Laccase-catalyzed dimerization of glycosylated lignols

Article abstract of DOI:10.1016/j.molcatb.2016.10.019

Phenylpropanoid glucosides (PPGs) are naturally occurring and bioactive phenolic derivatives, largely distributed in plants. In this work different PPGs have been chemically or enzymatically synthesized from the lignols coniferyl and p-coumaryl alcohols as substrates for a laccase-catalyzed oxidative coupling. The biooxidation of these PPGs has been investigated here and novel dihydrobenzofuran-based structurally modified analogues have been isolated and characterized. Specifically, the presence of a carbohydrate moiety increased the water solubility of these compounds and reduced the number of dimeric products, as pinoresinol-like structures could not be formed. Looking for a possible sugar-promoted stereochemical enrichment of the obtained diastereomeric mixtures of dimers, different carbohydrate moieties (D-glucose, L-glucose and the disaccharide rutinose) were considered and the respective d.e. values of the dimeric products were measured by ¹H NMR and HPLC. However, it was found that the sugar substituent had a minor effect on the stereochemical outcome of the radical coupling reactions, the best measured result being a d.e. value of 21%.

Full text of DOI:10.1016/j.molcatb.2016.10.019

Journal of Molecular Catalysis B: Enzymatic 134 (2016) 295–301
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Laccase-catalyzed dimerization of glycosylated lignols
Ivan Bassaninia,b, Paolo Gavezzottia, Daniela Montia, Jana Krejzovác, Vladimír Krˇenc,
Sergio Rivaa,∗
a Istituto di Chimica del Riconoscimento Molecolare (ICRM), CNR, Via Mario Bianco 9, 20131 Milano, Italy
b Università di Milano, Dipartimento di Chimica, Via Golgi 19, Milano, Italy
c Institute of Microbiology, Laboratory of Biotransformation, Czech Academy of Sciences, Vídenˇská 1083, CZ 142 20 Prague, Czechia
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 July 2016
Received in revised form 7 October 2016
Accepted 31 October 2016
Available online 2 November 2016
Phenylpropanoid glucosides (PPGs) are naturally occurring and bioactive phenolic derivatives, largely
distributed in plants. In this work different PPGs have been chemically or enzymatically synthesized
from the lignols coniferyl and p-coumaryl alcohols as substrates for a laccase-catalyzed oxidative cou-
pling. The biooxidation of these PPGs has been investigated here and novel dihydrobenzofuran-based
structurally modified analogues have been isolated and characterized. Specifically, the presence of a car-
bohydrate moiety increased the water solubility of these compounds and reduced the number of dimeric
products, as pinoresinol-like structures could not be formed. Looking for a possible sugar-promoted
stereochemical enrichment of the obtained diastereomeric mixtures of dimers, different carbohydrate
moieties (d-glucose, l-glucose and the disaccharide rutinose) were considered and the respective d.e.
values of the dimeric products were measured by 1H NMR and HPLC. However, it was found that the
sugar substituent had a minor effect on the stereochemical outcome of the radical coupling reactions,
the best measured result being a d.e. value of 21%.
Keywords:
Laccase
Rutinosidase
Biocatalysis
Phenylpropanoids
Biooxidation
© 2016 Elsevier B.V. All rights reserved.
1. Introduction
[6]. Specifically, we have shown that stilbenoids and vinyl phenols
are suitable substrates for these enzymes. Mixture of dimers and
Laccases (benzendiol:oxygen oxidoreductases, EC 1.10.3.2) are
copper-containing oxidases, distributed in plants, fungi and bac-
teria, that catalyze the formation of four highly reactive organic
radicals by reducing molecular oxygen to water [1]. It has been
reported that laccases play, i.a., an important role in the formation
of lignin by sequential oxidative couplings of monolignols, a family
of naturally occurring phenolic compounds [2]. p-Coumaryl (1) and
coniferyl (2) alcohols, Fig. 1, are two major examples of this class
of chemicals.
From a synthetic point of view, these ‘blue’ enzymes are known
to oxidize a wide range of organic molecules, both directly or in
the presence of a low-molecular-weight redox mediator [3]. Our
group has accordingly reported several examples dealing with the
oxidation of sugars [4], alkaloids [5] and, more extensively, phenols
oligomers are always obtained, with the 2,3-dihydrobenzofuran-
based ␤-5-type dimeric structures being isolated as the main
products [7]. These compounds carry one or two stereogenic car-
bons and are always obtained as racemates and, when two adjacent
stereocenters are present (like in the dimers obtained from resver-
atrol [7b]), only the trans-stereoisomers are formed.
The lack of enantioselectivity is a serious synthetic drawback
of these biocatalyzed oxidative couplings. Nature has solved this
problem developing the so-called “dirigent proteins”, a group of
peptides that act as chiral templates and direct the stereochemical
outcome of these processes. A significant example of these “diri-
gent proteins” was isolated from the plant Forsythia intermedia and
used by Davin et al. for the in vitro stereoselective biocatalyzed
synthesis of (+)-pinoresinol (3) by the laccase-catalyzed oxidation
of coniferyl alcohol [8]. Later on, in 2010, Pickel et al. isolated and
clonedan enantiocomplementarydirigentprotein, able to guide the
bio-oxidative coupling toward the formation of (−)-pinoresinol [9].
