Biocatalyzed synthesis of enantiomerically enriched β-5-like dimer of 4-vinylphenol

Article abstract of DOI:10.1016/j.molcatb.2012.03.020

The tandem use of laccases and lipases has been exploited for the preparative scale synthesis of enantiomerically enriched dimeric phenols. Laccase-catalyzed oxidation of 4-vinylphenol (3) in biphasic systems gave as main product the racemic compound 4. The enantiomerically enriched butanoate (+)-4b and acetate (-)-4a could be obtained by alcoholysis reactions catalyzed by porcine pancreatic lipase in organic solvent and subsequent acetylation.

Full text of DOI:10.1016/j.molcatb.2012.03.020

Journal of Molecular Catalysis B: Enzymatic 84 (2012) 115–120
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Biocatalyzed synthesis of enantiomerically enriched ␤-5-like dimer of
4
-vinylphenol
∗
∗
Cristina Navarra, Paolo Gavezzotti, Daniela Monti , Walter Panzeri, Sergio Riva
Istituto di Chimica del Riconoscimento Molecolare (ICRM), C.N.R., Via Mario Bianco 9, 20131 Milano, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 6 April 2012
The tandem use of laccases and lipases has been exploited for the preparative scale synthesis of enan-
tiomerically enriched dimeric phenols.
Laccase-catalyzed oxidation of 4-vinylphenol (3) in biphasic systems gave as main product the racemic
compound 4.
Keywords:
Laccase
Lipase
Enantioselectivity
Phenol oxidation
The enantiomerically enriched butanoate (+)-4b and acetate (−)-4a could be obtained by alcoholysis
reactions catalyzed by porcine pancreatic lipase in organic solvent and subsequent acetylation.
©
2012 Elsevier B.V. All rights reserved.
1
. Introduction
reproduced by Beifuss and coworkers, who were also able to get
the opposite enantiomer (−)-pinoresinol in the presence of an
enantiocomplementary dirigent protein obtained from the plant
Arabidopsis thaliana [10].
Laccases (benzendiol:oxygen oxidoreductases, EC 1.10.3.2) are
oxidative enzymes widely distributed in plants, fungi and bacteria
1] and are known to oxidize a broad range of molecules directly
[
Recently we have shown that enantiomerically enriched phe-
nolic dimers can be more easily produced from their racemates (in
turn obtained by laccase-catalyzed oxidations) by exploiting the
known enantioselectivity of commercially available lipases even
toward compounds possessing the so-called “remote” stereocen-
ters [11]. This approach has been successfully applied to the model
compounds isoeugenol (1) and 2-methoxy-4-vinylphenol (2) [12].
As a further exemplification we report here the results obtained
with the simplest phenolic derivative of this series, the unsubsti-
tuted 4-vinylphenol (3).
or in the presence of low-molecular-weight redox mediators [2].
On this respect, in recent years our group has reported several
examples dealing with the oxidation of sugars [3], alkaloids [4],
and phenols [5].
In the latter examples, dimeric compounds obtained by the
cross-coupling of the laccase-produced radical intermediates were
isolated and characterized. Specifically, different authors have
studied the laccase-catalyzed oxidation of propenylphenols, like
isoeugenol (1, Scheme 1) [6], coniferyl alcohol [6], trans-resveratrol
[
7] and other hydroxystilbenes [8].
These studies reproduced in vitro reactions that in nature are
2
2
2
. Experimental
key steps in the biosynthesis of polymeric lignin and of other nat-
ural compounds (i.e., lignans, flavonolignans, alkaloids), but with
a main difference. In fact, while in vivo the enzyme-catalyzed
dimerization of propenylphenols derivatives to form lignans gen-
erally occurs with high regio-, diastereo-, and enantioselectivity,
in vitro the enantioselectivity is negligible and the products are
isolated as racemates. In order to overcome this limitation and
to mimic natural processes in a more faithful way, in 1997 Lewis
and coworkers showed for the first time that, in the presence of a
so-called “dirigent” protein isolated from the plant Forsythia inter-
media, the laccase-catalyzed coupling of coniferyl alcohol produced
the enantiomerically pure (+)-pinoresinol [9]. This result was then
.1. Materials and methods
.1.1. General experimental procedures
Microwaves instrument: CEM Focused Microwave Synthesis
System, Model Discovery. Optical rotations were measured on
a Jasco P-2000 polarimeter (Cremella, IT). NMR spectra were
recorded on Bruker AC400 and AC500 spectrometers (400 and
5
00 MHz, respectively) in CDCl3 (copies of the NMR spectra are
available in Supplementary Materials). MS spectra were recorded
on Bruker Esquire 3000 Plus spectrometer. HPLC analyses were
carried out using a Jasco 880-PU pump equipped with a Jasco
8
75-UV/Vis detector. HPLC conditions: Chiralpak IA and Chiralcel
OD, Daicel Chemical Industries, 250 mm × 4.6 mm column, isocratic
∗ Corresponding author. Tel.: +39 02 28500038; fax: +39 02 28901239.
