Laccase-mediated oxidation of phenolic derivatives

Article abstract of DOI:10.1016/j.molcatb.2009.12.016

The laccase-catalyzed oxidation of para-alkyl phenols (p-cresol, 3,4-dimethylphenol, tyrosol, 2′-O-acetyl-tyrosol) in biphasic systems has been investigated. With all the substrates compounds similar to the so-called "Pummerer's ketone" could be isolated

Full text of DOI:10.1016/j.molcatb.2009.12.016

Journal of Molecular Catalysis B: Enzymatic 65 (2010) 52–57
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Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Laccase-mediated oxidation of phenolic derivatives
Cristina Navarraa, Candice Goodwinb,c, Stephanie Burtonb,c, Bruno Danielid, Sergio Rivaa,∗
a Istituto di Chimica del Riconoscimento Molecolare, C.N.R., Via Mario Bianco 9, I-20131 Milano, Italy
b Department of Chemical Engineering, University of Cape Town, Rondebosch 7700, Cape Town, South Africa
c Biocatalysis and Technical Biology Research Group, Cape Peninsula University of Technology, PO Box 7535, Bellville, Cape Town, South Africa
d Dipartimento di Chimica Organica ed Industriale, Università di Milano, Via Venezian 21, 20133 Milano, Italy
a r t i c l e i n f o
a b s t r a c t
The laccase-catalyzed oxidation of para-alkyl phenols (p-cresol, 3,4-dimethylphenol, tyrosol, 2ꢀ-O-acetyl-
tyrosol) in biphasic systems has been investigated. With all the substrates compounds similar to the so-
called “Pummerer’s ketone” could be isolated in reasonable yields. Accumulation of this kind of products
is due to the fact that these compounds, in contrast to other dimers, are “dead-end” products, as they
cannot be further oxidized by the enzyme.
Article history:
Available online 24 December 2009
Keywords:
Laccase
Phenol
Oxidation
© 2010 Elsevier B.V. All rights reserved.
Pummerer’s ketone
Radical coupling
1. Introduction
horseradish peroxidase gave either the dimer 2 or the so-called
“Pummerer’s ketone” (3) as the main product, depending upon the
Biocatalyzed oxidation of phenols, particularly for technological
applications, is a well-covered area of research. In fact, biooxidation
of these compounds at the expense of molecular oxygen or H2O2
generates reactive radical intermediates that, under suitable con-
ditions, can then undergo extensive polymerization. Accordingly,
these biotransformations have been exploited in bioremediation
processes [1] and for the production, under mild conditions, of new
polyphenolic polymers [2].
The isolation of low molecular weight products is a much more
difficult task, but it is a goal of synthetic relevance: for instance,
a large number of dimeric napthol derivatives are widely used for
the production of the famous and efficient Noyori’s organometallic
catalysts [3].
Oxygen-mediated oxidation of substituted phenols catalyzed by
tyrosinase produces cathecols first, but then these intermediates
are further oxidized to the corresponding quinones. The latter com-
pounds easily polymerize in water, whereas in organic solvents it
is possible either to trap the quinones with Diels–Alder condensa-
tions in a domino sequence [4], or to slow down the polymerization
processes so that they can be reduced back to the stable catechols
by extraction with an ascorbate solution [5].
H2O2-mediated oxidation of simple phenol derivatives cat-
alyzed by peroxidases has been described by different authors
[6–8]. Generally speaking, the yields of recovered dimers and
trimers are quite low. For instance, oxidation of p-cresol (1) with
reaction conditions used, in 27 or 12% yields, respectively [8].
Laccases are a third group of exocellular oxidoreductases [9],
whose synthetic exploitation has been quite neglected until few
years ago despite the fact that they are quite abundant in nature,
particularly in fungi. Laccases are ideally “green” catalysts, as they
can use air as oxidant and produce H2O as the byproduct of the oxi-
dation of four substrate molecules (phenols, aromatic or aliphatic
amines) to the corresponding reactive radicals; the redox processes
take place with the assistance of a cluster of copper atoms which is
the catalytic site of the enzyme [10].