However, this elegant biomimetic approach is of limited synthetic
appeal as, for any substrate and desired enantiomer of a dimeric
product, a specific dirigent protein should be isolated, providing
that it does exist in Nature.
Abbreviations: E.I., electron ionization; Py, pyridine; DMAP, dimethylaminopy-
ridine; THF, tetrahydrofuran; DBU, 1,5-diazabiciclo(5.4.0)undec-5-ene; TMSOTf,
trimethylsilyl trifluoromethanesulfonate; DCM, dichloromethane; TBAF, tetra-n-
butylammonium fluoride; TBDMS, tert-butyldimethylsilyl; d.e., diastereomeric
excess.
∗
Corresponding author.
E-mail address: Sergio.riva@icrm.cnr.it (S. Riva).
http://dx.doi.org/10.1016/j.molcatb.2016.10.019
1381-1177/© 2016 Elsevier B.V. All rights reserved.

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I. Bassanini et al. / Journal of Molecular Catalysis B: Enzymatic 134 (2016) 295–301
Fig. 1. Compounds 1–7.
To overcome this limitation, Urlacher and coworkers have
2. Experimental
recently proposed to couple the laccase-mediated oxidation of
coniferyl alcohol to a stereoselective reductase that is able to trans-
form the unwanted enantiomer of pinoresinol [10].
2.1. General methods and information
In a different and more general approach, we have exploited the
‘classical’ lipase-catalyzed kinetic resolution. The racemic mixtures
of ␤-5-type dimers derived from the laccase-mediated oxidation
of different vinyl phenols were separated obtaining both the possi-
ble enantiomers as enriched species. The target products could be
isolated with e.e. up to 98% [11].
More recently, both the enantiomers of the ␤-5-type dimers of
resveratrol could be isolated on a preparative scale starting from
the laccase-mediated oxidation of piceid (4), a natural d-glucoside
of resveratrol [12]. A mixture of trans-2R,3R and trans-2S,3S glu-
cosylated dimers was easily isolated in good yield, and the two
diastereomers were separated by preparative HPLC on a chiral
column. The target enantiomers of resveratrol dimers were eventu-
ally obtained with e.e. values up to 97% by glycosidases-catalyzed
hydrolysis of the glucose units.
We thought that this approach could be extended to other
compounds. Phenylpropanoid glucosides (PPGs, also known as glu-
cosylated lignols) are naturally occurring in various plants. As
secondary metabolites, these compounds are described as phy-
topharmaceuticals [13] and they are studied for their potential
biological activities [14]. Specifically, their antioxidant, antidepres-
sant and hypotensive effects have been extensively investigated
[15].
NMR spectra were recorded on Bruker AC400 spectrome-
ter (400 MHz) in MeOH-d4, DMSO-d6 or D2O, MS spectra were
recorded on a Bruker Esquire 3000 Plus Ion Trap spectrometer NMR
and the MS spectra in extenso are available in the Supplementary
materials.
HPLC analyses were carried out using a Jasco 880-PU pump
equipped with a Jasco 875-UV/vis detector. HPLC conditions:
Kinetex 5 ␮m Biphenyl 100 Å Phenomenex 150 × 4.6 mm column,
gradient of H2O: CH3CN as mobile phase (0–1 min 90% H2O, 10%
CH3CN; 1–20 min 80% H2O, 20% CH3CN; 20–22 min 80% H2O, 20%
CH3CN; 22–23 90% H2O, 10% CH3CN), flow rate 0.7 mL min−1 at
25 ◦C, detection at 270 nm. Reactions were monitored by TLC:
precoated silica gel 60 F254 plates (Merck, DE), developed with a
20% solution of H2SO4 in ethanol or using the molybdate reagent
((NH4)6Mo7O24·4 H2O, 42 g; Ce(SO4)2, 2 g; H2SO4 conc., 62 mL;
made up to 1 L of deionized water).
Flash chromatography: silica gel 60 (70–230 mesh, Merck, DE).
Biotransformations were carried out using a G24 Environmen-
tal Incubator New Brunswick Scientific Shaker (Edison, USA) or a
Thermomixer Comfort (Eppendorf, DE).
2.2. Enzymes and chemicals
In this work the laccase-catalyzed oxidative coupling of a small
family of glycosylated phenylpropanoids, previously synthesized
from coniferyl and p-coumaryl alcohol, has been investigated, and
novel, structurally modified analogues of these bioactive natural
compounds have been isolated and characterized. Looking for a
possible sugar-induced stereochemical enrichment of the obtained
diastereomeric mixtures of dimers, different carbohydrate moieties
(d-glucose, l-glucose and the disaccharide rutinose) were consid-
ered.