E-mail address: daniela.monti@icrm.cnr.it (D. Monti).
mobile phase petroleum ether: 2-propanol, flow rate 1.0 mL/min
◦
at 25 C, detection at 254 nm. Thin-layer chromatography (TLC):
1
381-1177/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2012.03.020

1
16
C. Navarra et al. / Journal of Molecular Catalysis B: Enzymatic 84 (2012) 115–120
yields), 8a and 8b (inseparable mixture of cis and trans isomers in
ratio 1:2, 56 mg, 0.22 mmol, 5.3% yields).
1
4:
H NMR ı (ppm, CDCl ): 7.31 (1H, s, H-4); 7.30 (2H, d,
3
ꢀ
ꢀ
J = 8.4 Hz, H-2 , H-6 ); 7.22 (1H, d, J = 8.0 Hz, H-6); 6.85 (2H, d,
J = 8.4 Hz, H-3 , H-5 ); 6.81 (1H, d, J = 8.0 Hz, H-7); 6.69 (1H, dd,
J = 17.6 Hz, J = 10.8 Hz, H-1 ); 5.73 (1H, t, J = 8.8 Hz, H-2); 5.61
1H, d, J = 17.6 Hz, H-2 trans); 5.12 (1H, d, J = 10.8 Hz, H-2 cis); 3.60
ꢀ
ꢀ
Scheme 1. Compounds 1–3.
ꢀ
ꢀ
1
(
2
ꢀ
ꢀ
ꢀꢀ
precoated silica gel 60 F254 plates (Merck), developed with
(1H, dd, J = 15.6 Hz, J = 9.2 Hz, H-3 ); 3.22 (1H, dd, J = 15.6 Hz,
1
2
a
1
1
3
the molybdate reagent ((NH ) Mo7O ·4H O, 42 g; Ce(SO ) , 2 g;
J = 8.0 Hz, H-3 ). C NMR ı (ppm, CDCl ): 159.46 (C-8); 155.53
b
2 3
4
6
24
2
4
2
ꢀ ꢀꢀ ꢀ
(C-4 ); 136.61 (C-1 ); 133.94 (C-5); 130.87 (C-9); 127.61 (C-2 , C-
H SO conc., 62 mL; made up to 1 L of deionized water); flash chro-
2
4
ꢀ
ꢀ
ꢀ
ꢀꢀ
matography: silica gel 60 (70–230 mesh, Merck). Reaction was
carried out using a G24 Environmental Incubator New Brunswick
Scientific Shaker (Edison, USA).
6 ); 127.00 (C-6); 122.37 (C-4); 115.52 (C-3 , C-5 ); 111.09 (C-2 );
109.22 (C-7); 84.46 (C-2); 38.06 (C-3). MS, m/z (E.I.) = 238 Da.