Synthetic exploitation of laccases can follow two different
approaches: the direct oxidation of the substrate or the oxidation of
an intermediate molecule (the so-called “mediator”) that then oxi-
dize the target substrate. The latter strategy is used in nature for the
oxidation of lignin which, being a polymeric material, cannot reach
the internal enzymatic copper cluster [11]. Examples of the in vitro
exploitation of this approach are given by the laccase-mediated
oxidative bleaching of indigo in the textile industry [12], or by
the oxidation of a series of primary alcohols to the corresponding
aldehydes or carboxylic acids mediated by TEMPO [13,14].
Few data are available, even today, on preparative scale laccase-
catalyzed direct oxidation reactions of these substrates. Most
reactions involve the modification of natural compounds, as, for
instance, the synthesis of actinocine (the chromophoric component
of the antibiotic actinomicin) from 3-hydroxy-4-methyl anthranilic
acid [15], the oxidation of penicillin G [16], the stilbenic phytoalexin
molecule resveratrol [17] or other hydroxy-stilbene derivatives
[18], the plant antibiotic totarol [19] and the steroid 17␤-estradiol
[20], to give dimeric products via radical coupling reactions. The
∗
Corresponding author. Tel.: +39 02 2850 0032; fax: +39 02 2890 1239.
E-mail address: sergio.riva@icrm.cnr.it (S. Riva).
1381-1177/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2009.12.016

C. Navarra et al. / Journal of Molecular Catalysis B: Enzymatic 65 (2010) 52–57
53
latter transformation, catalyzed by a laccase from Polyporus ver-
sicolor, was described more than 30 years ago and was one of the
first examples, if not the very first, of the use of enzymes in biphasic
systems [20a].
One of our present research directions is related to the exploita-
tion of enzymes for the selective formation of carbon–carbon
bonds. We reasoned that phenol-dimerization, catalyzed by lac-
cases, might be of interest in this respect and in this report, we
describe the results obtained using the laccases from Mycelyopthora
termophyla (MtL) and from Trametes versicolor (TvL) in the oxidation
of substituted phenol derivatives.
(b) 3,4-Dimethylphenol (4, 60 mg, 0.50 mmol) was dissolved in
AcOEt (2 ml), while the MtL laccase (30 mg, ∼30 U) was dis-
solved in 50 mM TRIS-buffer, pH 7.5 (2 mL). The biphasic
reaction was mildly shaken at 30 ◦C for 24 h. The phases were
separated, the water solution was extracted with AcOEt and
the crude residue was purified by silica flash chromatography
(petroleum ether–AcOEt, 8:2) to give 14 mg di 6 (23%).
TLC (petroleum ether–AcOEt, 8:2, RF 4, 0.44, RF 5, 0.32, RF 6, 0.20.
RP-HPLC (eluent, MeOH–H2O, 7:3; flow 0.5 mL/min, ꢀ: 254 nm):
tr 4: 4.20 min, tr 5: 14.30 min, tr 6: 8.45 min. 5 1H NMR ı (ppm,
CDCl3 + D2O): 7.02 (2 H, s, H-3 and H-3ꢀ); 6.85 (2 H, s, H-6 and H-
6ꢀ); 2.22 and 2.20 (6 H each, s each, two CH3 and two CH3ꢀ); MS:
m/z = 426 (M+). 6 1H NMR (CDCl3 + D2O) ı (ppm): 7.04 (1 H, s, H-
10); 6.65 (1 H, s, H-13); 5.88 (1 H, s, H-2); 4.63 (1 H, dd, J1 = 4.0 Hz,
J2 = 3.0 Hz, H-5); 3.02 (1 H, ddd, J1 = 17.5 Hz, J2 = 2.5 Hz, J3 = 1.0 Hz H-
6a); 2.75 (1 H, dd, J1 = 17.5 Hz, J2 = 3.5 Hz, H-6b); 2.25 (3 H, s, CH3);
2.23 (3 H, s, CH3); 1.93 (3 H, d, J = 1.5 Hz CH3); 1.59 (3 H, s, CH3);
13C NMR ı (ppm, CDCl3): ı = 194.72 (C-1); 158.65 (C-3); 157.30 (C-
8); 137.81 (C-9); 129.06, 129.04 (C-11, C-12); 126.01 (C-2); 125.12
(C-10); 111.71 (C-13); 87.87 (C-5); 47.93 (C-4); 37.07 (C-6); 20.52,
20.16, 19.57, 19.54 (C-14, C-15, C-16, C-17). MS: m/z = 426 (M+).
