Laccase-mediated oxidation of totarol

Article abstract of DOI:10.1002/adsc.200700005

In this study, novel dimers of the biologically active phenolic compound totarol were synthesized using the phenol oxidase enzyme lacease, obtained from Trametes pusbescens, in organic solvent medium. Two dimeric products, linked either by carbon-carbon or by carbon-oxygen bonds, were isolated and characterized. The effect of changes in various parameters such as solvent, temperature, pH and buffer concentration on the conversion of totarol by lacease was investigated. The nature of the organic solvent, in particular, was found to affect the nature and the ratio of the products obtained.

Full text of DOI:10.1002/adsc.200700005

FULL PAPERS
DOI: 10.1002/adsc.200700005
Laccase-Mediated Oxidation of Totarol
a
b
b
b
Sandile Ncanana, Lara Baratto, Lucia Roncaglia, Sergio Riva,
a,
and Stephanie G Burton *
Bioprocess Engineering Research Unit, Department of Chemical Engineering, Universityof Cape Town, Private Bag,
a
Rondebosch 7700, Cape Town, South Africa
Fax : (+27)-21-650-5501; e-mail: sburton@chemeng.uct.ac.za
Istituto di Chimica del Riconoscimento Molecolare, C.N.R., Via Mario Bianco 9, 20131 Milano, Italy
b
Received: January4, 2007
Supporting information for this article is available on the WWW under http://asc.wiley-vch.de/home/.
Abstract: In this study, novel dimers of the biologi- and buffer concentration on the conversion of totarol
callyactive phenolic compound totarol were s yn the-
bylaccase was investigated. The nature of the organ-
sized using the phenol oxidase enzyme laccase, ob- ic solvent, in particular, was found to affect the
tained from Trametes pusbescens, in organic solvent nature and the ratio of the products obtained.
medium. Two dimeric products, linked either by
carbon-carbon or bycarbon-ox yg en bonds, were iso-
lated and characterized. The effect of changes in var- Keywords: biocatalysis; biotransformations; laccase;
ious parameters such as solvent, temperature, pH oxidoreductases; phenols; totarol
Introduction
available commercially, but they can also be produced
simply, in the laboratory, by fermentation.
[
3]
Enzymes are increasingly becoming an important tool
(+)-Totarol (1) is a natural, phenolic, and highly
in the oxidation of various substrates to produce hydrophobic diterpenoid which has been found to be
novel derivatives for application as pharmaceuticals a potent antibacterial. It is extracted from the New
and, specifically, to modify natural compounds with Zealand endemic hardwood tree, Podocarpus totara.
[
1,2]
phenolic substructures.
Chemical methods maybe
This species is common and well known in New Zea-
used for the oxidation of these molecules, but these land and most of the wood used for totarol produc-
protocols are often complex, costly, and difficult to tion comes from discarded fencing, which, after as
predict or control. Thus, alternative methods which long as 150 years in the ground, is often found to be
are more selective and require more gentle conditions still in perfect condition at the core. The fact that the
[1]
are desirable. Laccases catalyze the one-electron ox- wood does not degrade is due to the valuable antimi-
idation of a wide varietyof organic substrates, includ- crobial properties of 1. The extract is presentlyused
ing mono-, di- and polyphenols, aminophenols, meth- in a varietyof cosmetic and personal care formula-
oxyphenols, aromatic amines and ascorbate, with the tions (i.e., toothpaste and mouthwash) in New Zea-
concomitant four-electron reduction of oxygen to land and Australia. More recently, totarol and its de-
[3]
water. Theyhave also been used in the biocatal yt ic
rivatives have been found to be highlyeffective as an-
oxidations of dyes, polymerization of lignin and ligno- tibacterial and antiplasmodial agents with potential
[
4,5]
[7]
sulfonates, bioremediation and bleaching.