1
ꢀꢀ
ꢀꢀ
5: H NMR ı (ppm, CDCl ): 7.34 (2H, d, J = 9.0 Hz, H-2 , H-6 );
3
7
.32 (1H, d, J = 8.5 Hz, H-5); 7.27 (1H, s, H-3); 6.86 (2H, d, J = 8.5 Hz,
ꢀ
ꢀ
ꢀꢀ
H-3 , H-5 ); 6.85 (1H, d, J = 8.5 Hz, H-6); 6.67 (1H, dd, J = 17.5 Hz,
2.1.2. Enzymes and chemicals
1
ꢀ
ꢀꢀ
ꢀ
J = 11.0 Hz, H-1 ); 6.10 (1H, dd, J = 7.5 Hz, J = 4.0 Hz, H-2 ); 5.62
Laccase from Trametes versicolor (10 U/mg) was from
2
1
2
ꢀ
ꢀꢀ
(1H, dd, J = 17.5 Hz, J = 1.0 Hz, H-2 trans); 5.14 (1H, dd, J = 11.0 Hz,
Sigma–Aldrich. Lipases from Pseudomonas cepacia (lipase PS),
Pseudomonas sp. (lipase AK), and Humicola lanuginosa (lipase
CE) were from Amano (Amano Enzyme Europe Ltd., Oxfordshire,
UK). Lipase from Chromobacterium viscosum (lipase CV) was from
Finnsugar. Lipases from Candida rugosa and porcine pancreas (PPL)
were from Sigma–Aldrich; Candida antarctica lipase B (Novozym
1
2
1
ꢀ
ꢀꢀ
ꢀ
); 4.27 (1H, dd, J = 10.5 Hz, J = 7.5 Hz, H-1 a); 4.13
1 2
J2 = 1.0 Hz, H-2
cis
ꢀ
1H, dd, J = 10.5 Hz, J = 4.0 Hz, H-1 b). MS, m/z (E.I.) = 238 Da due to
1 2
M−H O] .
(
[
+
2
1
ꢀ
ꢀ
6
: H NMR ı (ppm, CDCl ): 7.19 (2H, d, J = 8.4 Hz, H-2 , H-6 ); 6.81
3
ꢀ ꢀ
2H, d, J = 8.8 Hz, H-3 , H-5 ); 6.65 (1H, dd, J = 10.4 Hz, J = 2.4 Hz,
1 2
(
H-4); 6.27 (1H, d, J = 10.4 Hz, H-5); 5.90 (1H, dd, J = 17.6 Hz,
4
35) and Rhizomucor miehei lipase (Lipozyme) were a gift from
1
ꢀ
ꢀ
ꢀꢀ
J = 10.4 Hz, H-1 ); 5.29 (1H, d, J = 10.4 Hz, H-2 cis); 5.22 (1H, d,
Novozymes (Bagsvaerd, DK). Thermomyces lanuginosa lipase (Lipo-
lase) was from ChiralVision B.V. (Leiden, The Netherlands). The
enzymes were used in quantities based on respective activities,
as in previous investigations [12]. 4-Vinylphenol 3 was prepared
from 4-hydroxy benzaldehyde and malonic acid, following the
procedure described below. All other reagents were of the best
purity grade from commercial suppliers.
2
ꢀ
ꢀ
J = 17.6 Hz, H-2 trans); 4.86 (1H, dd, J = 11.2 Hz, J = 6.0 Hz, H-2);
1
2
4
.49 (1H, q, J = 2.8 Hz, H-8); 2.86 (1H, dd, J = 17.2 Hz, J = 3.2 Hz,
1 2
H-7a); 2.62 (1H, dd, J = 17.2 Hz, J = 3.2 Hz, H-7 ); 2.43 (1H, dd,
1
2
b
J = 12.8 Hz, J = 5.6 Hz, H-3a); 2.22 (1H, dd, J = 12.8 Hz, J = 10.8 Hz,
1
2
1
2
1
3
ꢀ
H-3 ). C NMR ı (ppm, CDCl ): 197.66 (C-6); 155.58 (C-4 ); 149.26
b
3
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
(
1
C-4); 137.59 (C-1 ); 133.94 (C-1 ); 130.43 (C-5); 127.22 (C-2 , C-6 );
ꢀꢀ ꢀ ꢀ
17.82 (C-2 ); 115.49 (C-3 , C-5 ); 80.79 (C-8); 80.03 (C-2); 51.26
(
C-9); 48.13 (C-3); 38.32 (C-7). MS, m/z (E.I.) = 256 Da.
2
.1.3. Synthesis of 4-vinylphenol (3)
To 2 g (16.4 mmol) of 4-hydroxybenzaldehyde dissolved in
2 mL of acetic acid (210 mmol), 6.82 g of malonic acid (65.6 mmol)
1
7:
H NMR ı (ppm, CDCl ): 7.36 and 7.34 (2H each, d each,
3
ꢀꢀ
ꢀꢀ
J = 8.5 Hz, H-3, H-5 and H-3 , H-5 ); 6.89 and 6.86 (2H each, d
each, J = 8.5 Hz, H-2, H-6 and H-2 , H-6 ); 6.67 (1H, dd, J = 17.5 Hz,
1
ꢀꢀ
ꢀꢀ
1
and 6.47 mL of piperidine (65.5 mmol) were added. The round bot-
tom flask with a suitable condenser was heated in the microwave
oven at 130 C and 150 W for 5 min. The reaction was controlled
by TLC (mobile phase, petroleum ether:AcOEt, 8:2). After complete
conversion of the substrate, 20 mL of H O and ice were added to
the mixture. The solution was extracted with AcOEt. Following dry-
ing by sodium sulfate addition, the solvent was evaporated under
reduced pressure and the crude residue was purified by flash chro-
matography (mobile phase, petroleum ether:AcOEt, 8:2) to give the
product 3; isolated yield: 820 mg (6.8 mmol, 41.6%).
