Laccase-Catalyzed Dimerization of Piceid, a Resveratrol Glucoside, and its Further Enzymatic Elaboration

Article abstract of DOI:10.1002/adsc.201500185

The laccase-catalyzed oxidation of piceid, the 3-β-D-glucopyranosyl derivative of the stilbenic phytoalexin resveratrol, allowed isolation of the corresponding β-5 like trans-dehydrodimer in good yield (45%) avoiding chromatography. This compound was then

Full text of DOI:10.1002/adsc.201500185

FULL PAPERS
DOI: 10.1002/adsc.201500185
Laccase-Catalyzed Dimerization of Piceid, a Resveratrol
Glucoside, and its Further Enzymatic Elaboration
a
a
b
c
Paolo Gavezzotti, Federica Bertacchi, Giovanni Fronza, Vladimír K rˇ en,
a,d
a,d,
Daniela Monti, and Sergio Riva *
Istituto di Chimica del Riconoscimento Molecolare (ICRM), C.N.R., via Mario Bianco 9, 20131 Milano, Italy
a
Fax: (+39)-02-2890-1239; phone: (+39)-02-2850-0032; e-mail: sergio.riva@icrm.cnr.it
Istituto di Chimica del Riconoscimento Molecolare (ICRM), UOS-Milano Politecnico, C.N.R., via Mancinelli 7, 20133
Milano, Italy
Laboratory of Biotransformation, Institute of Microbiology, Academy of Sciences of the Czech Republic, Víde nˇ skµ 1083,
CZ142 20 Prague, Czech Republic
The Protein Factory, Centro Interuniversitario di Biotecnologie Proteiche, Università degli Studi dell’Insubria, Politecnico
di Milano, ICRM-CNR, Milano, Italy
b
c
d
Received: February 23, 2015; Published online: May 15, 2015
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201500185.
Abstract: The laccase-catalyzed oxidation of piceid, glucosidase from almonds allowed the isolation of
the 3-b-d-glucopyranosyl derivative of the stilbenic a monoglycosylated intermediate in reasonable yield.
phytoalexin resveratrol, allowed isolation of the cor- The diastereoisomers and the enantiomers of the iso-
responding b-5 like trans-dehydrodimer in good yield lated dimeric products were separated using a semi-
(
45%) avoiding chromatography. This compound preparative chiral column. The basic antioxidant
was then submitted to the action of a small library of properties of these new dimers have been deter-
commercially available hydrolases. While the cellu- mined.
lase from Trichoderma viride was able to fully hydro-
lyze the diglycosylated dimer to give the correspond- Keywords: glycosidase; laccase; piceid; resveratrol;
ing b-5 like trans-dehydrodimer of resveratrol, the b- viniferin
Introduction
showed that a trans-dehydrodimer of resveratrol (d-
viniferin, 2) could be isolated in reasonable yield
[5]
trans-Resveratrol (3,5,4’-trihydroxystilbene, 1) is a stil- (31%) using a laccase from Trametes pubescens.
benic phytoalexin produced by plants, particularly by
At variance to the starting material, the dimer 2
grapevines, pines and legumes, via a metabolic se- possessed two stereogenic centers. The relative con-
quence induced in response to biotic or abiotic stress figuration of the two aromatic moieties was trans, but
[1]
factors. It has been proposed to be one of the com- the compound was obtained as a racemate and the
ponents in red wines with beneficial effects to human two enantiomers could be separated by chromatogra-
cardiovascular health (French paradox) and its use phy only using chiral stationary phases. As it is likely
has been suggested for treating obesity-related disor- that the two enantiomers interact differently with bio-
ders and neurodegenerative, inflammatory, cardiovas- logical receptors, it would be important to have in
[
2]
cular, and age-related diseases, like Alzheimer.
hand a suitable protocol to isolate both of them, in
Moreover, both monomeric and oligomeric stilbenes order to better understand the origin of the claimed
are reported to be potentially important cancer che- beneficial action of these compounds. Such a protocol
moprotective agents, being able to inhibit cellular was not available when this investigation started.
[
3]
events associated with carcinogenesis.
Only very recently a separation of 20 mg of racemic 2
Different studies have shown that resveratrol is was reported, exploiting the so-called high speed
transformed in vivo into various oxidized products counter-current chromatography with a biphasic sol-
[
4]
(
dimers and oligomers, which also possess significant vent system and a chiral selector (a substituted cyclo-
[
2,3]
[6]
biological activities ) whereas only few reports have dextrin) in the aqueous phase.
described the in vitro biocatalyzed oxidation of this In nature resveratrol is commonly isolated as the
molecule. In one of these works, years ago, we corresponding 3-O-glucoside, called piceid or polyda-
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Paolo Gavezzotti et al.
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tin
(3,5,4’-trihydroxystilbene-3-O-b-d-glucopyrano-
[7]
side, 3). Different studies have shown that piceid
possesses pharmacological actions similar to those of
[
8]
resveratrol, but it is more stable to light and heat. It
can be considered as a pro-drug of 1 and there are lit-
erature reports that describe the deglucosylation of 3
[
9]
to 1 by the action of suitable b-glucosidases.