In recent for application in treating malaria. Site-specific deri-
years, there has been an increasing interest in the ap- vatization of totarol has proved to be a challenging,
plication of laccases in organic solvents, the advantage but promising, route to new biologicallyactive prod-
[
8]
being the increased solubilityof their substrates in the
reaction medium. More recently, organic solvents
ucts.
These observations prompted us to investigate the
have also been shown to have an influence on the lac- potential of biocatalysis for the conversion of totarol
case enzyme selectivity, by changing the relative ratio to other novel derivatives, as part of our current study
of the dimers obtained in laccase-catalyzed oxida- aimed to the production of new biologicallyactive
[6]
tions. Among the different laccases found in nature, compounds. As an outcome of this study, we report
the enzymes isolated from Trametes strains are gener- for the first time the oxidation of totarol (1) catalyzed
allystable biocata lsyt s. Some of them are readily
bya laccase in the presence of significant amounts
Adv. Synth. Catal. 2007, 349, 1507 – 1513
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Sandile Ncanana et al.
FULL PAPERS
(
50% v/v) of organic cosolvents, to produce novel de- these parameters would affect enzyme stability and
[
9]
rivatives identified as carbon-carbon or carbon- activity. No significant improvements in the biocon-
oxygen linked dimers (1a and 1b).
version were observed as a result of using either
lower or higher salt concentrations. However, the pH
of the reaction mixture did affect the bioconversion
of totarol, the best performances being achieved at
pH 4.5–5 (a result consistent with other reports on
Results and Discussion
[
10]
Extracellular laccase was produced byfermentation
this laccase).
The oxidation of totarol at various
of the white rot fungal strain Trametes pubescens temperatures was also investigated, and it was shown
using an airlift reactor at a scale of 5 L. The laccase to increase with temperature up to an optimum tem-
was produced as an extracellular enzyme and was perature 308C, beyond which the conversion de-
readilyisolated from the fermentation medium by
precipitation with ammonium sulfate or acetone.
The freeze-dried laccase produced in this waywas
creased. Under these optimized reaction conditions a
62.6% conversion of totarol was observed after 24 h,
as demonstrated byHPLC. The incomplete transfor-
[
3b]
used for the oxidation of totarol (1) dissolved in a ho- mation was mainlydue to substrate precipitation even
À1
mogeneous solution comprising sodium acetate buffer at the low concentration used (2 mgmL ). In order
and a water miscible organic solvent (MeOH was ar- to improve products yields, several other water-misci-
bitrarilychosen for the initial experiments) in a 1:1
v/v ratio.
ble cosolvents were considered and, among them, ace-
tonitrile and acetone proved to be the most suitable
Using this reaction medium, totarol was converted ones. Reactions were performed on a semi-prepara-
bythe laccase into two products ( 1a and 1b), as re- tive scale (100 mg of 1) in the presence of 50% v/v of
solved byTLC and HPLC. The more polar compound
these solvents as well as of MeOH (for the sake of
(
1a, TLC R of 0.15) was the main product and was comparison). As reported in Table 1, the best results
f
obtained at a 8 : 1 ratio with the minor one (1b, TLC were obtained in the presence of acetone, which al-
R of 0.19). These compounds were purified byflash
chromatographyand anal yz ed using mass spectrome-
tryand NMR. The ESI-MS values of their molecular
lowed an almost quantitative conversion of totarol
(96.3%), as shown bythe HPLC chromatogram
(Figure 2). Even more remarkable, and unexpected,
f
peaks (m/z=593.43389 and 593.43290, respectively) was the effect of the organic solvents on the products
suggested that both the products were dimers of 1, composition: the 1a:1b ratio moved from 8.2 with
whereas their complete structure elucidation was ach- MeOH to 16.8 with CH CN and to 25.8 with acetone
3
1
13
ieved by H NMR and C NMR. Figure 1 compares (Table 1). In each case, we observed that the ratio
the signals due to the aromatic protons (Figure 1A) 1a:1b was not altered if the reaction was stopped at 3,
and to some of the aliphatic protons (Figure 1B) of 6 or 24 h.