Laccase from Trametes versicolor was from Sigma-Aldrich. ␣-
l-Rhamnosyl-␤-d-glucosidase (rutinosidase) from Aspergillus niger
was produced according to the listed reference [16]. The enzymes
were used based on their respective activities evaluated according
to literature assays [16,6b].
TBDMS-phenol protected phenylpropanoid alcohols were pre-
pared from their correspondent acids or aldehydes according to
literature [19]. All other reagents were of the best purity grade from
commercial suppliers.

I. Bassanini et al. / Journal of Molecular Catalysis B: Enzymatic 134 (2016) 295–301
297
2.3. Glucose trichloroacetimidate. General experimental
procedure
in vacuo, affording the desired product that was used without
any further purifications.
(i) Peracetylated glucose (1 eq, final concentration 0.1 M) was
added at room temperature to a solution of glacial acetic acid
(3.0 eq) and ethylenediamine (2.5 eq) in tetrahydrofuran. The
resulting mixture was stirred for 3 h in a round bottom flask
cupped with a calcium chloride guard tube. The conversion
of the starting material was checked by TLC (mobile phase
petroleum ether: AcOEt; 4:6). Once the peracetylated glucose
was fully consumed, the reaction mixture was diluted with two
volumes with water and extracted twice with DCM. The com-
bined organic layers were washed with of a 5% aqueous solution
of hydrochloric acid, a saturated aqueous solution of sodium
bicarbonate and water, until pH 7 was reached. After drying
over sodium sulfate, the solvent was concentrated in vacuo
affording the desired product used without any further purifi-
cations.
(iii) Phenol-deprotected phenylpropanoid glucoside (1 eq) was
suspended in MeOH (0.03 M) and, under vigorous stirring, a
0.5 M solution of MeONa in MeOH (2 eq) was added slowly and
dropwise. The resulting mixture was stirred for 30 min. After
that, the reaction was diluted three times with MeOH and a
Dowex® 50WX8 hydrogen form resin was added. The mixture
was then stirred until pH 7.0 was reached. The resin was then
filtered and the solvent concentrated in vacuo, affording the
desired product that was used without any further purifica-
tions.
2.6. Compound (D)-5
According to general procedures 2.5, (D)-5 (463 mg, 1.48 mmol,
oil, isolated yield: 28%) was obtained from phenol-protected
coumaric alcohol (1.4 g, 5.30 mmol) and d-glucose trichloroacetim-
idate (3.6 g, 7.42 mmol).
(ii) 1,5-Diazabicyclo(5.4.0)undec-5-ene (0.4 eq) and trichloroace-
tonitrile (1.1 eq) were added at room temperature and
under nitrogen atmosphere to
a
solution of 2,3,4,6-
1H NMR (400 MHz; D2O): ı 7.32 (d, J = 8.0 Hz, 2H: H-3, H-5), 6.83
(d, J = 8.0 Hz, 2H: H-2, H-4), 6.58 (d, J = 15.6 Hz, 1H: Ha), 6.16-6.12
(m, 1H: Hb), 4.47-4.40 (m, 2H: H-7A, H-8), 4.31-4.27 (m, 1H: H-7B),
3.86 (d, J = 12.6 Hz, 1H: H-13A), 3.69–3.66 (m, 1H: H-13B), 3.44-3.24
(m, 4H: H-9, H-10, H-11, H-12).
tetraacetylglucopyranose (1 eq) in dry DCM (0.2 M). The
resulting mixture was stirred for 45 min. After checking the
complete conversion of the starting material by TLC (mobile
phase petroleum ether: AcOEt; 6:4), the crude product was
concentrated in vacuo and purified by flash column chromatog-
raphy (mobile phase petroleum ether: AcOEt; 7:3), affording
the desired compound.
13C NMR (101 MHz; D2O): ı 155.6, 133.6, 128.9, 128.2, 122.3,
115.7, 101.1, 75.92, 75.90, 73.2, 70.5, 69.7, 60.8, 38.8, 30.3.
MS, m/z ESI = 335.1 Da [M+Na]+.
2.4. Peracetylated d- and l-glucose trichloroacetimidate
2.7. Compounds (D)-6 and (L)-6
d-Glucose: according to the general procedure 2.3, d-glucose
trichloroacetimidate (8.2 g, 17.16 mmol, yellow solid, isolated
yield: 67%) was obtained from d-glucose tetraacetate (10.0 g,
25.62 mmol).
(D)-6: according to general procedure 2.5, fully deprotected
(D)-6 (500 mg, 1.46 mmol, oil, isolated yield: 30%) was obtained
from phenol-protected coniferyl alcohol (1.5 g, 5.10 mmol) and d-
glucose trichloroacetimidate (3.6 g, 7.42 mmol).
1H NMR (400 MHz; CDCl3): ı 5.61–5.55 (m, 1H: H-1), 5.22–5.07
(m, 2H: H-6), 4.31–4.11 (m, 4H: H-2, H-3, H-4, H-5), 2.09 (s, 3H:
H-7), 2.06 (s, 3H: H-7), 2.05 (s, 3H: H-7), 2.03 (s, 3H: H-7).The NMR
data were in accordance to the literature values [17].
l-Glucose: according to general procedure 2.3, l-glucose
trichloroacetimidate (8.0 g, 16.39 mmol, yellow solid, isolated
yield: 64%) was obtained from l-glucose tetraacetate (10.0 g,
25.62 mmol).