2. Experimental
2.1. Materials and methods
2.1.1. Enzymes and chemicals
The lipases from Candida antarctica (Novozyme 435®) and
the laccase from M. termophyla (MtL) were from Novozymes.
T. versicolor (TvL) laccase was purchased from Sigma–Aldrich.
The enzymes were each used in quantities based on respective
activities, as in previous investigations [19,21]. All chemicals and
solvents were purchased from Sigma–Aldrich.
2.1.4. Oxidation of tyrosol (7) by laccase from T. versicolor
Tyrosol (7, 500 mg) dissolved in 50 mL AcOEt was added to 50 mL
acetate buffer 20 mM pH 3.5 in which the laccase from T. versicolor
(500 U) had been previously dissolved. The solution was incubated
at 30 ◦C under gentle shaking, and the conversions was followed by
TLC (eluent: AcOEt). After 48 h the organic phase was separated and
the water phase was extracted with AcOEt. Following drying over
sodium sulfate, the solvent was evaporated under reduced pres-
sure and the crude residue was purified by flash chromatography
(eluent: AcOEt) to give the product 9 (43 mg, 8.7% yields).
1H NMR ı (ppm, MeOD): ı = 7.21 (1H, d, J = 2.0 Hz, H-13);
7.07 (1H, dd, J1 = 8.5 Hz, J2 = 2.0 Hz, H-15); 6.73 (1H, d, J = 8.5 Hz,
H-16); 4.80 (1H, dt, J1 = 3.0 Hz, J2 = 1.0 Hz, H-5); 4.15 (1H, dt,
J1 = 9.5 Hz, J2 = 6.5 Hz, H-8a); 4.06 (1H, dt, J1 = 9.5 Hz, J2 = 6.5 Hz, H-
8b); 3.95 (1H, m, H-3); 3.74 (2H, t, J = 7.0 Hz, CH2-18); 2.88 (1H, dd,
J1 = 18.5 Hz, J2 = 3.0 Hz, CH2-6a); 2.83 (1H, dd, J1 = 18.5 Hz, J2 = 3.0 Hz,
CH2-6b); 2.82 (1H, m, H-9a); 2.80 (2H, t, J = 7.0 Hz, CH2-17); 2.59 (1H,
dd, J1 = 18.0 Hz, J2 = 3.5 Hz, H-2a); 2.23 (1H, m, H-9b); 2.22 (1H, dd,
J1 = 18.0 Hz, J2 = 2.5 Hz, H-2b). 13C NMR ı (ppm, MeOD): ı = 207.52
(C-1); 158.02 (C-11); 132.38 (C-14); 129.75 (C-12); 129.54 (C-15);
123.64 (C-13); 109.15 (C-16); 87.70 (C-5); 83.24 (C-3); 66.90 (C-8);
63.05 (C-18); 52.73 (C-4); 39.37 (C-6); 38.83 (C-2); 38.33 (C-17);
37.84 (C-9). MS, m/z = 274 Da. (The NMR data were in accordance
to literature [23].)
2.1.2. Laccase-catalyzed oxidation of p-cresol (1)
(a) p-Cresol (50 mg, 0.46 mmol) was dissolved in a mixture of
ethanol (1.5 mL) and 50 mM TRIS-buffer, pH 7.5 (3.5 mL) and a
lyophilized sample of laccase from Myceliophtora thermophyla
(30 mg, ∼30 U evaluated using syringaldazine as a substrate)
was added. The homogeneous solution was gently shaken at
30 ◦C for 2 h, until complete conversion of the substrate was
achieved. The solution was extracted with AcOEt and the crude
residue was purified by silica flash chromatography (petroleum
ether–AcOEt, 8:2) to give 3 (10 mg, 20% isolated yields).