We thought that, due to the presence of the glucose
moiety, a laccase-catalyzed oxidation of piceid 3
might be suitable to produce a diastereomeric mixture
of the corresponding dimeric products 4. The separa-
tion of such diastereoisomers, which have an impor-
tance by themselves and have never been isolated
before, could be more feasible, at least in principle,
than the separation of the two corresponding enantio-
mers. Moreover, subsequent deglucosylation would
furnish the desired pure enantiomers of 2. In the fol-
lowing we present the results that have been obtained
in this respect.
Figure 1. TLC of the laccase-catalyzed oxidation of 3. From
left to right: a) 3; b) crude reaction mixture plus 3; c) crude
reaction mixture; d) AcOEt extract of the crude reaction
mixture.
pletely oxidized. As shown by the TLC reproduced in
Figure 1, the work-up was straightforward, as a simple
extraction of the water phase with AcOEt gave the
almost pure trans-dimeric derivative 4 in up to 45%
isolated yield.
The positive ESI mass spectrum of the isolated
product had the expected molecular peak at 801.6 Da.
1
The H NMR spectrum showed the typical signals due
to the presence of a b-5 like dimeric structure (as in
2
). Moreover, 4 being the expected diastereomeric
mixture, some of the signals were clearly distinguisha-
ble. For instance, there were two signals due to H-2
(both doublets with J=7.6 Hz resonating at 5.456 and
Results and Discussion
5
.448 ppm), and to the two anomeric protons of the
[
10]
Laccases, the so-called blue oxidases, are a group of two sugar moieties: H-1’’’ (both doublets with J=
copper-containing enzymes, which catalyze the oxida- 7.7 Hz resonating at 4.800 and 4.777 ppm) and H-1’’’’
tion of a variety of compounds at the expense of mo- (both doublets with J=7.7 Hz resonating at 4.793 and
[
11]
lecular oxygen that is reduced to water. Therefore, 4.727 ppm).
in principle, they are ideal catalysts for sustainable
The presence of a 1:1 product mixture was demon-
chemical and technological processes. As more and strated by HPLC elution with an analytical chiral
more laccases are becoming available, their synthetic column (Lux 5u Cellulose-1, Phenomenex), as clearly
exploitation is significantly growing, as documented shown in Figure 2 (A).
[
12]
by the numerous patents and literature reports. In
Unfortunately, despite all our attempts, it was not
recent years our group has exploited these biocata- possible to find a suitable chromatographic achiral
lysts, i.e., for the selective oxidation of phenolic deriv- system that could allow the separation of the two dia-
[13]
[6,14]
atives and specifically of phenolic stilbenes.
stereoisomers. Their isolation was eventually obtained
Following a preliminary optimization of the reac- with a semi-preparative chiral column (Lux 5u Cellu-
tion parameters, a 20 mM solution of piceid (3, 1 g lose-1, Figure 2 (B) shows a typical elution pattern),
dissolved in 140 mL of a buffered solution containing and in this way 4a [165 mg, de >99%, Figure 2 (C)]
3
0% v/v MeOH to improve its solubility) was oxi- and 4b [175 mg, de 98.4%, Figure 2 (D)] were ob-
dized by the commercially available laccase from Tra- tained.
metes versicolor. The reaction was gently shaken at The neat separation of the two compounds was also
58C for 2–3 h until the starting material was com- confirmed by a comparison of the respective H NMR
1
2
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Laccase-Catalyzed Dimerization of Piceid, a Resveratrol Glucoside
1
Figure 3. H NMR spectra of 4 (A), 4a (B), and 4b (C). Se-
lected signals due to the sugars anomeric protons (top, 4.86–
4
3
.62 ppm) and to the C-6 methylene protons (bottom, 3.80–
.39 ppm).
To the best of our knowledge neither the diastereo-
isomers 4a and 4b nor the mixture of trans-dehydro-
dimers 4 have been isolated before. These compounds
are relevant by themselves, as examples of potential
metabolites of 3, but another transformation that they
might undergo in vivo is a progressive deglucosylation
to give, eventually, the two enantiomers of the trans-
dehydrodimer 2. In order to reproduce this biotrans-
formation in vitro, the 1:1 diastereomeric mixture 4
was submitted to the catalytic action of a small library
of commercially available b-glucosidases. To our satis-
faction, with two of these enzymes complementary re-
sults were obtained: while the b-glucosidase from al-
monds allowed the isolation of the monoglucosylated
intermediate 5 in reasonable yield, the cellulase from
Trichoderma viride was able to fully hydrolyze 4 to
give 2 (Figure 4).
Figure 2. HPLC chromatograms (Lux 5u Cellulose-1, Phe-
nomenex) of: (A) “racemic” 4, analytical chiral column; (B)
The almond b-glucosidase-catalyzed reaction was
repeated on a preparative scale either on 4 to give the
mixture of diastereomeric trans-monoglucosides 5,
which were then separated by semi-preparative chiral
“
racemic” 4, semi-preparative chiral column; (C) 4a; (D)
b.
4
spectra. For instance, Figure 3 compares some of the HPLC to give 5a and 5b, or on the previously purified
signals due to the two sugar moieties in the mixture 4 4a and 4b, which allowed the direct isolation of 5a
and in the two isolated compounds 4a and 4b. A table and 5b. The positive ESI mass spectrum of 5 showed
1
13
with the full set of data (both H and C NMR) as a molecular peak at 639.5 Da, thus confirming the
well as copies of the respective NMR spectra are loss of a single glucose moiety. Structure determina-
available in the Supporting Information.
tion was established by careful NMR investigations
Adv. Synth. Catal. 2015, 357, 1831 – 1839
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Paolo Gavezzotti et al.