1H NMR (400 MHz; MeOD): ı 7.03 (d, J = 1.6 Hz, 1H: H-3), 6.87
(dd, J = 8.4, 1.6 Hz, 1H: H-5), 6.76 (d, J = 8.4 Hz, 1H: H-6), 6.59 (d,
J = 16.0 Hz, 1H: Ha), 6.21 (ddd, J = 16.0, 6.8, 6.0 Hz, 1H: Hb), 4.51 (ddd,
J = 12.4, 6.0, 1.2 Hz, 1H: H-7A), 4.39 (d, J = 7.8 Hz, 1H: H-8), 4.32 (ddd,
J = 12.4, 6.8, 1.2 Hz, 1H: H-7B), 3.90 (dd, J = 11.9, 2.0 Hz, 1H: H-13A),
3.88 (s, 3H: OCH3), 3.70 (dd, J = 11.9, 5.4 Hz, 1H: H-13B), 3.41–3.23
(m, 4H: H-9, H-10, H-11, H-12).
13C NMR (101 MHz; MeOD): ı 147.7, 146.3, 132.9, 129.0, 122.4,
119.8, 114.8, 109.3, 101.8, 76.74, 76.57, 73.7, 70.3, 69.6, 61.4, 55.0.
MS, m/z ESI = 365.2 Da [M + Na]+.
The NMR data were in accordance to the values listed for the
d-glucoside.
(L)-6: according to general procedure 2.5, fully deprotected (L)-
6 (280 mg, 1.13 mmol, oil, isolated yield: 20%) was obtained from
phenol-protected coniferyl alcohol (1.6 g, 5.44 mmol) and l-glucose
trichloroacetimidate (3.6 g, 7.42 mmol).
The NMR data were in accordance to the values listed for the
d-glucoside.
2.5. Chemical glucosylation. General experimental procedure
(i) Under nitrogen atmosphere, glucose trichloroacetimidate
(5 eq) and crushed 4 Å molecular sieves were added to a stirring
solution of TBSM-protected phenylpropanoid alcohol (1 eq) in
dry DCM (0.1 M). After cooling to −15 ◦C, trimethylsilyl tri-
fluoromethanesulfonate (0.03 eq), dissolved in dry DCM, was
added dropwise. The resulting mixture was stirred for 30 min
at −15 ◦C. The reaction was subsequently quenched with tri-
ethylamine (0.5 eq), filtered through a Celite®545 pad and
the solvent concentrated in vacuo. The desired fully-protected
product was purified by flash column chromatography (mobile
phase petroleum ether: AcOEt; 8:2).
2.8. Biocatalyzed rutinosylation: compound 7
Rutin (2.5 g, 4.16 mmol) was added step by step (625 mg, 0.5 eq
per time) within a reaction time of 8 h, to a solution of coniferyl
alcohol (500 mg, 2.81 mmol, 0.128 M) and rutinosidase (0.037 U) in
22 mL of a 85:15 mixture of citrate-phosphate buffer (0.05 M, pH
5.0) and DMSO. The obtained suspension was incubated at 35 ◦C
and 1000 rpm. The reaction was monitored by TLC (MeOH: AcOEt:
HCOOH; 2:8:0.1). When all the starting alcohol was consumed,
the reaction mixture was cooled to room temperature, diluted ten
times with citrate-phosphate buffer and centrifuged, recovering
only the supernatant. After changing the pH from 5.0 to 7.5–7.7,
the aqueous layer was extracted twice with AcOEt and then, after
removing the residual AcOEt via concentration in vacuo, subjected
to an XAD4 solid phase extraction (mobile phase H2O: MeOH, from
(ii) Under nitrogen atmosphere and at 0 ◦C, the fully-protected
phenylpropanoid glucoside (1 eq) was dissolved in dry THF
(0.03 M). A 1 M solution of tetra-n-butylammonium fluoride
(1 eq) in THF was then added and the reaction stirred for
20 min. After checking the conversion of the starting material
by TLC (mobile phase petroleum ether: AcOEt, 1:1), the mix-
ture was diluted with diethyl ether, washed with water, brine
and water again, dried over sodium sulfate and concentrated

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I. Bassanini et al. / Journal of Molecular Catalysis B: Enzymatic 134 (2016) 295–301
100:0 to 0:100), affording 7 (716 mg, 1.72 mmol, yellow solid, iso-
lated yield: 60%).
87.5, 77.33, 77.24, 77.07, 77.01, 73.9, 70.9, 70.47, 70.40, 69.26, 69.20,
61.5, 56.20, 56.17, 56.10, 51.1, 50.8.