(b) p-Cresol (50 mg, 0.46 mmol) was dissolved in AcOEt (2 mL),
while the MtL laccase (30 mg, ∼30 U) was dissolved in 50 mM
TRIS-buffer, pH 7.5 (2 mL). The biphasic reaction was gen-
tly shaken at 30 ◦C for 24 h. The phases were separated,
the water solution was extracted with AcOEt and the crude
residue was purified by silica flash chromatography (petroleum
ether–AcOEt, 8:2) to give 8 mg of a mixture of 2 and 3 (16%
isolated yields).
TLC (petroleum ether–AcOEt, 8:2, Rf 1, 0.50, Rf 2, 0.35, Rf 3, 0.35.
RP-HPLC (eluent, MeOH–H2O, 7:3; flow 0.5 mL/min, ꢀ: 254 nm):
tr 1: 3.63 min, tr 2: 8.41 min, tr 3: 6.08 min. 2 1H NMR ı (ppm,
CDCl3 + D2O): 7.10 (2 H, dd, J1 = 8.7 Hz, J2 = 1.0 Hz, H-4 and H-4ꢀ);
7.05 (2 H, s, H-6 and H-6ꢀ); 6.90 (2 H, d, J = 8.2 Hz, H-3 and H-3ꢀ);
5.40 (2 H, s, phenolic OHs); 2.30 (6 H, s, CH3 and CH3ꢀ). 3 1H NMR
(CDCl3 + D2O) ı (ppm): 6.95 (1 H, s, H-10); 6.94 (1 H, d, J = 10.2 Hz, H-
3); 6.70 (1 H, d, J = 8.0 Hz, H-13); 6.45 (1 H, dd, J1 = 8.0 Hz, J2 = 1.8 Hz,
H-12); 5.90 (1 H, d, J = 10.2 Hz, H-2); 4.70 (1 H, t, J = 3.2 Hz, H-5); 3.05
(1 H, dd, J1 = 17.5 Hz, J2 = 2.8 Hz, H-6a); 2.78 (1 H, dd, J1 = 17.5 Hz,
J2 = 3.8 Hz, H-6b); 2.31 (3 H, s, CH3-15); 1.62 (3 H, s, CH3-14).
2.1.5. Acetylation of tyrosol by lipase from C. antarctica
(Novozym 435)
Tyrosol (7, 200 mg), vinyl acetate (5 ml) and the enzymatic
preparation Novozym 435 (75 mg) were added to a vial. The solu-
tion was incubated at 45 ◦C, under vigorous shaking (200 rpm),
and the conversion was followed by TLC (eluent: petroleum ether,
AcOEt 3:7). After 3 h the enzyme was eliminated by filtration, the
solvent was evaporated under reduced pressure and the crude
material was purified by flash chromatography (eluent: petroleum
ether–AcOEt, 9:1) to give the product 10 (210 mg, 80.5% yields):
1H NMR ı (ppm, MeOD): ı = 7.05 (2H, d, J = 8.5 Hz, H-3, H-5); 6.72
(2H, d, J = 8.5 Hz, H-2, H-6); 4.21 (2H, t, J = 7.0 Hz, CH2-8); 2.83 (2H,
t, J = 7.0 Hz, CH2-7); 2.01 (3H, s, CH3).
2.1.3. Laccase-catalyzed oxidation of 3,4-dimethylphenol (4)
(a) 3,4-Dimethylphenol (4, 60 mg, 0.50 mmol) was dissolved in a
mixture of ethanol (1.5 mL) and 50 mM TRIS-buffer, pH 7.5
(3.5 mL) and a lyophilized sample of laccase from M. ther-
mophyla (30 mg, ∼30 U evaluated using siringaldazine as a
substrate) was added. The homogeneous solution was mildly
shaken at 30 ◦C for 4 h, till complete conversion of the sub-
strate. The solution was extracted with AcOEt and the crude
residue was purified by silica flash chromatography (petroleum
ether–AcOEt, 8:2) to give 5 and 6 (32 mg, 53% isolated yields).