FULL PAPERS
moiety are attributed from that of the natural (À)-e-
viniferin, an isomeric resveratrol dimer of 2 isolated,
[
15a]
i.e., from the plant Carex pumila.
By analysis of its
electronic circular dichroism spectrum, the absolute R
configurations were assigned to its two chiral cen-
[
15]
ters.
The purified enantiomers 2a and 2b were subjected
to electronic circular dichroism (ECD) measurements
to establish their absolute configuration at the stereo-
genic centers C-2 and C-3. The absolute configura-
tions of 2a and 2b were determined by comparison of
their ECD spectra with that reported for (À)-e-vini-
ferin. Both 2a and (À)-e-viniferin showed a negative
Cotton effect in the range 230–240 nm, and conse-
quently, the 2R,3R configuration was assigned to 2a
and the 2S,3S configuration to 2b. Figure 6 reproduces
the ECD spectra of the enantiomers 2a and 2b.
Figure 4. TLC of the glucosidases-catalyzed hydrolysis of 4.
A) Reaction catalyzed by the b-glucosidase from almonds.
(
From left to right: a) 4; b) reaction mixture plus 4; c) reac-
tion mixture. (B) Reaction catalyzed by the cellulase from
Trichoderma viride. From left to right: a) 4 and 5 standards;
b) reaction mixture plus 4 and 5; c) reaction mixture; d) d-
glucose standard.
that clearly proved the almond b-glucosidase-cata-
lyzed deglucosylation at C-11.
Specifically, from the NOESY spectrum it was ob-
served that H-2 and H-3 had an NOE connection
with the signals at 6.40 and 6.33 ppm, due to H-6’’ and
H-2’’. The residual anomeric proton also showed an
NOE connection with the already mentioned signal at
6.40 (H-6’’) and with the signal at 6.49 ppm (H-4’’).
These observations allowed us to establish unequivo-
cally that the residual glucose unit was linked to the
phenolic C-5’’. The full set of NMR data of 5a and 5b
as well as copies of NMR spectra and chiral HPLC
chromatograms are also presented in the Supporting
Information.
Due to solubility problems, which hampered the
separation of racemic 2 with the chiral semi-prepara-
tive column, the enantiomers of the trans-dehydro-
dimer of resveratrol 2a and 2b were obtained by the
T. viride cellulase-catalyzed hydrolysis of the previ-
ously isolated diastereoisomers 4a and 4b. As shown
in the HPLC chromatograms reported in Figure 5,
both the compounds were obtained with very high ee
values (>99% and 98.3%, respectively, for 2a and 2b.
With a different chiral column, ChiralPack IA, the re-
tention time was inverted and therefore the enantio-
mer 2b, obtained from 4b, was eluted first). Mass
spectra and NMR data were in accordance with the
proposed structures.
In the literature the absolute configurations of Figure 5. HPLC chromatograms (Chiralpack IA column) of:
structures containing the 1,2-diaryldihydrobenzofuran (A) racemic 2; (B) 2a; (C) 2b.
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Laccase-Catalyzed Dimerization of Piceid, a Resveratrol Glucoside
glucosylated dimer 2. This low value, that is an indica-
tion of its higher stability towards radical oxidation,
might provide a reason for the good yield in 4 of the
laccase-catalyzed oxidation of 3.
Conclusions
We have shown that the sequential combination of
biocatalyzed reactions (oxidation, hydrolysis) and of
semi-preparative HPLC chromatography is a suitable
methodology to isolate diasteromerically (4a, 4b, 5a,
5
b) or enantiomerically (2a, 2b) enriched derivatives
of the phytoalexin resveratrol. The protocol is reliable
and easily scalable. Moreover, from an experimental
point of view, the treatment of the glucosylated deriv-
atives of resveratrol and of its b-5 like dimer is more
efficient than the manipulation of the corresponding
“naked” compounds. Further investigations of the
properties of the reported derivatives, particularly of
the two enantiomers 2a and 2b, in more specific bio-
logical studies are currently in progress and the re-
sults will be reported in due course.
Experimental Section
Figure 6. Electronic circular dichroism spectra and chemical
structures of 2a and 2b (2a c; 2b g; MeOH ---).
