 0.468 mM). A methanolic solution of 2a
(100 ml) was added to 3.9 ml of a 0.06 mM DPPH
methanolic solution. The decrease in absorbance at
515 nm was evaluated until the reaction reached a plateau
(about 3 h). The percentage of residual DPPH at the steady
state was reported onto a graph as a function of the molar
ratio of antioxidant to DPPH. Antiradical activity was
defined as the amount of antioxidant necessary to decrease
the initial DPPH concentration by 50% (efficient concen-
tration).
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Acknowledgements
We thank Professor D. Haltrich and Dr. Y. Galante for a
generous gift of the laccases used in this work and Ministero
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dell’Istruzione, dell’Universita e della Ricerca (MIUR) for
financial support.
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