ꢀꢀꢀ
ꢀꢀꢀ
J = 10.5 Hz, H-1 ); 5.63 (1H, d, J = 17.5 Hz, H-2 trans); 5.15 (1H, d,
2
ꢀꢀꢀ
ꢀ
); 5.07 (1H, dd, J = 8.5 Hz, J = 3.0 Hz, H-1 ); 4.09
1 2
J = 10.5 Hz, H-2
cis
◦
ꢀ ꢀ
1H, dd, J = 9.5 Hz, J = 3.0 Hz, H-2 a); 4.02 (1H, br t, J = 9.0 Hz, H-2 b).
1 2
(
13
ꢀꢀ
C NMR ı (ppm, MeOD): 158.72 (C-1 ); 156.98 (C-1); 136.21 (C-
2
ꢀꢀꢀ
ꢀꢀ
1
); 131.89 (C-4); 130.67 (C-4 ); 127.37 and 126.99 (C-3, C-5 and
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
C-3 , C-5 ); 114.79 and 114.34 (C-2, C-6 and C-2 , C-6 ); 110.29
(
ꢀꢀꢀ
ꢀ
ꢀ
C-2 ); 73.02 (C-2 ); 71.83 (C-1 ). MS, m/z (E.I.) = 256 Da.
1
8a and 8b: 8a: H NMR ı (ppm, DMSO): 7.18 (4H, d, J = 8.4 Hz,
ꢀꢀꢀ
ꢀꢀꢀ
ꢀꢀꢀ
ꢀꢀꢀ
H-3, H-3 , H-5, H-5 ); 6.72 (4H, d, J = 8.4 Hz, H-2, H-2 , H-6, H-6 );
4
ꢀ ꢀꢀ ꢀ ꢀꢀ
.77 (2H, t, J = 5.2 Hz, H-1 , H-1 ); 2.26 (2H, m, H-2 a, H-2 a); 1.77
1
H NMR (CDCl ): ı (ppm, CDCl ): 7.3 (2H, d, J = 8.6 Hz, H-2); 6.8
ꢀ
ꢀ
ꢀ
13
ꢀ
ꢀ
ꢀ
3
3
(2H, m, H-2 b, H-2 b). C NMR ı (ppm, DMSO): 157.35 (C-1, C-1 );
(
2H, d, J = 8.6 Hz, H-1); 6.7 (1H, dd, J = 17.6 Hz, J = 10.9 Hz, H-3);
ꢀꢀꢀ ꢀꢀꢀ ꢀꢀꢀ ꢀꢀꢀ
34.51 (C-4, C-4 ); 128.44 (C-3, C-3 , C-5, C-5 ); 116.12 (C-2, C-2 ,
1
2
1
5
.6 (1H, dd, J = 17.5 Hz, J = 0.71 Hz, H-5); 5.1 (1H, dd, J = 11.0 Hz,
ꢀꢀꢀ
ꢀ
ꢀꢀ
ꢀ
ꢀꢀꢀ
ꢀꢀ
1
1
2
1
C-6, C-6 ); 81.25 (C-1 , C-1 ); 34.97 (C-2 , C.2 ). 8b: H NMR ı (ppm,
DMSO): 7.15 (4H, d, J = 8.4 Hz, H-3, H-3 , H-5, H-5 ); 6.71 (4H, d,
J = 8.4 Hz, H-2, H-2 , H-6, H-6 ); 4.98 (2H, t, J = 6.8 Hz, H-1 , H-1 );
J2 = 0.66 Hz, H-4). The NMR data were in accordance to the literature
values.