General Experimental Procedures
Optical rotations were measured on a Jasco P-2000 polarim-
eter (Cremella, IT). Electronic circular dichroism spectra
were recorded on a Jasco J-600 spectropolarimeter. NMR
spectra were recorded on Bruker AC500 spectrometer
Having this set of dehydrodimers (2, 4, 5) in hands,
the first obvious investigation was an estimation of
their antiradical activity in comparison with their pa- (500 MHz) in DMSO-d and CD OD. Mass spectra were re-
6
3
rents resveratrol 1 and piceid 3, activity evaluated corded on a Bruker Esquire 3000 Plus spectrometer. HPLC
spectrophotometrically as the ability of the tested analyses were carried out using a Jasco 880-PU pump
equipped with a Jasco 875-UV/Vis detector. HPLC condi-
substances to reduce 1,1-diphenyl-2-picrylhydrazyl
[16]
tions: Lux 5u Cellulose-1 Phenomenex 150”4.60 mm analyt-
ical column, Lux 5u Cellulose-1 Phenomenex 250”10.0 mm
semi-preparative column, isocratic mobile phase water/ace-
tonitrile (relative ratio reported in the following para-
(
DPPH) radical. Compounds 1–5 were all able to
scavenge the stable model DPPH radical with various
efficiencies. The results, reported in Table 1, showed
the expected similar IC50 values for resveratrol 1 and
piceid 3 and a lower antiradical activity for the trans-
dehydrodimers 2, 4, 5 (in agreement with data that
À1
À1
graphs), flow rate 1.0 mLmin and 4.0 mLmin respective-
ly at 258C, detection at 310 nm; ChiralPack IA Daicel
Chemical Industries 250”4.60 mm analytical column, iso-
had previously been obtained with other stilbenic de- cratic mobile phase petroleum ether/2-propanol (relative
[14a]
rivatives ). Quite unexpectedly, however, the IC50 ratio reported in the following paragraphs), flow rate
À1
of the diglucosylated dimer 4 was more than one 1.0 mLmin at 258C, detection at 310 nm. Thin-layer chro-
matography (TLC): precoated silica gel 60 F254 plates
order of magnitude higher than that of the fully de-
(
Merck), developed with the molybdate reagent
[
(NH ) Mo O ·4H O, 42 g; Ce(SO ) , 2 g; H SO conc.,
4 6 7 24 2 4 2 2 4
6
2 mL; made up to 1 L with deionized water]; flash chroma-
Table 1. DPPH scavenging by hydroxystilbenes and their re-
spective dimers.
tography: silica gel 60 (70–230 mesh, Merck). Reactions
were carried out using a G24 Environmental Incubator New
Brunswick Scientific Shaker (Edison, USA).
Compound
IC50 (mM)
1
2
3
4
5
28.7Æ2.9
95.1Æ4.5
Enzymes and Materials
33.2Æ2.1
Laccase from Trametes versicolor (20 U/mg), b-glucosidase
from almonds (ꢀ2 U/mg) and cellulase from Trichoderma
viride (3–10 U/mg) were from Sigma–Aldrich. 3,5,4’-Trihy-
1395.9Æ102.4
624.1Æ32.6
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Paolo Gavezzotti et al.
FULL PAPERS
droxystilbene-3-O-b-d-glucopyranoside (piceid 3, 97%
purity) was from Sigma–Aldrich. Piceid 3: H NMR
tention time of 58.2 min, was isolated: yield:165 mg,
1
1
0.212 mmol; de >99%. H NMR (DMSO-d ): d=7.41 (1H,
6
(
(
DMSO-d ): d=7.41 (2H, d, J=8.5 Hz, H-2’, H-6’), 7.04
1H, d, J=16.5 Hz, H-b), 6.87 (1H, d, J=16.5 Hz, H-a),
br.d, J=8.5 Hz, H-6), 7.23 (1H, br.s, H-4), 7.15 (2H, d, J=
8.5 Hz, H-2’ and H-6’), 7.05 (1H, d, J=16.5 Hz, H-8), 6.89
(1H, d, J=8.5 Hz, H-7), 6.86 (1H, d, J=16.5 Hz, H-9), 6.73
(2H, d, J=8.5 Hz, H-3’ and H-5’), 6.68 (1H, br.t, J=1.7 Hz,
H-10), 6.54 (1H, br.t, J=1.7 Hz, H-14), 6.36 (1H, t, J=
2.2 Hz, H-4’’), 6.33 (1H, t, J=2.2 Hz, H-12), 6.26 (1H, br.t,
J=1.7 Hz, H-6’’), 6.23 (1H, br.t, J=1.7 Hz, H-2’’), 5.44 (1H,
d, J=7.5 Hz, H-2), 4.79 (1H, d, J=7.7 Hz, H-1’’’’), 4.77 (1Ha,
d, J=7.7 Hz, H-1’’’), 4.50 (1H, d, J=7.5 Hz, H-3), 3.70
6
6
.77 (2H, d, J=8.5 Hz, H-3’, H-5’), 6.74 (1H, br.s, H-2), 6.58
1H, br.s, H-6), 6.35 (1H, t, J=2 Hz, H-4), 4.81 (1H, d, J=
.5 Hz, H-1’’), 3.74 (1H, d, J=12 Hz, H-6’’), 3.51 (1H, dd,
J =12 Hz, J =6 Hz, H-6’’), 3.33 (1H, dd, J =9.6 Hz, J =
(
7
1
2
1
2
2
.4 Hz, H-2’’), 3.34–3.21 (2H, m, H-3’’ and H-4’’), 3.19 (1H,
td, J =4.2 Hz, J =3.6 Hz, J =4.8 Hz H-5’’). The NMR data
1
2
3
[17]
were in accordance to the literature values. All other re-
agents were of the best purity grade from commercial sup-
pliers.