1H NMR (400 MHz; MeOD): ı 7.04 (d, J = 2.0 Hz, 1H: H-3), 6.89
(dd, J = 8.4, 2.0 Hz, 1H: H-5), 6.76 (d, J = 8.4 Hz, 1H: H-6), 6.60 (d,
J = 16.0 Hz, 1H: Ha), 6.21 (ddd, J = 16.0, 6.8, 6.0 Hz, 1H: Hb), 4.80 (d,
J = 1.6 Hz, 1H: H-14), 4.48 (ddd, J = 12.3, 6.0, 1.6 Hz, 1H: H-7A), 4.37
(d, J = 7.6 Hz, 1H:H-8), 4.28 (ddd, J = 12.3, 6.8, 0.8 Hz, 1H: H-7B), 4.01
(dd, J = 11.2, 1.6 Hz, 1H: H-13A), 3.88 (s, 3H: = OCH3), 3.73-3.64 (m,
3H: H-13, H-16, H-18), 3.44–3.21 (m, 5H: H-9, H-10, H-11, H-12,
H-17), 1.30 (d, J = 6.4 Hz, 3H: H-19).
MS, m/z ESI = 705.3 Da [M+Na]+.
2.12. Compound 10
According to general procedure 2.9, 10 (109 mg, 0.16 mmol, oil,
isolated yield: 37%) was obtained from compound (L)-6 (380 mg,
0.89 mmol) and laccase (1.90 U).
1H NMR (400 MHz; DMSO-d6): ı 7.14 (s, 0.5H), 7.04 (s, 0.5H),
6.98–6.95 (m, 2H), 6.81–6.74 (m, 2H), 6.58 (d, J = 16.0 Hz, 1H: Ha),
6.27–6.18 (m, 1H: Hb), 5.55 (d, J = 6.0 Hz, 0.5H: Ha’A), 5.50 (d,
J = 7.3 Hz, 0·5H:Ha’B), 4.41 (dd, J = 12.8, 5.5 Hz, 1H: H-7), 4.26–4.16
(m, 3H: H-GlcanomerA, H-GlcanomerB, H-7), 4.08–3.97 (m, 2H), 3.82
(s, 1.5H: OCH3A), 3.81 (s, 1.5H: OCH3B), 3.76 (s, 1.5H: OCH3A), 3.75
(s, 1.5H: OCH3B), 3.72–3.60 (m, 4H), 3.46 (dq, J = 12.0, 6.0 Hz, 3H),
3.21–2.98 (m, 8H).
13C NMR (101 MHz; MeOD): ı 147.7, 146.3, 133.1, 128.9, 122.1,
120.0, 114.8, 109.2, 101.6, 100.9, 76.7, 75.5, 73.7, 72.6, 70.97, 70.83,
70.3, 69.5, 68.4, 54.9, 16.7.
MS, m/z ESI = 511.3 Da [M+Na]+.
2.9. Laccase-mediated dimerization. General experimental
procedure
1C NMR (101 MHz; DMSO-d6): ı 148.02, 147.97, 147.84, 147.75,
146.93, 146.80, 144.22, 144.15, 132.6, 132.33, 132.27, 132.11,
130.78, 130.70, 129.8, 129.2, 124.08, 124.06, 119.2, 118.8, 116.14,
116.05, 115.77, 115.72, 111.16, 111.07, 110.9, 103.45, 103.31, 102.4,
87.57, 87.56, 77.45, 77.39, 77.36, 77.32, 77.26, 74.00, 73.94, 70.61,
70.54, 69.26, 69.20, 61.60, 61.57, 61.54, 56.22, 56.19, 56.11, 51.1,
50.8.
To a solution of phenylpropanoid glycoside (0.05 M) in acetate
buffer (0.02 M, pH 5.0), the needed amount of a 1 mg mL−1
(4.6 U mL−1) laccase solution, prepared in the same buffer, was
added to achieve the value of 2.5 U mmolsubstrate−1. The resulting
mixture was incubated at 30 ◦C and 300 rpm and monitored by TLC
(mobile phase MeOH: chloroform: H2O; 6:4:0.5, or MeOH: AcOEt:
H2O; 6:4:0.5). Once the starting glycoside was consumed, the pH of
the medium was changed to 7.0 and the mixture was lyophilized.
The crude product was purified by flash column chromatography
(mobile phase MeOH: chloroform: H2O; 7:3:0.3 or MeOH: AcOEt:
H2O; 7:3:0.3), affording the desired dimers as a mixtures of trans-
diastereoisomers.
MS, m/z ESI = 705.3 Da [M+Na]+.
2.13. Compound 11
According to general procedure 2.9, 11 (74 mg, 0.07 mmol, oil,
isolated yield: 33%) was obtained from compound 7 (225 mg,
0.46 mmol) and laccase (1.12 U).
1H NMR (400 MHz; D2O, mixture of diastereoisomers): ı 7.01
(br s, 1H), 6.96 (dd, J = 8.5, 1.4 Hz, 1H), 6.86-6.80 (m, 2H), 6.58
(d, J = 15.8 Hz, 1H: Ha), 6.24-6.17 (m, 1H: Hb), 5.60 (d, J = 6.0 Hz,
0.5H: Ha’A), 5.55 (d, J = 6.8 Hz, 0.5H: Ha’B), 4.49–4.42 (m, 2H: H-
GlcanomerA, H-7), 4.38–4.29 (m, 2H: H-GlcanomerB, H-7), 4.08–4.01
(m, 1H), 3.96–3.91 (m, 3H), 3.89-3.83 (m, 5H), 3.80-3.76 (m, 4H),
3.73–3.56 (m, 7H), 3.53–3.23 (m, 11H), 1.25–1.15 (m, 6H).