ꢀꢀꢀ
ꢀꢀꢀ
ꢀꢀꢀ
ꢀ
ꢀꢀ
ꢀ ꢀꢀ ꢀ ꢀꢀ
13
2
.30 (2H, m, H-2 a, H-2 a); 1.80 (2H, m H-2 b, H-2 b). C NMR ı
ꢀꢀꢀ ꢀꢀꢀ
(ppm, DMSO): 157.25 (C-1, C-1 ); 135.21 (C-4, C-4 ); 128.19 (C-3,
2.1.4. Oxidation of 4-vinylphenol 3 by the laccase from T.
ꢀ
ꢀꢀ
ꢀꢀꢀ
ꢀꢀ
ꢀꢀꢀ
ꢀꢀꢀ
ꢀ
ꢀꢀ
versicolor
C-3 , C-5, C-5 ); 116.12 (C-2, H-2 , H-6, H-6 ); 81.48 (C-1 , C-1 );
36.29 (C-2 , C.2 ). MS, m/z (E.I.) = 256 Da.
ꢀ
4
-Vinylphenol (1 g, 8.33 mmol, 3) dissolved in 100 mL of AcOEt
was added to 100 mL 20 mM acetate buffer, pH 3.5, in which the
laccase from T. versicolor (1430 U, 143 mg) had been previously dis-
solved. The solution was incubated at 30 C under mild shaking,
2.1.5. Esterification of the dimer 4 to the corresponding
butanoate 4b
◦
following the conversion by TLC (eluent, petroleum ether:AcOEt,
To 53 mg (0.22 mmol) of 4 dissolved in 3 ml of anhydrous THF,
5 equiv. of TEA (0.15 mL, 106 mg, 1.05 mmol) and 3 mg (0.02 mmol)
of DMAP were added. The mixture was cooled at 0 C and 2 equiv.
of butyric anhydride were slowly added under stirring. Conver-
sions were monitored by TLC (eluent, petroleum ether:AcOEt, 9:1).
After 12 h the reaction was submitted to the usual work-up and the
crude residue purified by flash chromatography (eluent, petroleum
ether:AcOEt, 9:1) to give 65 mg (0.21 mmol, 95% yields) of product
4b. A sample of 4b was submitted to HPLC analyses: (a) Chiralpak
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 pres-
sure and the crude residue was purified by flash chromatography
(
0
eluent, petroleum ether:AcOEt, 9:2) to give the products 4 (114 mg,
.48 mmol, 11.4% yields) and 5 (6 mg, 0.03 mmol, 0.6% yields). Then
the eluent was changed to CHCl :MeOH, 95:5, to isolate the prod-
3
ucts 6 (29 mg, 0.11 mmol, 2.7% yields), 7 (10 mg, 0.04 mmol, 0.9%

C. Navarra et al. / Journal of Molecular Catalysis B: Enzymatic 84 (2012) 115–120
117
IA column, mobile phase petroleum ether:2-propanol, 75:25; (b)
Chiralcel OD column, mobile phase petroleum ether:2-propanol,
81%, [˛]D −73.2, c 0.0038 in MeOH). E.e. were determined using
a Chiralcel OD column, mobile phase petroleum ether:2-propanol,
9:1.
9
:1.
1
1
H NMR ı (ppm, CDCl ): 7.42 (2H, dt, J = 2.0 Hz, J = 9.0 Hz,
(−)-4a. H NMR ı (ppm, CDCl ): 7.42 (2H, dt, J = 2.0 Hz,
3
1
2
3
1
ꢀ
ꢀ
ꢀ
ꢀ
H-2 , H-6 ); 7.30 (1H, d, J = 1.5 Hz, H-4); 7.22 (1H, dd, J = 1.5 Hz,
J = 9.0 Hz, H-2 , H-6 ); 7.30 (1H, d, J = 1.5 Hz, H-4); 7.22 (1H, dd,
1
2
ꢀ
ꢀ
J = 8.0 Hz, H-6); 7.10 (2H, dt, J = 2.0 Hz, J = 9.0 Hz H-3 , H-5 ); 6.83
1H, d, J = 8.0 Hz, H-7); 6.68 (1H, dd, J = 11.0 Hz, J = 17.5 Hz, H-1 );
J = 1.5 Hz, J = 8.0 Hz, H-6); 7.10 (2H, dt, J = 2.0 Hz, J = 9.0 Hz H-
2
(
1
2
1