a
(1H, dd, J =11.9 Hz, J =2.4 Hz, H-6a’’’), 3.60 (1H, dd,
1
2
J =11.9 Hz, J =2.0 Hz, H-6a’’’’), 3.48 (2H, dd, J =11.9 Hz,
1
2
1
J =5.2 Hz, H-6b’’’ and H-6b’’’’), 3.32–3.15 (8H, m, H-2’’’, H-
2
a
3
’’’, H-4’’’, H-5’’’, H-2’’’’, H-3’’’’, H-4’’’’ and H-5’’’’) [ =assign-
Laccase-Catalyzed Oxidation of Piceid 3 to 4
13
ments may be interchanged]; C NMR (DMSO-d ): d=
6
a
a
a
Piceid (3, 1000 mg, 2.56 mmol), dissolved in 40 mL of meth-
anol was added to 100 mL of 20 mM sodium acetate buffer,
pH 5.0, in which the laccase from Trametes versicolor
1
1
1
1
1
1
1
7
7
6
59.31 (br) (C-11), 158.90 (br) (C-13), 158.87 (C-7a),
a
a
a
58.74 (C-4’), 158.60 (C-3’’), 158.48 (br) (C-5’’), 143.74 (C-
’’), 139.05 (C-9a), 131.08 (C-3a), 130.28 (C-5), 129.50 (C-1’),
28.23 (C-8), 127.72 (C-6), 127.56 (C-2’,6’), 126.04 (C-9),
22.82 (C-4), 115.46 (C-3’,5’), 109.22 (C-7), 108.52 (C-2’’),
07.47 (C-14), 106.39 (C-6’’), 104.46 (C-10), 103.11 (C-12),
02.06 (C-4’’), 100.54 (C-1’’’’), 99.95 (C-1’’’), 92.42 (C-2),
6.97 (C-3’’’), 76.77 (C-5’’’), 76.68 (C-3’’’’), 76.55 (C-5’’’’),
3.25 (C-2’’’), 73.12 (C-2’’’’), 69.77 (C-4’’’), 69.47 (C-4’’’’),
0.69 (C-6’’’), 60.43 (C-6’’’’), 55.51 (C-3) [
(
18 mg, 86 U) had been previously dissolved. The solution
was incubated at 258C in an open flask under moderate stir-
ring and the conversion was monitored by TLC (mobile
phase: chloroform-methanol-water 8:4:0.5). After 210 min
the reaction was stopped by adding 50 mL of 3M NaCl solu-
tion and the water phase was extracted with AcOEt. Follow-
ing drying by sodium sulfate addition and washing of the
salt with methanol, the solvent was evaporated under re-
duced pressure to give the dimeric product 4 as an amor-
phous brownish powder, obtained without any further pu-
b
b
b
b
c
c
d
d
e
e
a,b,c,d,e
=assign-
ments may be interchanged].
The almost pure diastereoisomer 4b, eluted with a reten-
tion time of 80.7 min, was also isolated: yield: 175 mg,
rification; isolated yield: 455 mg (0.58 mmol, 45.7%); R =
1
f
0.225 mmol, de 98.4%. H NMR (DMSO-d ): d=7.40 (1H,
6
1
0
8
.31. H NMR (DMSO-d ): d=7.419, 7.415 (1H, d, J=
6
dd, J =8.4 Hz, J =2.0 Hz H-6), 7.23 (1H, br.s, H-4), 7.14
1
2
.5 Hz, H-6), 7.217, 7.206 (1H, br.s, H-4), 7.183, 7.179 (2H,
(
2H, d, J=8.5 Hz, H-2ꢁ and H-6’), 7.05 (1H, d, J=16.3 Hz,
m, H-2’ and H-6’), 7.058, 7.054 (1H, d, J=16.4 Hz, H-8),
H-8), 6.88 (1H, d, J=8.4 Hz, H-7), 6.86 (1H, d, J=16.3 Hz,
H-9), 6.71 (2H, d, J=8.5 Hz, H-3’ and H-5’), 6.66 (1H, br.t,
J=1.7 Hz, H-10), 6.54 (1H, br.dd, J =2.1 Hz, J =1.5 Hz,
H-14), 6.35 (1H, t, J=2.2 Hz, H-4’’), 6.31 (1H, t, J=2.2 Hz,
H-12), 6.23 (1H, br.t, J=1.7 Hz, H-6’’), 6.21 (1H, br.t, J=
6
1
6
1
6
1
5
7
.897 (1H, d, J=8.4 Hz, H-7), 6.865, 6.858 (1H, d, J=
6.4 Hz, H-9), 6.762, 6.758 (2H, m, H-3’ and H-5’), 6.703,
.699 (1H, br.t, J=1.8 Hz, H-10), 6.563, 6.559 (1H, br.t, J=
.7 Hz, H-14), 6.369, 6.365 (1H, t, J=2.1 Hz, H-4’’), 6.328,
.322 (1H, t, J=2.3 Hz, H-12), 6.305, 6.291 (1H, br.t, J=
.7 Hz, H-6’’), 6.263, 6.244 (1H, br.t, J=1.7 Hz, H-2’’), 5.456,
.448 (1H, d, J=7.6 Hz, H-2), 4.800, 4.777 (1H, d, J=
1
2
1
.7 Hz, H-2’’), 5.42 (1H, d, J=7.7 Hz, H-2), 4.80 (1H, d, J=
7
.7 Hz, H-1’’’), 4.72 (1H, d, J=7.7 Hz, H-1’’’’), 4.49 (1H, d,
a
J=7.7 Hz, H-3), 3.70 (1H, dd, J =11.9 Hz, J =2.2 Hz, H-