totarol and of the two dimers. In compound 1a the ar-
Laccase-mediated oxidation of totarol was also at-
omatic AB system present in 1 (two doublets at d= tempted in biphasic systems, using reaction media
7
7
.03 and 6.54) was missing and onlya singlet at d= comprising 1:1 v/v sodium acetate buffer with the
.03 was present. Instead the signals between d=1.90 non-miscible solvents AcOEt, CHCl , methyl tert-
3
and 3.60, due to some of the aliphatic protons of 1 butyl ether, t-amyl alcohol or toluene. The reactions
and 1a, were almost superimposible, suggesting the were observed to proceed veryslowl y, with far lower
presence of the symmetric CÀC dimeric structure levels of conversion being achieved even after 4 days.
shown in Scheme 1. In contrast, the aromatic portion Specifically, no products were observed using methyl
1
of the H NMR spectrum of 1b showed both the pres- tert-butyl ether, conversions were lower than 10%
ence of two doublets and of one singlet (overall three using AcOEt, CHCl or toluene, and acceptable re-
3
protons), whereas all the signals between d=1.90 and sults (56% conversion, 1a:1b ratio of 3.9) were
3
.60 were duplicated, thus suggesting the formation of achieved onlywith the reaction medium that con-
a CÀO linkage between two totarol units. The tained t-AmOH. The slow rate of oxidation in a bi-
C NMR spectra of 1a and 1b, as well as their bidi- phasic system can be attributed to insufficient mass
13
mensional COSY and HSQC spectra further support- transfer of the reactants to and products from the
ed the proposed structures (spectra not shown; see enzyme and between two phases, as suggested by
[
11]
[12]
Supporting Information). As shown in Scheme 1, the Carrea and Faber.
formation of dimeric products is attributed to the cou-
pling of the radical intermediates produced bythe lac-
The influence of the solvent on enzyme selectivity
has been widelyexploited to optimize the stereoselec-
case-mediated oxidation of the phenolic sub- tive performances of hydrolytic enzymes, mainly li-
[
3a,6b]
strate.
pases and proteases, with the so-called “medium engi-
[
13]
In order to optimize the reaction conditions, the ef- neering”.
More recentlywe observed a similar
fects of buffer composition, pH and concentration effect on the reaction outcomes of the oxidation of
were investigated, based on previous reports that tetrahydro-2-naphthol or 17b-estradiol catalyzed by
1508
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Adv. Synth. Catal. 2007, 349, 1507 – 1513

Laccase-Mediated Oxidation of Totarol
FULL PAPERS
1
Figure 1. H NMR spectra of compounds 1, 1a and 1b, showing (A) the aromatic protons and (B) a portion of the aliphatic
protons.
the laccases from Trametes pubescens or Myceliop- case oxidative action. If this had been the case, a simi-
thora thermophyla, where the relative ratios of the di- lar effect would have been observed using chemical
meric products were stronglyaffected bythe nature
oxidants. To verifythis h py othesis, control experi-
of the organic solvent present in the reaction solutio- ments were run (in triplicate) using FeCl (homogene-
3
[6a]
n. However, the values of the 1a:1b ratio obtained ous solutions) or MnO (heterogeneous system) as
2
with totarol, bychanging the solvent (3.9, 8.2, 16.8,
catalysts. The results, summarized in Table 2, (visual-
and 25.8 with t-AmOH, MeOH, CH CN and acetone, ized bythe TLC) clearlyshowed that the laccase-cata-
3
respectively) could have simply originated by differ- lyzed reaction was much more efficient and “cleaner”,
ent interactions of the different solvent molecules and that the “solvent effect” on the products ratio
with the radical intermediates during their coupling was either absent (MnO ) or veryminor (FeCl ) in
2
3
reactions, without anyreal interference with the lac-
comparison with the data obtained byenz ym atic cat-
Adv. Synth. Catal. 2007, 349, 1507 – 1513
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Sandile Ncanana et al.