2
1
2
ꢀ
ꢀ
ꢀ
ꢀ
3 , H-5 ); 6.82 (1H, d, J = 8.0 Hz, H-7); 6.68 (1H, dd, J = 11.0 Hz,
1
2
1
ꢀꢀ
J = 18.0 Hz, H-1 ); 5.78 (1H, dd, J = 8.0 Hz, J = 9.0 Hz, H-2); 5.60
2 1 2
5
.78 (1H, dd, J = 8.0 Hz, J = 9.0 Hz, H-2); 5.60 (1H, dd, J = 1.0 Hz,
1
2
1
ꢀ
ꢀ
ꢀꢀ
ꢀꢀ
(1H, dd, J = 1.0 Hz, J = 18.0 Hz, H-2 trans); 5.12 (1H, dd, J = 1.0 Hz,
1 2 1
J = 17.5 Hz, H-2 trans); 5.12 (1H, dd, J = 1.0 Hz, J = 11.0 Hz, H-2 cis);
2
1
2
ꢀ
J2 = 11.0 Hz, H-2 ); 3.64 (1H, dd, J = 9.0 Hz, J = 15.5 Hz, H-3a);
1 2
ꢀ
3
.63 (1H, dd, J = 9.0 Hz, J = 15.5 Hz, H-3a); 3.21 (1H, dd, J = 8.0 Hz,
1
2
1
cis
1
3
J = 15.5 Hz, H-3 ); 2.55 (2H, t, J = 7.5 Hz, 2H␣); 1.81 (2H, sextet,
3.21 (1H, dd, J = 8.0 Hz, J = 15.5 Hz, H-3 ); 2.31 (3H, s, CH ).
C
2
b
1
1
2
b
3
1
3
ꢀ ꢀꢀ
NMR ı (ppm, CDCl ): 159.78 (C-8); 150.72 (C-4 ); 139.74 (C-1 );
3
J = 7.5 Hz, 2H␤); 1.06 (3H, t, J = 7.5 Hz, 3H␥). C NMR ı (ppm,
1
1
ꢀ
ꢀꢀ
ꢀ ꢀ
136.86 (C-5); 131.34 (C-9); 127.31 (C-6); 127.21 (C-2 , C-6 ); 127.10
CDCl ): 159.79 (C-8); 150.81 (C-4 ); 139.60 (C-1 ); 136.86 (C-5);
3
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀꢀ
1
31.32 (C-9); 127.31 (C-6); 127.19 (C-2 , C-6 ); 127.13 (C-1 ); 122.71
(C-1 ); 122.71 (C-4); 122.09 (C-3 , C-5 ); 111.44 (C-2 ); 109.52 (C-
7); 84.18 (C-2); 38.53 (C-3). Acyl moiety: 169.66 (carbonyl group);
21.40 (C␣). MS: m/z (ESI) = 303.1 Da (M+Na ); m/z (E.I.) = 280.1 Da.
ꢀ ꢀ ꢀꢀ
(
C-4); 122.11 (C-3 , C-5 ); 111.43 (C-2 ); 109.52 (C-7); 84.22 (C-
+
2
1
); 38.55 (C-3). Acyl moiety: 172.34 (carbonyl group); 36.55 (C␣);
8.76 (C␤); 13.93 (C␥). MS: m/z (ESI) = 331.2 Da (M+Na ); m/z
+
(
E.I.) = 308.2 Da.
3. Results and discussion
2.1.6. Esterification of the dimer 4 to the corresponding acetate
4
a
As 4-vinylphenol (3) is not commercially available, its syn-
5
mg (0.02 mmol) of 4 were dissolved in 0.7 mL of MTBE and,
thesis was pursued via condensation of p-hydroxybenzaldehyde
and malonic acid, followed by decarboxylation. Best results were
achieved by performing the reaction under microwave irradiation
[13], which after just 5 min allowed the isolation of the target 3
in 57% isolated yield (Scheme 2). In turn, 4-vinylphenol was sub-
mitted to the action of the commercially available laccase from T.
versicolor in a biphasic system, following the previously described
reaction protocol [12].
◦
under stirring at 0 C, 2 equiv. of acetic anhydride were added.
After 12 h, complete conversion was shown by TLC (mobile phase,
petroleum ether:AcOEt, 9:1) and a sample of the reaction mixture
was submitted to HPLC analysis (mobile phase, petroleum ether:2-
propanol, 9:1, Chiralcel OD column): (−)-4a, tR = 8.2 min; (+)-4a,
tR = 10.6 min.