1
2
.7 Hz, H-1’’’), 4.793, 4.727 (1H, d, J=7.7 Hz, H-1’’’’), 4.507
a
6
a’’’), 3.65 (1H, dd, J =11.9 Hz, J =2.0 Hz, H-6a’’’’), 3.48
1
2
a
(
1H, br.d, J=7.6 Hz, H-3), 3.691 (1H, dd, J =11.8 Hz, J =
1
2
(2H, dd, J =11.9 Hz, J =5.3 Hz, H-6b’’’ and H-6b’’’’), 3.33–
1
2
a
a
2
2
.3 Hz, H-6a’’’), 3.647 , 3.592 (1H, dd, J =11.9 Hz, J =
.1 Hz, H-6a’’’’), 3.34–3.13 (4H, m, H-2’’’, H-3’’’, H-4’’’ and
1
2
3.15 (8H, m, H-2’’’, H-3’’’, H-4’’’, H-5’’’, H-2’’’’, H-3’’’’, H-4’’’ꢁ
a 13
and H-5’’’’) [ =assignments may be interchanged]; C NMR
H-5’’’), 3.32–3.15 (4H, m, H-2’’’’, H-3’’’’, H-4’’’’ and H-5’’’’),
a
a
(
DMSO-d ): d=159.91 (br) (C-4’), 159.32 (br) (C-3’’),
6
a
b
a
(
H-6b’’’ and H-6b’’’’) [ =assignments may be interchanged;
1
5
5
1
59.16 (br) (C-13), 158.87 (C-7a), 158.73 (C-11), 158.67 (C-
’’), 143.47 (C-1’’), 138.96 (C-9a), 131.15 (C-3a), 130.23 (C-
), 128.92 (C-1’), 128.08 (C-8), 127.66 (C-6), 127.66 (C-2’,6’),
26.07 (C-9), 122.77 (C-4), 115.46 (C-3’,5’), 109.21 (C-7),
b
=
obscured by water]. MS (ESI): m/z=801.6 Da (M+
Na ). A sample of 4 was submitted to HPLC analysis using
+
a chiral column (Lux 5u Cellulose-1 Phenomenex, 150”
4
.6 mm, mobile phase: water/acetonitrile 8:2): 4a, t =
R
108.70 (C-6’’), 107.69 (C-14), 106.10 (C-2’’), 104.04 (C-10),
103.16 (C-12), 102.27 (C-4’’), 100.45 (C-1’’’’), 100.19 (C-1’’’),
1
1.7 min; 4b, t =16.9 min.
R
b
b
b
9
2.52 (C-2), 76.93 (C-3’’’), 76.87 (C-5’’’), 76.70 (C-3’’’’),
b c c d
Preparative Scale HPLC Separation of the Two trans
Diastereoisomers 4a and 4b
76.59 (C-5’’’’), 73.26 (C-2’’’), 73.18 (C-2’’’’), 69.79 (C-4’’’),
d e e
69.64 (C-4’’’’), 60.69 (C-6’’’), 60.58 (C-6’’’’), 55.54 (C-3)
a,b,c,d,e
[
=assignments may be interchanged].
The separation of the two trans diastereoisomers 4a and 4b
was carried out by HPLC-UV using a chiral column (Lux 5u
Cellulose-1 Phenomenex, 250”10.0 mm) and an isocratic
Screening of Glycosidases for the Selective
Deglucosylation of the Dimer 4
À1
elution (water/acetonitrile, 83:17; flow rate 4.0 mLmin at
2
9
58C; detection at 310 nm). 15 mg of 4 were dissolved in
00 mL of the same mixture of eluents and injected. The sep-
Piceid dimer 4 (25 mg, 0.032 mmol), dissolved in 800 mL of
methanol, was added to 5.8 mL of 10 mM sodium acetate
buffer, pH 5.0, in which the b-glucosidase from almonds
aration protocol was repeated in order to separate 500 mg
of 4. The almost pure diastereoisomer 4a, eluted with a re-
1836
asc.wiley-vch.de
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Synth. Catal. 2015, 357, 1831 – 1839

FULL PAPERS
Laccase-Catalyzed Dimerization of Piceid, a Resveratrol Glucoside
(
34 mg, 18 U) had been previously dissolved. The solution
8:3:0.3) to give the corresponding monoglycoside 5; isolated
was incubated at 258C and the conversion was monitored by
TLC (mobile phase: chloroform-methanol-water 6:4:0.5).
The reaction was stopped before complete conversion and
the water phase was extracted with AcOEt. Following
drying by sodium sulfate addition and washing of the salt
with methanol, the solvent was evaporated under reduced
pressure and the crude residue was purified by flash chro-
matography (mobile phase: chloroform-methanol-water
yield: 116.0 mg (0.188 mmol, 48.9%); R =0.65.