13C NMR (101 MHz; D2O, mixture of diastereoisomers): ı 147.6,
147.2, 143.8, 133.7, 131.1, 128.4, 123.1, 119.0, 115.86, 115.78, 115.6,
110.9, 110.3, 102.9, 101.4, 100.61, 100.45, 96.1, 88.0, 75.8, 74.7, 73.2,
72.1, 70.28, 70.10, 69.6, 68.7, 66.7, 56.12, 55.97, 50.7, 48.9, 16.7.
MS, m/z ESI = 997.3 Da [M+Na]+.
2.10. Compound 8
According to general procedure 2.9, 8 (31 mg, 0.07 mmol, oil,
isolated yield: 35%) was obtained from compound (D)-5 (63 mg,
0.20 mmol) and laccase (0.32 U).
1H NMR (400 MHz; MeOD, mixture of diastereoisomers): ı
7.54 (s, 0.5H), 7.47 (s, 0.5H), 7.25–7.23 (m, 2H), 6.79–6.74 (m,
3H), 6.64 (d, J = 15.6 Hz, 1H: Ha), 6.29–6.20 (m, 1H: Hb), 5.57 (d,
J = 6.0 Hz, 0.5H: Ha’A), 5.53 (d, J = 6.4 Hz, 0.5H: Ha’B), 4.54-4.48 (m,
1H), 4.40–4.37 (m, 2H: H-anomerA+B), 4.36–4.30 (m, 1H), 4.23–4.15
(m, 1H), 3.92–3.79 (m, 3H), 3.72–3.60 (m, 4H), 3.42–3.22 (m, 14H).
13C NMR (101 MHz; MeOD, mixture of diastereoisomers): ı
181.9, 179.3, 159.6, 157.07, 156.99, 132.86, 132.80, 132.59, 132.39,
130.0, 128.3, 127.06, 126.94, 122.8, 122.4, 114.83, 114.81, 108.6,
103.0, 101.69, 101.64, 87.38, 87.22, 76.74, 76.63, 76.59, 73.7, 70.34,
70.24, 69.6, 61.4.
3. Results and discussion
The convenient use of piceid (4) as starting substrate to obtain
the enantiomeric trans ␤-5-type dimers of resveratrol prompted
us to extend this approach to other phenolic glucosides. Specif-
ically, it was decided to study the laccase-mediated coupling of
phenylpropanoid glucosides, i.e. (D)-5 and (D)-6. To the best of our
knowledge, these compounds were never submitted to the oxida-
tive action of laccases. Moreover, the presence of a sugar moiety
linked to the primary alcohol of the propenol side chain, preventing
the formation of pinoresinol-like structures, would have allowed
to limit the number of dimeric products obtained by radical cou-
pling (a detailed description of the dimeric products that could
be obtained by the laccase/catalyzed oxidation of the aglycone
coniferyl alcohol 2 was reported years ago [18]). In addition, it was
intriguing to verify the effect of the sugar moiety on the stereo-
chemical outcome of the coupling reactions. In fact, at variance to
piceid 4, in these compounds glucose was linked very close to the
carbons (C-a and C-b, Scheme 2) that were going to become stere-
ocenters (C-a’ and C-b’, Scheme 2). Our hypothesis was that bulky
multichiral moiety in the vicinity of the reaction sites would induce
a discrimination among the possible diastereomeric products.
MS, m/z ESI = 645.3 [M+Na]+.
2.11. Compound 9
According to general procedure 2.9, 9 (150 mg, 0.22 mmol, oil,
isolated yield: 34%) was obtained from compound (D)-6 (470 mg,
1.4 mmol) and laccase (2.35 U).
1H NMR (400 MHz; DMSO-d6, mixture of diastereoisomers A
and B): 7.12 (s, 0.5H), 7.03 (s, 0.5H), 6.97–6.94 (m, 2H), 6.81-6.74
(m, 2H), 6.57 (d, J = 16.0 Hz, 1H: Ha), 6.26-6.17 (m, 1H: Hb), 5.55 (d,
J = 6.0 Hz, 0.5H: Ha’A), 5.50 (d, J = 7.2 Hz, 0.5H: Ha’B), 4.40 (dd, J = 12.8,
5.7 Hz, 1H: H-7), 4.27–4.15 (m, 3H: H-GlcanomerA, H-GlcanomerB, H-
7), 4.06–3.96 (m, 2H), 3.81 (s, 2H: OCH3A), 3.80 (s, 1H: OCH3B), 3.75
(s, 1H: OCH3A), 3.74 (s, 2H: OCH3B), 3.72–3.56 (m, 4H), 3.50–3.42
(m, 2H), 3.20–2.98 (m, 9H).