2.1.6. Oxidation of acetyl-tyrosol (10) by laccase from T.
versicolor
Tyrosyl acetate (10, 50 mg) dissolved in 5 mL AcOEt was added
to 5 mL acetate buffer 20 mM pH 3.5 in which the laccase from T.

54
C. Navarra et al. / Journal of Molecular Catalysis B: Enzymatic 65 (2010) 52–57
versicolor (50 U) had been previously dissolved. The solution was
incubated at 30 ◦C under mild shaking, and the conversion was fol-
lowed by TLC (eluent: petroleum ether, AcOEt 7:3). After 48 h the
organic phase was separated and the water phase was extracted
with AcOEt. Following drying over sodium sulfate, the solvent was
evaporated under reduced pressure and the crude residue was puri-
fied by flash chromatography (eluent: petroleum ether, AcOEt 8:2,
then petroleum ether, AcOEt 7:3) to give the products 11 (2.7 mg,
5.4% yields) and 12 (10 mg, 20.1% yields).
2.1.7. Oxidation of 4-vinylphenol (13) by laccase from T.
versicolor
4-Vinylphenol (13, 1000 mg) dissolved in 100 mL of AcOEt was
added to 100 mL acetate buffer 20 mM pH 3.5 in which the lac-
case from T. versicolor (1430 U) had been previously dissolved.
The solution was incubated at 30 ◦C under mild shaking, follow-
ing the conversion by TLC (eluent: petroleum ether–AcOEt, 9:2).
After 48 h the organic phase was separated and the water phase
was extracted with AcOEt. Following anhydrification over sodium
sulfate, the solvent was evaporated under reduced pressure and
the crude residue was purified by flash chromatography (eluent:
petroleum ether–AcOEt, 9:2) to give the product 15 (114 mg, 11.4%
yields). Then the eluent was changed to CHCl3/MeOH 95:5 to isolate
the product 14 (29 mg, 2.7% yields).
11: 1H NMR ı (ppm, MeOD): ı = 7.19 (2H, d, J = 9.0 Hz, H-3, H-
5); 6.88 (4H, m, Har); 6.77 (1H, d, J = 1.5 Hz, H-14); 4.25 and 4.17
(2H each, t each, J = 7.0 Hz, CH2-8 and CH2-16); 2.90 and 2.80 (2H
each, t each, J = 7.0 Hz, CH2-7 and CH2-15); 2.01 (3H, s, CH3); 1.96
(3H, s, CH3). MS, m/z = 358, 298 due to [M−AcOH]+, 238 due to [M-2
AcOH]+.
15: 1H NMR ı (ppm, CDCl3): 7.31 (1H, s, H-4); 7.30 (2H,
d, J = 8.4 Hz, H-2ꢀ, H-6ꢀ); 7.22 (1H, d, J = 8.0 Hz, H-6); 6.85 (2H,
d, J = 8.4 Hz, H-3ꢀ, H-5ꢀ); 6.81 (1H, d, J = 8.0 Hz, H-7); 6.69 (1H,
dd, J1 = 17.6 Hz, J2 = 10.8 Hz, H-1ꢀꢀ); 5.73 (1H, t, J = 8.8 Hz, H-2);
5.61 (1H, d, J = 17.6 Hz, H-2ꢀꢀtrans); 5.12 (1H, d, J = 10.8 Hz, H-
12: 1H NMR
ı
(ppm, MeOD): ı = 7.23 (1H, dd,
J1 = 2.0 Hz, J2 = 0.5 Hz, H-10); 7.08 (1H, dd, J1 = 8.0 Hz, J2 = 2.0 Hz,
H-12); 6.73 (1H, d, J = 8.0 Hz, H-13); 6.65 (1H, dd,
J1 = 10.5 Hz, J2 = 2.0 Hz, H-3); 5.97 (1H, dd, J1 = 10.5 Hz, J2 = 1.0 Hz,
H-2); 5.00 (1H, m, H-5); 4.32 (1H, dt, J1 = 11.5 Hz, J2 = 7.0 Hz, H-17a);
4.25 (2H, t, J = 7.0 Hz, CH2-15); 4.24 (1H, dt, J1 = 11.5 Hz, J2 = 7.0 Hz,
H-17b); 3.01 (1H, dd, J1 = 17.5 Hz, J2 = 4.0 Hz, H-6a); 2.94 (1H, ddd,
J1 = 17.5 Hz, J2 = 3.0 Hz, J3 = 0.5 Hz, H-6b); 2.91 (2H, t, J = 7.0 Hz,
CH2-14); 2.47 (1H, dt, J1 = 15.0 Hz, J2 = 7.0 Hz, H-16a); 2.32 (1H,
dt, J1 = 15.0 Hz, J2 = 7.0 Hz, H-16b); 2.01 (6H, s, 2 CH3). 13C NMR
ı (ppm, MeOD): ı = 150.35 (C-3); 130.76 (C-12); 127.12 (C-2);
124.91 (C-10); 110.75 (C-13); 85.73 (C-5); 66.25 (C-15); 61.75
(C-17); 38.98 (C-6); 35.42 (C-14); 35.01 (C-16); 20.70 (2 CH3). MS,
m/z = 298 due to [M−AcOH]+.