FULL PAPERS
Scheme 1. Synthesis and structures of the dimeric products 1a and 1b obtained bythe laccase-catal yz ed oxidation of totarol
1).
(
Figure 2. HPLC profile of the T. pubescens laccase-catalyzed oxidation of totarol in acetate buffer-acetone 50% v/v after
4 h reaction time.
2
alysis (Table 2). Additionally, it should be noted that did not occur in the presence of the laccase). There-
the formation of considerable amounts of unidentified fore it could reasonablybe concluded that the solvent
by-products using chemical oxidants makes less signif- used for the laccase-catalyzed oxidation of totarol had
icant the comparison of the relative ratio of just two indeed a significant effect on the course of the enzy-
of them (1a and 1b), as one of these two dimers could matic oxidation, both in terms of degree of conversion
have undergone a preferential subsequent oxidation and of products composition.
bythe chemical catal ys t (something that, apparentl ,y
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Laccase-Mediated Oxidation of Totarol
FULL PAPERS
Table 1. Conversion of totarol and products composition of
pubescens mycelial blocks were aseptically inoculated on the
plates and incubated at 288C for 6 days. T. pubescens was
also cultured in Trametes Defined Medium (liquid medium)
containing 10 g glucose, 10 g peptone bacteriological, and
the laccase-catalyzed oxidation in the presence of different
[a]
cosolvents.
[15]
Cosolvent
Conversion
[%]
Products [%]
Ratio of
1a:1b
2
g KH PO per liter. The autoclaved medium was distrib-
2 4
(
50% v/v)
1a
1b
uted into 1000-mL Erlenmeyer flasks and inoculated with a
homogenized mycelial plug taken from a solid dish precul-
ture (see above). Cultures were incubated at 288C with agi-
tation, for 9 days. These cultures were used to inoculate two
MeOH
62.6
71.3
96.3
49.1
60.3
85.0
6.0
3.6
3.3
8.2
16.8
25.8
CH CN
3
Acetone
4
-L airlift bioreactors each containing 10 g/L glucose, 10 g/L
[
a]
[14]
Detected byHPLC after 24 h.
peptone, 0.03% antifoam and 2 g/L KH PO . After 5 days
2 4
the medium in the bioreactors was supplemented with
3
3
0 mL of phenol mixture and 1 g of glucose. Subsequently
0 mL of phenol mixture and 1 g of glucose were added to
the medium daily. The laccase activity was assayed after 6
days (see below). After 13 days, the medium, containing lac-
case with the activityof 2–3 UmL , was harvested from the
Table 2. Relative ratio of the dimers 1a and 1b obtained by
totarol oxidation with different catalysts.
[a]
À1
Cosolvent (50% v/v)
Ratio of 1a:1b
T. pubescens laccase MnO2 FeCl3
bioreactors and kept at À208C or 48C till further use. Some
samples were freeze- dried and stored at À208C until
needed.
MeOH
8.2
5.2
5.2
6.6
n.d.
13.8
18.6
18.9
n.d.
CH CN
16.8
25.8
3.9
3
Laccase Assay
Acetone
t-AmOH
Laccase activitywas determined with 2,2 ’-azino-bis(3-ethyl-
[16]
[
a]
benzthiazoline-6-sulfonate) (ABTS) as the substrate. The
assaymixture contained 0.33 mL of 5 mM ABTS, 2.5 mL of
Detected byHPLC after 24 h.