13C NMR (101 MHz; DMSO-d6, mixture of diastereoisomers):
ı 147.95, 147.91, 147.80, 147.72, 144.21, 144.14, 132.7, 132.34,
132.27, 132.18, 130.79, 130.71, 129.8, 129.2, 124.1, 119.1, 118.8,
116.10, 116.00, 115.7, 111.10, 111.08, 111.04, 110.85, 103.4, 102.3,

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299
Scheme 1. Synthesis of the glucopyranosides (D)-5 and (D)-6, reagents and conditions: (a) Py, Ac2O, DMAP, r.t., 24 h; (b) AcOH glacial, NH2(CH2)2NH2, THF, r.t., 3 h; (c) N2,
DBU, CCl3CN, DCM dry, r.t.; (d) TMSOTf, powdered sieves 4 Å, DCM dry, −15 ◦C, 30 min; (e) TBAF, THF dry, 0 ◦C, 2 h; (f) MeONa/MeOH, r.t., 3 h.
Scheme 2. Laccase-mediated synthesis of compounds 8–11, reagents and conditions: Phenylpropanoid glycoside (0.05 M), acetate buffer (0.02 M, pH 5.0), laccase
(0.005 U mgsubstrate−1), at 30 ◦C and 300 rpm, 6–8 h. Isolated yields: 8 = 35%; 9 = 34%; 10 = 37%; 11 = 33%. All products were obtained as mixtures of trans-diastereoisomers.
As shown in Scheme 1, compounds (D)-5 and (D)-6 were
obtained following standard chemical glycosylation protocols,
the key step being the coupling of the trichloroacetimidate of
tetraacetylglucopyranoside to a TBDMS-protected (at the phenolic
moiety) alcohol [19].
tered at 4.263 and the second one at 4.256 ppm (J = 7.6 Hz). Both the
two singlets due to the two methoxy substituents were also split,
resonating respectively at 3.807 and 3.800 ppm and at 3.751 and
3.742 ppm, respectively.
Being intrigued by the observed difference in the relative ratio of
the two diastereoisomers combined in 9, it was decided to synthe-
size the phenylpropanoid glucoside (L)-6, carrying an enantiomeric
sugar moiety, that is l-glucose. The previously described synthetic
protocol (Scheme 1) allowed the isolation of the target product (L)-
6, which was then submitted to the laccase-catalyzed oxidation.
The main product 10 was isolated in yield similar to the previ-
ously described products (37%) and the structure was confirmed
by mass spectrometry and 1H NMR. As expected, the spectra were
generally similar to those previously obtained with 9. Specifically,
the presence of two diastereomeric substituted benzodihydrofuran
moieties was confirmed by the two doublets resonating at 5.554
and 5.504 ppm (J = 6.8 Hz). However, at variance to 9, the relative
ratio of the area of the two doublets of 10 was lower (1.1:1) and,
moreover, the most abundant dimer was the one whose doublet
resonated at higher fields (5.504 ppm, Fig. 2), whereas with 9 it
was the one resonating at lower fields (5.547 ppm). A result, that
was coherent with the presence of two enantiomeric sugars in the
starting substrates (D)-6 and (L)-6.
The water-soluble phenylpropanoid glucosides (D)-5 and (D)-
6 were then submitted to the catalytic action of the laccase from
Trametes versicolor and the corresponding main products, 8 and 9,
were isolated in 35 and 34% yield, respectively (Scheme 2. These
compounds were the less polar UV-active spots in the respective
TLC. A minor, slightly more polar UV-active spot was also observed
in both reactions, but the corresponding products were not iso-
lated. In addition, the formation of a continuous series of spots due
to more polar UV-active and UV-inactive byproducts was observed
at longer reaction times). Their mass spectra showed the expected
values for dimeric products, the quasimolecular peaks (M+Na+)
being registered at 645.3 at 705.3 Da respectively. The trans ␤-
5-type dimeric structures shown in formula 8 and 9 were then
confirmed by the corresponding 1H NMR spectra (full spectra in
Supplementary materials).
In addition to the signals due to the expected seven aromatic
and two olefinic protons, the 1H NMR spectrum of 8 showed
the diagnostic signal due to H-a’. Provided 8 is a mixture of
diastereoisomers, the signals were split in two baseline sepa-
rated doublets (relative ratio almost 1:1, Fig. 2) centered at 5.574
and 5.234 ppm (J = 6.4 Hz). Similarly, the signals due to the two
anomeric protons (doublets) were split in two doublets each
(resonating between 4.403 and 4.347 ppm). All the other signals
appeared as multiplets, due to the duplication of the expected
peaks.
Fig. 2 shows the relative abundance of the signals due to H-a’ in
compounds 8–11.
The difference of reactions outcomes, in terms of diastereomeric
ratio of the dimeric products 9 and 10, was confirmed by HPLC.