2ꢀꢀ ); 3.60 (1H, dd, J1 = 15.6 Hz, J2 = 9.2 Hz, H-3a); 3.22 (1H, dd,
cis
J1 = 15.6 Hz, J2 = 8.0 Hz, H-3b). 13C NMR ı (ppm, CDCl3): 159.46 (C-
8); 155.53 (C-4ꢀ); 136.61 (C-1ꢀꢀ); 133.94 (C-5); 130.87 (C-9); 127.61
(C-2ꢀ, C-6ꢀ); 127.00 (C-6); 122.37 (C-4); 115.52 (C-3ꢀ, C-5ꢀ); 111.09
(C-2ꢀꢀ); 109.22 (C-7); 84.46 (C-2); 38.06 (C-3). MS, m/z = 238 Da.
14: 1H NMR ı (ppm, CDCl3): 7.19 (2H, d, J = 8.4 Hz, H-2ꢀ, H-
6ꢀ); 6.81 (2H, d, J = 8.8 Hz, H-3ꢀ, H-5ꢀ); 6.65 (1H, dd, J1 = 10.4 Hz,
J2 = 2.4 Hz, H-4); 6.27 (1H, d, J = 10.4 Hz, H-5); 5.90 (1H, dd,
J1 = 17.6 Hz, J2 = 10.4 Hz, H-1ꢀꢀ); 5.29 (1H, d, J = 10.4 Hz, H-2ꢀꢀcis); 5.22
(1H, d, J = 17.6 Hz, H-2ꢀꢀtrans); 4.86 (1H, dd, J1 = 11.2 Hz, J2 = 6.0 Hz, H-
Scheme 1. Laccase-catalyzed oxidation of p-cresol (1).

C. Navarra et al. / Journal of Molecular Catalysis B: Enzymatic 65 (2010) 52–57
55
Scheme 2. Laccase-catalyzed oxidation of tyrosol (7).
2); 4.49 (1H, q, J = 2.8 Hz, H-8); 2.86 (1H, dd, J1 = 17.2 Hz, J2 = 3.2 Hz,
H-7a); 2.62 (1H, dd, J1 = 17.2 Hz, J2 = 3.2 Hz, H-7b); 2.43 (1H, dd,
J1 = 12.8 Hz, J2 = 5.6 Hz, H-3a); 2.22 (1H, dd, J1 = 12.8 Hz, J2 = 10.8 Hz,
H-3b). 13C NMR ı (ppm, CDCl3): 197.66 (C-6); 155.58 (C-4ꢀ); 149.26
(C-4); 137.59 (C-1ꢀꢀ); 133.94 (C-1ꢀ); 130.43 (C-5); 127.22 (C-2ꢀ, C-6ꢀ);
117.82 (C-2ꢀꢀ); 115.49 (C-3ꢀ, C-5ꢀ); 80.79 (C-8); 80.03 (C-2); 51.26
(C-9); 48.13 (C-3); 38.32 (C-7). MS, m/z 256 Da.
NMR data were fully in accordance with those reported by Delle
Monache and coworkers [23], who investigated the oxidation of
7 catalyzed by a laccase from the basidyomicete Lentinus edodes.