2
.1.7. Screening of lipases for alcoholysis of 4b
To a solution of 4b (3 mg, 0.01 mmol) in 0.6 mL of MTBE, 0.03 mL
As the laccase-catalyzed oxidation of this molecule had not been
reported before, a detailed investigation of the reaction dimeric
products was performed. As shown in Scheme 3, six dimers (4–8)
could be isolated and characterized. The main product, albeit with
lower isolated yield in comparison to other substituted phenolic
precursors, was the “usual” ˇ-5-like dimer 4, whose structure was
easily confirmed by mass spectrometry (m/z at 238 Da), 1H NMR
(signals due to para-substituted and trisubstituted aromatic rings,
to a vinyl moiety and to an ABX system were clearly identified, see
Section 2), and 13C NMR analysis.
(
0.33 mmol) of n-BuOH and the respective lipase preparation were
added (Novozym 435, Lipolase, lipase PS, lipase CV, C. rugosa lipase
and PPL, 3.6 mg; lipase AK, 30 mg; lipase CE, 60 mg; Lipozyme,
6
◦
mg). The mixtures were incubated at 30 C and 250 rpm and mon-
itored by TLC (mobile phase, petroleum ether:AcOEt, 9:1) and HPLC
mobile phase, petroleum ether:2-propanol, 75:25, Chiralpak IA
(
column, 4b, tR = 6.5 min; (+)-4, tR = 9.7 min; (−)-4, tR = 10.5 min) at
scheduled times.
All the other products showed a molecular peak at 256 m/z, sug-
gesting a dimeric structure with the formal addition of a water
molecule. The structure of compounds 5 (“ˇ-1”-like dimer) and 7
(“ˇ-O-4”-like dimer) could be assigned on the basis of the clear
differences in the signals due to their aromatic rings. A struc-
ture similar to the so-called Pummerer’s ketone could be assigned
to compound 6 by comparison with the literature data [5a,14].
Additionally, NOESY experiments clearly showed the relative rela-
tionship between the three stereogenic centers of the molecule:
the p-hydroxyphenyl and the vinyl moieties being on the same side
(cis) of the tetrahydrofuran ring and the proton being on the oppo-
site (trans) site. Finally, the 1H NMR spectrum of the last isolated
dimeric product indicated the presence in a 2:1 ratio (by comparing
the area of the signals due to two sets of benzylic protons – triplet at
4.98 and 4.77 ppm) of symmetrical structures that, on the contrary
to compounds 4–7, were not carrying a vinyl substituent anymore.
These evidences suggested the presence of the inseparable mixture
of the cis and trans isomers 8a–8b.
2
.1.8. Preparative alcoholysis of 4b catalyzed by porcine pancreas
lipase (PPL) and subsequent acetylation
To a solution of 4b (35 mg, 0.11 mmol) in 5 mL of MTBE, 0.3 mL
of n-BuOH (3.27 mmol) and 150 mg of PPL were added. The mixture
was incubated at 30 C and 190 rpm and monitored by TLC (mobile
phase, petroleum ether:AcOEt, 9:1) and HPLC (mobile phase,
petroleum ether:2-propanol, 75:25, Chiralpak IA column). After
◦
1
.5 h the enzyme was removed by filtration and 1 mL (10.4 mmol)
of acetic anhydride and 5 mg (0.04 mmol) of DMAP were slowly
added. After additional 12 h, complete acetylation of residual 4
was shown by TLC. The reaction mixture was extracted with H O,
5
anhydrification by sodium sulfate, the solvent was evaporated and
the crude residue purified by flash chromatography (mobile phase,
petroleum ether:AcOEt, 95:5) to give enantiomerically enriched
2
% (w/v) NaHCO3 solution, and again H O (5 mL each). Following
2
(+)-4b (17 mg, 0.061 mmol, e.e. 61%, [˛]D +27.4, c 0.0038 in MeOH),
and enantiomerically enriched (−)-4a (12.2 mg, 0.040 mmol, e.e.
O
COOH
AcOH, piperidine
H
2
CO2
H O
2
+
+
+
MW (150 W)
130 °C, 5 min
COOH
HO
HO
(3)
Scheme 2. Synthesis of 4-vinylphenol 3.

1
18
C. Navarra et al. / Journal of Molecular Catalysis B: Enzymatic 84 (2012) 115–120
Scheme 3. Laccase-catalyzed oxidation of 4-vinylphenol 3.