f
Preparative Scale HPLC Separation of the Two trans
Diastereoisomers 5a and 5b
The separation of the two trans diastereoisomers 5a and 5b
was carried out by HPLC-UV using a chiral column (Lux 5u
Cellulose-1 Phenomenex, 250”10.0 mm) and an isocratic
elution (water/acetonitrile, 78.5:21.5; flow rate 4.0 mLmin
at 258C; detection at 310 nm). 5 mg of 5 were dissolved in
À1
8
:3:0.3) to give the product 5 as an amorphous brownish
powder; isolated yield: 8.6 mg (0.014 mmol, 43.8%); R =
f
+
9
00 mL of the same mixture of eluents and injected. The sep-
0
.65. MS (ESI): m/z=639.5 Da (M + Na ). A sample of 5
was submitted to HPLC analysis using a chiral column (Lux
u Cellulose-1 Phenomenex, 150”4.6 mm, mobile phase:
water/acetonitrile 75:25): 5a, t =11.8 min; 5b, t =15.3 min.
aration protocol was repeated in order to separate 100 mg
of 5. The almost pure diastereoisomer 5a, eluted with a re-
5
tention time of 53.5 min, was isolated: yield: 42.4 mg,
R
R
1
0
.069 mmol; de 95.8%. H NMR (CD OD): d=7.36 (1H,
Piceid dimer 4 (25 mg, 0.032 mmol), dissolved in 500 mL
of methanol, was added to 6.5 mL of 10 mM sodium acetate
buffer, pH 5.0, in which the cellulase from Trichoderma
viride (18 mg, 108 U) had been previously dissolved. The so-
lution was incubated at 308C and the conversion was moni-
tored by TLC (mobile phase: chloroform-methanol-water
3
ddt, J =8.2 Hz, J =1.9 Hz, J =0.5, H-6), 7.16 (2H, m, H-2ꢁ
1
2
3
and H-6’), 7.14 (1H, d, J=1.9 Hz, H-4), 6.96 (1H, d, J=
1
1
2
6.3 Hz, H-8), 6.84 (1H, d, J=8.3 Hz, H-7), 6.77 (1H, d, J=
6.3 Hz, H-9), 6.77 (2H, m, H-3’ and H-5’), 6.48 (1H, t, J=
.2 Hz, H-4’’), 6.42 (2H, dd, J =2.2 Hz, J =0.5 Hz, H-10
1
2
and H-14), 6.39 (1H, dd, J =2.2 Hz, J =1.4 Hz, H-6’’), 6.29
6
:4:0.5). The reaction was stopped after complete conver-
1
2
(
1H, dd, J =2.2 Hz, J =1.4 Hz, H-2’’), 6.14 (1H, t, J=
sion and the water phase was extracted with AcOEt. Follow-
ing drying by sodium sulfate addition, the solvent was
evaporated under reduced pressure to give the product 2 as
1
2
2
7
.2 Hz, H-12), 5.40 (1H, d, J=8.1 Hz, H-2), 4.86 (1H, d, J=
.5 Hz, H-1’’’), 4.45 (1H, d, J=8.1 Hz, H-3), 3.78 (1H, dd,
J =12.0 Hz, J =2.4 Hz, H-6a’’’), 3.68 (1H, dd, J =12.0 Hz,
an amorphous brownish powder; isolated yield: 14.3 mg
1
2
1
1
J =4.8 Hz, H-6b’’’), 3.45–3.38 (4H, m, H-2’’’, H-3’’’, H-4’’’,
(
0.031 mmol, 98.0%); R =0.85. H NMR (CD OD): d=7.35
2
f
3
1
3
H-5’’’); C NMR (CD OD): d=160.96 (C-7a), 160.41 (C-
(
1H, dd, J =8.2 Hz, J =1.9 Hz, H-6), 7.17 (1H, br.d, J=
3
1
2
5
1
5
1
1
1
7
’’), 160.29 (C-3’’), 159.73 (C-11,13), 158.95 (C-4’), 145.55 (C-
1.9 Hz, H-4), 7.16 (2H, m, H-2ꢁ and H-6’), 6.96 (1H, d, J=
16.3 Hz, H-8), 6.84 (1H, d, J=8.2 Hz, H-7), 6.78 (1H, d, J=
16.3 Hz, H-9), 6.77 (2H, m, H-3ꢁ and H-5’), 6.42 (2H, dd,
a
a
’’), 141.13 (C-9a), 132.64 (C-1’), 132.41 (C-3a), 132.34 (C-
), 129.34 (C-8), 128.72 (C-6), 128.60 (C-2’,6’), 127.60 (C-9),
24.13 (C-4), 102.88 (C-12), 116.45 (C-3’,5’), 110.52 (C-2’’),
10.39 (C-7), 108.66 (C-6’’), 105.89 (C-10,14), 103.82 (C-4’’),
J =2.2 Hz, J =0.5 Hz, H-10 and H-14), 6.19 (1H, t, J=
1
2
2
2
.2 Hz, H-4’’), 6.14 (1H, t, J=2.2 Hz, H-12), 6.13 (2H, d, J=
.2 Hz, H-2’’ and H-6’’), 5.38 (1H, d, J=8.5 Hz, H-2), 4.39
b
b
01.85 (C-1’’’), 94.79 (C-2), 77.96 (C-3’’’), 77.92 (C-5’’’),
a,b
1
3
4.81 (C-2’’’), 71.09 (C-4’’’), 62.26 (C-6’’’), 58.65 (C-3) [
=
(
1H, d, J=8.5 Hz, H-3); C NMR (CD OD): d=161.01 (C-
3
assignments may be interchanged].