Scheme 2 shows the plausible reaction mechanism (as proposed
in [23]), which implies the coupling of two radicals, followed by
two intramolecular Michael additions (compound 9 was a racemic
mixture of the two trans enantiomers, only one of which is shown
in Scheme 2).
A more polar intermediate (likely to be the dimer 8) could be
detected by TLC, but any attempt to isolate it failed, as it was con-
verted into the final product 9 during the chromatography step.
In order to unambiguously confirm the reaction mechanism
hypothesis, we prepared the monoacetylated derivative 10 by
lipase-catalyzed acylation in organic solvent [24]. TvL-catalyzed
oxidation of 10 worked smoothly and two compounds could be
isolated in 5.4 and 20.1% yields, respectively. The minor product
could be easily identified as the C–O dimer 11 (Scheme 3) by mass
spectrometry (m/z at 358 Dalton) and its very simple 1H NMR spec-
trum.
3. Results and discussion
As starting model substrates, we chose the already men-
tioned p-cresol (1) and its analogue 3,4-dimethylphenol (4). The
commercially available laccases MtL or TvL were used as the
catalysts in two different reaction systems: (a) a homogeneous
solution of 1 or 4 in TRIS buffer containing EtOH (10%, v/v) as
a cosolvent; (b) a biphasic system TRIS buffer–AcOEt (1:1, v/v).
Reactions were much faster in the homogeneous solutions and
complete conversions were obtained after few hours, whereas
reactions in biphasic systems were stopped after 24 h. These
first experiments indicated that even with laccases it is not easy
to exploit these oxidative reactions to produce low molecular
weight compounds, the main products being brown-red polymeric
materials.
Fig. 1 shows the 1H NMR spectrum of the major product. The
presence of an olefinic double bond was clearly shown by the two
doublets at 6.65 and 5.97 p.p.m. (J = 10.5 Hz), while the other signals
between 7.2 and 6.7 p.p.m. were diagnostic for a three-substituted
aromatic ring. COSY and HMQC analysis allowed the complete
assignments of all the other signals of the 1H- and 13C NMR spec-
tra to the corresponding proton and carbon atoms, thus confirming
the proposed structure 12 (compound 12 was a racemic mixture
of the two trans enantiomers, only one of which is shown in the
scheme).
However, oxidized derivatives could be isolated by chromatog-
raphy and fully characterized by NMR and mass spectrometry.
Specifically, the dimers 2 and 3 (from 1, see Scheme 1) and the
corresponding analogues 5 and 6, (from 4) were clearly identified.
In a further investigation we have synthesized and submitted
to laccase-catalyzed oxidation a series of substituted p-vinyl phe-
nols, i.e. 13. Due to the more extensive possible delocalization of
the radical intermediate, the coupling pathways were much more
complex. “Pummerer’s ketone” structures (such as 14) could be iso-
lated, but as very minor byproducts, whereas the main products
In a next step we turned our attention to the more interest-
ing phenol tyrosol (7). This is one of the natural phenols which
are present in olive oil and wine and are believed to contribute
to their beneficial effects (cardioprotection and anti-atherogenic
activity, antioxidant skin photoprotection and anti-inflammatory
activity, . . .) [22]. Laccase-catalyzed oxidation of 7 was performed
under varied conditions and the best results were obtained using
the laccase from Trametes in a biphasic system. In this system,
the compound 9 could be isolated and characterized. Its mass and
Scheme 3. Laccase-catalyzed oxidation of acetyl-tyrosol (10).

56
C. Navarra et al. / Journal of Molecular Catalysis B: Enzymatic 65 (2010) 52–57
Fig. 1. 1H NMR spectrum of compound 12.
Scheme 4. Laccase-catalyzed oxidation of 4-vinyl-phenol (13).
were dimers such as 15 (Scheme 4). These results will be discussed
Acknowledgments
in more details in due course.
The authors are grateful for research support via the inter-
national Scientific Liason Agreement between Italy and South
Africa (2008–2010, significant bilateral project, MAE-Italy) and the
National Research Foundation, South Africa.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.molcatb.2009.12.016.
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