0
.1M sodium acetate buffer (pH 5), and 0.17 mL aliquots of
culture fluid. Oxidation of ABTS was monitored byfollow-
ing the increase in absorbance at 420 nm (e=36
Conclusions
À1
À1
0
00 M cm ). One unit of laccase activitywas defined as
the amount of enzyme required to oxidize 1 mmol of ABTS
This studyhas described the s yn thesis of novel C ÀC per min at 258C.
and CÀO linked dimers of the biologicallyactive com-
pound totarol via oxidative reactions catalyzed by a HPLC Analysis
laccase from Trametes pubescens in biphasic or homo-
Laccase-catalyzed oxidations of totarol were analyzed by
genous aqueous-organic media. The dominant prod-
HPLC using a JASCO 880-PU instrument equipped with a
Jasco 870 UV detector. Samples were analyzed on a Li-
chrosperꢁ 100 RP-18 column (4”125 mm Particle SizePore
uct was a symmetrical CÀC linked dimer, which was
obtained in unusuallyhigh yi elds under optimized re-
action conditions and exploiting the “medium engi-
5
.0 mm; Merck). Eluent: from acetonitrile-H O 80:20 to ace-
2
neering” approach; to the best of our knowledge tonitrile-H O 100:0 in 10 min; same eluent (100% CH CN)
2
3
À1
these are byfar the best results obtained in the bio-
for 40 min; flow rate 1 mLmin ; UV detection at 280 nm.
catalyzed coupling of phenols to give a product with a Retention times: 1, 6.72 min; 1a, 22.34 min; 1b, 28.87 min.
defined chemical structure. The laccase-catalyzed oxi-
dation of 1 was achieved under verymild reaction
conditions and gave significantlybetter results in
comparison with similar reactions obtained with
chemical oxidants. It can be considered a significant
example of “sustainable chemistry” performed with
environmentallybenign protocols.
Laccase-Catalyzed Oxidation of Totarol
A) In methanol/buffer medium under non-optimized condi-
tions: Totarol (1, 200 mg) dissolved in 32 mL methanol was
added to 32 mL acetate buffer 20 mM pH 4.5 in which the
laccase from Trametes pubescens (100 mg, 60 U) had been
previouslydissolved. The milkysolution was incubated at
2
(
4
58C under mild shaking, following the conversion byTLC
eluent: petroleum ether-AcOEt, 19:1) and HPLC. After
8 h a 39% conversion to two products in an 8:1 ratio was
Experimental Section
achieved, with the formation of a precipitate that was recov-
ered byfiltration. The solution was extracted with AcOEt
and the solvent was evaporated to give a residue that
showed a composition similar to the precipitate. The overall
crude material (200 mg) was purified byflash chromatogra-
phy(eluent: petroleum ether) to give the products 1a (yield:
37 mg; 18.6%) and 1b (yield: 2.5 mg; 1.3%), together with
Growth of Trametes Pubescens for Laccase
Production
T. pubescens (CBS 696.94) was grown on agar plates con-
taining 50 g/L malt extract agar, supplemented with laccase
inducer [1% phenol mixture (phenol: 82.8 mM, p-cresol:
unreacted 1 (109 mg). R in TLC (eluent: petroleum ether-
f
[14]
24.99 mM, m-cresol: 25.8 mM, o-cresol: 77.03 mM)].
T.
AcOEt, 100:0.5): 1, 0,09; 1a, 0,15; 1b, 0,19.
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Sandile Ncanana et al.
FULL PAPERS
B) In other monophasic reaction media and under iopti-
mized conditions (see Table 1): Totarol (1, 100 mg) dissolved
in 16 mL water-miscible organic solvent (methanol, acetoni-
trile or acetone) was added to 16 mL acetate buffer 20 mM
pH 4.5 in which the laccase from Trametes pubescens
contained 1 mg totarol, 10 mg (6 U) Trametes pubescens lac-
case, 1 mL methanol and 1 mL 20 mM sodium acetate
pH 4.5. The reactions were incubated at 20, 30, 40, and
508C , with all other conditions remaining the same.