Several chiral and achiral columns were tested and the best results
were obtained with a Kinetex 5 ␮m Biphenyl 100 Å column. Fig. 3
shows the baseline separated HPLC peaks of the diastereomeric
mixtures 9 and 10. The low d.e. (18% and 6.0%, respectively) and
the stereocomplementarity of the two reactions outcomes were
confirmed.
As a last substrate of this investigation, a bulkier sugar moiety
was linked to coniferyl alcohol. The choice was on the rutinoside
derivative 7, as the rutinosidase from Aspergillus niger [16] was in
our hands. It has been reported that this enzyme has a strong trans-
glycosylation activity and we could confirm this property. As shown
Similarly, the 1H NMR spectrum of 9 showed the presence of the
five aromatic and two olefinic protons. The signal due to H-a’ was
again split in two baseline separated doublets centered at 5.547 and
5.503 ppm (J = 6.4 Hz, Fig. 2). However, the relative ratio of these
two doublets, evaluated by signals integration, was approximately
1.5:1. Among the two anomeric protons, the first one resonated
as a doublet at 4.217 ppm (J = 8.0 Hz). The second one was split in
two doublets, again in a 1.5:1 ratio, the most abundant being cen-

300
I. Bassanini et al. / Journal of Molecular Catalysis B: Enzymatic 134 (2016) 295–301
Fig. 2. Diastereomeric excesses evaluation: 1H NMR spectra expansions of compounds 8–11.
Scheme 3. Enzymatic transglycosylation to give the disaccharide derivative 7, reagents and conditions: Rutinosidase from A. niger (0.013 U mgalcohol−1), coniferyl alcohol
(0.13 M, 1 eq), rutin (1.25 eq), citrate-phosphate buffer (0.05 M, pH 5.0): dimethyl sulfoxide = 85%: 15%, 35 ◦C and 750 rpm. Isolated yield: 60%.
Table 1
Laccase-catalyzed oxidation of 7 in the presence of water miscible co-solvents.a
Buffer % v/v
Co-solvent % v/v
d.e. of 11 (%)
–
100
70
70
–
5.5
4.5
3.8
8.5
MeOH
CH3CN
Acetone
30
30
30
70
a
All the reactions were stopped after 3 h and analyzed by HPLC (Kinetex 5 ␮m
Biphenyl 100 Å, Phenomenex).
Fig. 3. Diastereomeric excesses evaluation: HPLC chromatograms of compounds 9
and 10.
relative ratio of the two diastereoisomers in 11 did not change
significantly (Table 1).
in Scheme 3, using rutine as a cheap rutinose donor, the desired
product 7 was isolated in one step in 60% yield.
4. Conclusions
Laccase-catalyzed dimerization of 7 was performed following
the usual protocol, and the main product 11 was isolated in 33%
yield. The mass spectrum was in accordance with the structure
of a dimeric product (M+Na+ m/z = 997.3 Da) and the 1H NMR cor-
roborated the structure. The spectrum was quite complex, due to
the presence of two disaccharide moieties and two diastereoiso-
mers. The anomeric protons of the two glucopyranose units were
doublets centered at 4.489 and 4.373 ppm. The presence of the
rhamnopyranose units was confirmed, i.a., by the signals due to the
C-18 methyl groups resonating at 1.174 and 1.238 ppm. The diag-
nostic signals due to H-a’ were two baseline separated doublets at
5.602 and 5.552 ppm (J = 6.4 Hz, Fig. 2) with a relative ratio of 1.16:
1.
It is well-known that the solvent composition can have a deep
influence on the stereoselectivity of the biocatalyzed reactions
[20], and this effect was observed by us also with laccases [6c,6d].
Accordingly, the biooxidation of 7 was also performed in the pres-
ence of different water-miscible co-solvents, in v/v percentages
that do not significantly affect the laccase activity [4a], but the
It has been shown that coumaryl and coniferyl glycosides are
suitable substrates for the laccase from Trametes versicolor. The
presence of a carbohydrate moiety increased the water solubility
of these compounds and reduced the number of dimeric products,
as pinoresinol-like structures could not be formed. However, the
sugar substituents had a minor effect on the stereochemical out-
come of the radical coupling reactions, the best results being the
21% d.e. observed in ␤-5-like dimer 9 obtained by the laccase-
catalyzed dimerization of (D)-6.
Future work will focus on the preparative scale isolation of enan-
tiomerically enriched dimers of coumaryl and coniferyl alcohols
following the protocol previously exploited with resveratrol glu-
coside [12].
Acknowledgments
The authors acknowledge Fondazione Cariplo (Progetto INBOX,
2015–2017) for financial support. The Czech-Italian academic col-

I. Bassanini et al. / Journal of Molecular Catalysis B: Enzymatic 134 (2016) 295–301
301
laborative project between CNR and AVCR (2016–2018) and the
COST Action “Systems Biocatalysis” CM-1303 (MSMT LD15085) are
also acknowledged for mobility support of D.M., S.R., J.K. and I.B.
[6] (a) C. Navarra, C. Goodwin, S. Burton, B. Danieli, S. Riva, J. Mol. Catal. B: Enzym.
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Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.molcatb.2016.10.
019.
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