The almost pure diastereoisomer 5b, eluted with a reten-
7
a), 159.94 (C-3’’,5’’), 159.63 (C-11,13), 158.71 (C-4’), 145.41
a
a
(C-1’’), 141.19 (C-9a), 132.89 (C-1’), 132.40 (C-3a), 132.36
C-5), 129.42 (C-8), 128.67 (C-6), 128.63 (C-2’,6’), 127.49 (C-
tion time of 69.4 min, was isolated: yield: 48.2 mg,
(
1
0
.078 mmol; de 92.3%. H NMR (CD OD): d=7.36 (1H,
9), 124.17 (C-4), 116.33 (C-3’,5’), 110.35 (C-7), 107.77 (C-
3
ddt, J =8.2 Hz, J =1.9 Hz, J =0.6, H-6), 7.17 (1H, br.d, J=
2
’’,6’’), 105.86 (C-10,14), 102.77 (C-4’’), 102.53 (C-12), 94.88
1
2
3
a
1
1
1
2
.9 Hz, H-4), 7.16 (2H, m, H-2ꢁ and H-6’), 6.96 (1H, d, J=
6.3 Hz, H-8), 6.84 (1H, d, J=8.2 Hz, H-7), 6.77 (1H, d, J=
6.3 Hz, H-9), 6.77 (2H, m, H-3ꢁ and H-5’), 6.49 (1H, t, J=
(
(
C-2), 58.77 (C-3) [ =assignment may be interchanged]. MS
ESI): m/z=477.4 Da (M+Na ). A sample of 2 was submit-
+
ted to HPLC analysis using a chiral column (ChiralPack IA
Daicel Chemical Industries 250”4.6 mm, mobile phase: pe-
troleum ether/2-propanol 8:2): 2a, t =54.1 min; 2b, t =
.2 Hz, H-4’’), 6.42 (2H, dd, J =2.2 Hz, J =0.5 Hz, H-10
1
2
and H-14), 6.40 (1H, dd, J =2.2 Hz, J =1.4 Hz, H-6’’), 6.33
1
2
R
R
(
1H, dd, J =2.2 Hz, J =1.4 Hz, H-2’’), 6.14 (1H, t, J=
33.1 min.
1
2
2
7
.2 Hz, H-12), 5.40 (1H, d, J=8.0 Hz, H-2), 4.78 (1H, d, J=
.6 Hz, H-1’’’), 4.45 (1H, d, J=8.0 Hz, H-3), 3.82 (1H, dd,
J =12.1 Hz, J =2.4 Hz, H-6a’’’), 3.69 (1H, dd, J =12.1 Hz,
b-Glucosidase-Catalyzed Deglucosylation of 4 to the
Monoglycoside 5
1
2
1
J =5.2 Hz, H-6b’’’), 3.44–3.37 (4H, m, H-2’’’, H-3’’’, H-4’’’,
2
1
3
H-5’’’); C NMR (CD OD): d=160.96 (C-7a), 160.49 (C-
3
Piceid dimer 4 (300 mg, 0.385 mmol), dissolved in 10 mL of
methanol, was added to 70 mL of 10 mM sodium acetate
buffer, pH 5.0, in which the b-glucosidase from almonds
5
1
5
1
1
1
7
’’), 160.00 (C-3’’), 159.65 (C-11,13), 158.81 (C-4’), 145.36 (C-
a
a
’’), 141.14 (C-9a), 132.66 (C-1’), 132.37 (C-3a), 132.28 (C-
), 129.36 (C-8), 128.77 (C-6), 128.70 (C-2’,6’), 127.54 (C-9),
24.07 (C-4), 102.82 (C-12), 116.38 (C-3’,5’), 110.31 (C-2’’),
10.39 (C-7), 108.99 (C-6’’), 105.88 (C-10,14), 103.93 (C-4’’),
(
410 mg, 219 U) had been previously dissolved. The solution
was incubated at 258C and the conversion was monitored by
TLC (mobile phase: chloroform-methanol-water 6:4:0.5).
The reaction was stopped before complete conversion and
the water phase was extracted with AcOEt. Following
drying by sodium sulfate addition and washing of the salt
with methanol, the solvent was evaporated under reduced
pressure and the crude residue was purified by flash chro-
matography (mobile phase: chloroform-methanol-water
b
b
02.20 (C-1’’’), 94.72 (C-2), 78.02 (C-3’’’), 77.97 (C-5’’’),
a,b
4.83 (C-2’’’), 71.21 (C-4’’’), 62.35 (C-6’’’), 58.65 (C-3) [
=
assignments may be interchanged].
Adv. Synth. Catal. 2015, 357, 1831 – 1839
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
asc.wiley-vch.de
1837

Paolo Gavezzotti et al.
FULL PAPERS
Cellulase-Catalyzed Deglucosylation of 4a and 4b to
À)-2a and (+)-2b
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Acknowledgements
This work was supported by the program “Suschem Lombar-
dia: prodotti e processi chimici sostenibili per l’industria lom-
barda”, Accordo Quadro Regione Lombardia-CNR (proto-
col no. 18096/RCC). The Czech-Italian collaborative project
between CNR and AVCR (2013–2015) and the COST Action
“System Biocatalysis” CM-1303 (MSMT LD13041) are also
acknowledged. Moreover S.R. acknowledges support from
MIUR, Italy, Progetto PRIN 2011 “PROxy”.
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ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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