(
100 mg, 60 U) had been previouslydissolved. The solution
was incubated at 308C under mild shaking, following the
conversion byTLC and HPLC. After 24 h the solution was
extracted with AcOEt (which dissolved also most of the
solid precipitate) and the solvent was evaporated. The crude
material (~100 mg) obtained using acetone as cosolvent was
purified byflash chromatography(eluent: petroleum ether-
AcOEt, 100:0.5) to give the products 1a (62 mg) and 1b
Product Characterization
NMR spectra were recorded with
a Bruker AC400
(
400 MHz). High resolution electrospraymass spectra (HR-
ESI-MS) were acquired with an FT-ICR (Fourier Transfer
Ion Cyclotron Resonance) APEX II model (Bruker Dal-
tonics) equipped with a 4.7 Tesla cryo-magnet (Magnex).
Samples were dissolved in CH CN and injected into the in-
strument equipped with its own ESI source. Spectra were re-
corded in the HR mode with resolutions ranging from 20000
to 30000.
TM
(
2 mg) with a yield of 64% and 2%, respectively.
3
C) In biphasic system: The reaction mixture contained
10 mg totarol, 40 U Trametes pubescens laccase, 0.5 mL
AcOEt or CHCl or methyl tert-butyl ether or t-AmOH or
3
toluene and 0.5 mL acetate buffer 20 mM pH 4.5. The reac-
tions were incubated at 308C with shaking at 200 rpm, the
progress of the reactions being monitored byTLC and
HPLC. After 5 days the degrees of conversion (evaluated by
Totarol (1): TLC, Rf (eluent: petroleum ether-AcOEt
1
1
00:0.5): 0.09; HPLC, R : 6.72 min; H NMR (400 MHz,
t
CDCl ): d=7.03 (1H, d, J=8.5 Hz, H-11), 6.54 (1H, d, J=
3
8
.5 Hz, H-12), 3.31 (1H, sept, J=7.0 Hz, H-15), 2.96 (1H,
HPLC) were 7, 7, 0, 56 or 8% in AcOEt, CHCl , methyl
3
dd, J =17.1 Hz, J =6.3 Hz, H-7 ), 2.78 (1H, ddd, J =
1
2
eq
1
tert-butyl ether, t-AmOH or toluene, respectively. The reac-
tion with t-AmOH was scaled-up. Totarol (100 mg) was dis-
solved in 8 mL t-AmOH and added to 16 mL acetate buffer
1
1
7.1 Hz, J =11.5 Hz, J =7.8 Hz, H-7 ), 2.25 (1H, dt, J =
2 3 ax 1
2.7 Hz, J =3.7 Hz, H-1 ) and 1.36 (1H, td, J =12.8 Hz,
2
eq
1
J =3.8 Hz, H-1 ), 1.94 (1H, ddt, J =13.3 Hz, J =7.8 Hz,
2
ax
1
2
2
(
2
0 mM pH 4.5 with the laccase from Trametes pubescens
400 U). The reaction was incubated at 308C byshaking at
20 rpm for 4 days. Following phase separation and extrac-
J =1.8 Hz, H-6 ) and 1.67 (1H, m, H-6 ), 1.75 (1H, qt,
3
eq
ax
J =13.7 Hz, J =3.4 Hz, H-2 ) and 1.61 (1H,d quint, J =
1
2
ax
1
1
3
4
1
3.8 Hz, J =3.7 Hz, H-2 ), 1.49 (1H, ddt, J =13.2 Hz, J =
2 eq 1 2
tion of the water phase with CHCl , the organic phases were