All the compounds were isolated as racemates. Specifically, as
of enantiocomplementary lipases which acted on the enantiomer-
ically enriched residual substrates, thus allowing overall efficient
kinetic resolutions. At variance, all the lipases acting on 4b showed
the same enantiopreference for the levorotatory enantiomer (−)-
4b (the only enzyme showing a minor preference for the opposite
enantiomer – lipase CE – was basically non-selective: E = 1.2). How-
ever, we were pleased to note that the crude porcine pancreatic
lipase indeed showed a significant E-value (18.8), which was not
expected for such a molecule (the sterocenter was 6 bonds distant
from the acyl moiety that suffered alcoholysis cleavage and, more-
over, no other substituents were present close to the reactive ester).
Eventually, the kinetic resolution of 4b was scaled-up and, to avoid
racemization during the reaction work-up, the released phenolic
product was in situ acetylated to give the acetate 4a (Scheme 4).
The two enantiomerically enriched esters (+)-4b and (−)-4a could
be easily purified by silica chromatography to give the products in
61% and 81% enantiomeric excess, respectively (as judged by chiral
HPLC with a Chiralcel OD column, Fig. 2).
far as the dimer 4 concerned, the two enantiomers could be easily
separated by chiral HPLC using a Chiralpak IA column (Fig. 1).
Following our described methodology [12], dimer 4 was con-
verted to the racemic butanoate 4b to be submitted to the action
of a panel of commercially available lipases for enzyme-mediated
resolution. Table 1 summarizes the results obtained in the lipase-
catalyzed alcoholysis of 4b dissolved in methyl t-butyl ether in
the presence of a large excess of n-BuOH. Reactions were moni-
tored by chiral HPLC, which allowed the evaluation of the degree
of conversion and of the e.e. values of the released phenolic prod-
uct 4 (the racemic starting material 4b was eluted as a single peak,
Fig. 1), parameters that were then used to calculate the values of
the enantiomeric ratio “E” of the tested lipases [15].
In the previously described examples based on the dimers of
compounds 1 and 2 [12], the E-values were quite low (E < 8), as it
might be expected. This problem was overcome by finding couples
In conclusion this short report further exemplifies the synthetic
usefulness of the tandem use of laccases and lipases for the prepar-
ative synthesis of enantiomerically enriched dimeric phenols. Once
again it is really noteworthy and surprising the ability of crude and
commercially available lipases preparations to discriminate enan-
tiomers possessing remote stereocenters.
Table 1
Screening of lipases for the enantioselective alcoholysis of 4b.
Enzyme
Conversion (%)
Product e.e. (%)
0
E
Lipase AK
Lipase CE
Lipase PS
Novozym 435
Lipase CV
Lipozyme
Lipolase
Porcine pancreas lipase
Candida rugosa lipase
41.4
39.4
20.3
39.0
30.2
40.3
42.2
46.7
23.2
1.0
1.2
2.6
3.5
1.3
3.4
1.1
18.8
1.0
b
7.1
a
40.8
a
45.2
a
12.0
43.7
3.4
a
a
a
80.1
a
1.5
a
Fig. 1. HPLC chromatogram of the PPL-catalyzed alcoholysis of racemic 4b (column:
Chiralpak IA).
Major product: (−)-4.
b
Major product: (+)-4.

C. Navarra et al. / Journal of Molecular Catalysis B: Enzymatic 84 (2012) 115–120
119
Scheme 4. Lipase-catalyzed alcoholysis of compound 4b, followed by in situ acetylation.
Fig. 2. HPLC chromatogram of isolated enantiomerically enriched (+)-4b (left) and (−)-4a (right). Column: Chiralcel OD.
Acknowledgments
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Mol. Life Sci. 67 (2010) 369–385;
(
d) O.V. Morozova, G.P. Shumakovich, S.V. Shleev, Y.I. Yaropolov, Appl. Biochem.
This work was performed in the frame of the COST Action
Microbiol. 43 (2007) 523–535;
CM0701 “Cascade chemoenzymatic processes
–
new synergies
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Chem. Res. 40 (2007) 445–452;
between chemistry and biochemistry” and of the Project “VEL-
ICA”. The contribution by Regione Lombardia (Italy) is gratefully
acknowledged. We thank Dr. Gabriella Roda (Università di Milano)
for technical assistance in the use of the microwave instrument.
(
f) S. Riva, Trends Biotechnol. 24 (2006) 219–226.
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