3
.7 Hz, J =1.4 Hz, H-3 ) and 1.22 (1H, td, J =13.4 Hz, J =
3
eq
1
2
mixed, and the solvent was evaporated. The crude material
was purified byflash chromatography(eluent: petroleum
ether-AcOEt, 25:1) to give a mixture of 1a and 1b (34 mg;
.1 Hz, H-3 ), 1.29 (1H, dd, J =12.7 Hz, J =2.2 Hz, H-5),
ax
1
2
.38 and 1.36 (3 H each, d each, J=7.1 Hz, CH -16 and
3
CH -17), 1.20 (3H, s, CH -20), 0.97 (3H, s, CH -18) 0.94
3
3
3
34% overall yield) and two additional new minor by-prod-
1
3
(
3H, s, CH -19); C NMR (100 MHz, CDCl ): d=152.6 (C-
3
3
ucts (a yellow one, ~1 mg, R 0.34 in TLC with the eluent:
f
1
1
3
2
3), 144.0 (C-9), 134.7 (C-8), 131.7 (C-14), 123.7 (C-11),
15.0 (C-12), 50.3 (C-5), 42.3 (C-3), 40.4 (C-1), 38.4 (C-10),
3.9 (C-18), 29.4 (C-7), 27.8 (C-15), 25.8 (C-20), 22.3 (C-19),
1.1 (C-16 and C-17), 20.2 (C-2), 20.1 (C-6) [for COSY and
petroleum ether-AcOEt, 19:1; an orange one, ~1.5 mg, Rf
0,45 in TLC), whose attempted structural characterization
resulted unsuccessful.
HSQC spectra see Supporting Information].
Effect of Buffer Salts Concentration on the
Bioconversion of Totarol
Totarol main product 1a (symmetrical CÀC dimer): TLC,
R (eluent: petroleum ether-AcOEt, 100:0.5): 0.15; HPLC,
f
1
R : 22.34 min; H NMR (400 MHz, CDCl ): d=7.03 (2H, s,
The buffer concentrations were varied from 20 to 100 mM.
The reaction mixture contained 1 mg totarol, 10 mg (6 U)
Trametes pubescens laccase, 1 mL methanol and 1 mL
sodium acetate buffer with concentration of 20, 50 or
t
3
H-11 and H-11’), 3.35 (2H, m, H-15 and H-15’), 3.02 (2H,
dd, J =17.2 Hz, J =6.4 Hz, H-7 and H-7’ ), 2.82 (2H, m,
1
2
eq
eq
H-7ax and H-7’ ), 2.25 (2H, br d, J=12.6 Hz, H-1 and H-
ax
eq
1
6
’ ), 1.97 (2H, br dd, J =13.2 Hz, J =7.9 Hz, H-6 and H-
100 mM at pH 5. The reactions were incubated at 308C with
eq 1 2 eq
’ ), 1.72 (4H, m, H-6 , H-6’ , H-2 , H-2’ ), 1.60 (2H, br d
shaking at 200 rpm. The progress of the reaction was moni-
tored byHPLC.
eq
ax
ax
ax
ax
quint, J =13.9 Hz, J =3.4 Hz, H-2 and H-2’ ), 1.49 (2H,
1
2
eq
eq
br d, J=13.1 Hz, H-3eq and H-3’ ), 1.40–1.20 (6 H , m, H-
eq
1ax, H-1’ , H-3 , H-3’ , H-5, H-5’), 1.40 and 1.38 (6 H each,
ax
ax
ax
Effect pH on the Bioconversion of Totarol
d each, J=7.1 Hz, CH -16 and CH -16’, CH -17 and CH -
3
3
3
3
The pH of the reaction medium was varied from 4 to 7
using 20 mM sodium acetate buffer. The reaction mixture
contained 1 mg totarol, 10 mg (6 U) Trametes pubescens lac-
case, 1 mL methanol and 1 mL 20 mM sodium acetate
buffer.
17’), 1.22 (6H, s, CH
and CH
(100 MHz, CDCl
and C-9’), 135.3 (C-8 and C-8’), 132.7 (C-14 and C-14’),
125.1 (C-11 and C-11’), 121.5 (C-12 and C-12’), 50.3 (C-5
and C-5’), 42.3 (C-3 and C-3’), 40.3 (C-1 and C-1’), 38.5 (C-
3
-20 and CH
3
-20’), 0.99 (6H, s, CH
3
-18
1
3
-18’), 0.95 (6H, s, CH
-19 and CH -19’); C NMR
3 3
3
): d=150.4 (C-13 and C-13’), 144.0 (C-9
3
10 and C-10’), 34,0 (C-4 and C-4’), 33.9 (C-18 and C-18’),
29.5 (C-7 and C-7’), 28.4 (C-15 and C-15’), 26.0 (C-20 and C-
20’), 22.3 (C-19 and C-19’), 20.9 (C-16 and C-16’, C-17 and
Effect of Reaction Temperature on the Bioconversion
of Totarol
In order to determine the impact of temperature on totarol
conversion bya particular dose of enz ym e, reactions were
conducted at various temperatures. The reaction mixture
C-17’), 20.2 (C-2 and C-2’), 20.1 (C-6 and C-6’) [for COSY
and HSQC spectra see Supporting Information]; ESI-MS,
+
positive mode: m/z=593.43389 [M+Na] , calcd.: 593.43290.
1512
asc.wiley-vch.de
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Synth. Catal. 2007, 349, 1507 – 1513

Laccase-Mediated Oxidation of Totarol
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nol. 2006, 24, 219–226.
f
troleum ether-AcOEt, 100:0.5): 0.19; HPLC, R : 28.87 min;
t
1
H NMR (400 MHz, CDCl , selected data): d=7.04 (1H, d,
3
J=8.8 Hz, H-11), 6.67 (1H, s, H-11’), 6.56 (1H, d, J=
8
3
.8 Hz, H-12), 3.38 and 3.30 (1 H each, m, H-15 and H-15’),
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[4] M. Lorenzo, D. Moldes, C. S. Rodriguez, A. Sanroman,
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eq
1
1
1
6.9 Hz, J =9.1 Hz, J =5.3 Hz„ H-7 and H-7’ ), 1.42 and
2 3 ax ax
.41, 1.40 and 1.38 (3 H each, d each, J=7.1 Hz, CH -16 and
3
CH -16’, CH -17 and CH -17’), 1.22 and 1.14 (3 H each, s,
3
3
3
CH -20 and CH -20’), 0.99 and 0.96 (3 H each, s, CH -18
3
3
3
and CH -18’), 0.95 and 0.91 (3H each, s, CH -19 and CH -
3
3
3
13
1
9’); C NMR (100 MHz, CDCl ): d=148.2 and 146.1 (C-13
3
and C-13’), 145.0 and 143.0 (C-9 and C-9’), 135.7 and 134.8
C-8 and C-8’), 132.4 (C-14). 128,9 (C-14’), 123.9 (C-11),
(
115.7 (C-12), 113.3 (C-11’), 50.5 and 50.3 (C-5 and C-5’),
42.3 (C-3 and C-3’), 40.3 (C-1 and C-1’), 38.6 (C-10 and C-
10’), 34.0 (C-4 and C-4’), 33.9 (C-18 and C-18’), 29.5 and
29.1 (C-7 and C-7’), 28.5 and 28.2 (C-15 and C-15’), 25.8 (C-
20 and C-20’), 22.3 (C-19 and C-19’), 21.8 and 20.9 (C-16
and C-16’, C-17 and C-17’), 20.1 (C-2 and C-2’, C-6 and C-
’) [for COSY and HSQC spectra see Supporting Informa-
6
+
tion]; ESI-MS, positive mode: m/z=593.43130 [M+Na] ,
calcd.: 593.43290.
[9] Y.-J. , Kim, A. J. Nicell, Bioresource Technology 2006,
9
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Acknowledgements
1
274–1279.
The authors are grateful for research support via the Interna-
tional Scientific Liason agreement(2005–2007) between the
National Research Foundation, South Africa and Italy.
[
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1513