Unexpected different chemoselectivity in the aerobic oxidation of methylated planar catechin and bent epicatechin derivatives catalysed by the Trametes villosa laccase/1-hydroxybenzotriazole system

Article abstract of DOI:10.1039/c3ra47753c

Unreported methylated catechin and epicatechin derivatives 5 and 6 were synthesized by an oxa-Pictet-Spengler reaction. Catechin 5 shows the B and C rings coplanar because of the formation of a trans junction between the C ring and the newly generated six

Full text of DOI:10.1039/c3ra47753c

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Unexpected different chemoselectivity in the
aerobic oxidation of methylated planar catechin
and bent epicatechin derivatives catalysed by the
Trametes villosa laccase/1-hydroxybenzotriazole
system†
Cite this: RSC Adv., 2014, 4, 8183
a
a
b
Roberta Bernini, Fernanda Crisante, Patrizia Gentili, Sergio Menta,c
*
*
Fabio Moranab and Marco Pierinic
Unreported methylated catechin and epicatechin derivatives 5 and 6 were synthesized by an oxa-Pictet-
Spengler reaction. Catechin 5 shows the B and C rings coplanar because of the formation of a trans
junction between the C ring and the newly generated six-term cycle D, in turn condensed to ring B. In
contrast, epicatechin 6 presents a bent geometry due to the establishment of a cis junction between the
C ring and the newly formed cycle D. The oxidation of compounds 5 and 6 in the presence of the
Trametes villosa laccase/1-hydroxybenzotriazole (HBT) system was investigated under aerobic conditions
in both a biphasic system and a reverse micelle. The unexpected different chemoselective oxidation at
the benzylic position of catechin and epicatechin derivatives 5 and 6 has been rationalized using a
molecular modelling approach. These results demonstrate that the Trametes villosa laccase/HBT system
represents a useful tool to functionalize the C-2 or C-4 position of phenolic compounds depending on
the structural features.
Received 5th August 2013
Accepted 6th January 2014
DOI: 10.1039/c3ra47753c
www.rsc.org/advances
Being biodegradable and able to perform oxidations under
mild experimental conditions, their use presents several
Introduction
advantages. Laccases can catalyse the monoelectronic oxidation
of both organic compounds3 and inorganic ions4 with the
simultaneous reduction of molecular dioxygen to water without
the intermediate production of hydrogen peroxide. Laccases'
potential use in oxidation reactions can be increased by
combination with low molecular weight compounds termed
mediators. These compounds enable these enzymes to oxidize
substrates with a redox potential above 1.3 V (e.g. benzylic
alcohols and ethers) by means of an indirect way where the
active species is the oxidized form of the mediator.5
Mediators can act as an “electron shuttle”: once oxidized by
laccases and converted to more or less stable radicals, they
diffuse far away from the enzymatic pocket and, by mechanisms
different from the enzymatic one, enable the oxidation of target
compounds that in principle are not substrates of laccases
because of their large size or high redox potential (Scheme 1).6
Laccases (benzenediol: oxygen oxidoreductase, EC 1.10.3.2)
belong to a small group of enzymes known as the blue copper
oxidases. These enzymes contain four catalytic copper atoms
distributed in three redox sites. Originally described in plants,1
laccases can also be found in fungi and bacteria.2 Fungal lac-
cases have higher redox potentials than bacterial or plant lac-
cases, and their action appears to be relevant in nature, namely
in the degradation of lignin – a nding with important impli-
cations for the eld of biotechnology. Laccases are highly
regarded as environmentally friendly catalysts in oxidation
reactions.
aDipartimento di Scienze e Tecnologie per l'Agricoltura, le Foreste, la Natura e
`
l'Energia (DAFNE), Universita degli Studi della Tuscia, Via S. Camillo De Lellis,
01100 Viterbo, Italy. E-mail: berninir@unitus.it; Fax: +39 0761 357242; Tel: +39
0761 357452
b
`
Dipartimento di Chimica and IMC-CNR Sezione Meccanismi di Reazione, Universita
degli Studi La Sapienza, P. le A. Moro 5, 00185 Roma, Italy. E-mail: patrizia.gentili@
uniroma1.it; Fax: +39 06 49913692; Tel: +39 06 49913082
c
`
Dipartimento di Chimica e Tecnologia del Farmaco, Universita degli Studi La
Sapienza, P. le A. Moro 5, I-00185 Roma, Italy
† Electronic supplementary information (ESI): 1H-NMR and 13C-NMR spectra of
compounds 5, 6, 7, 8 and 9; computational methods and Cartesian coordinates
of all the optimized molecular structures. See DOI: 10.1039/c3ra47753c
Scheme 1 The catalytic cycle of the laccase-mediator (Med) system.
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(+)-Catechin 1 and (ꢀ)-epicatechin 2 are representative of oxidation of methylated planar catechin 5 and bent epi-
members of avonoids, a naturally occurring class of plant catechin 6 has been rationalized using a molecular modelling
phenolic compounds widely distributed in foods and beverages, approach.
mainly in green and black tea (Fig. 1).7 Their biological and
pharmacological effects, in particular radical scavenger activity,
are well known. In the last few years, a synthetic catechin
Results and discussion
1. Synthetic aspects
analogue in which the chroman and catechol moieties of 1 are
coplanar by formation of a bridge between the 3-OH group on
ring C and C-60 on ring B has been identied as a novel and
efficient antioxidant with reduced pro-oxidant activity poten-
tially useful for the prevention and treatment of free radical-
associated diseases.8
Catechin and epicatechin derivatives 5 and 6 were synthesized
via an oxa-Pictet-Spengler reaction from the corresponding
tetramethylated catechin 3 and epicatechin 4 in acetone using
BF3–Et2O as Lewis acid (Scheme 2) according to Fukuhara's
protocol.8,13
The enzymatic oxidations of polyphenols, and in particular
of catechins, are widely described in the literature, being
implicated in numerous biological processes in the vegetal
world.9 In contrast, only a few articles have reported on the
oxidative enzyme-catalysed chemistry of catechin derivatives
for synthetic purposes.10 This lack of literature data is mainly
due to the complexity of the reaction mixtures and the
corresponding difficulties in isolating nal oxidation prod-
ucts that are generally dimeric, oligomeric and polymeric
compounds.
Taking into consideration our recent results regarding the
oxidation of methylated catechins 3 and 4 with Trametes villosa
laccase using 1-hydroxybenzotriazole (HBT) as mediator11 and
as part of our research devoted to the selective oxidation of
natural compounds in order to obtain ne chemicals and
bioactive compounds,12 we projected the synthesis of new taxi-
folin-like compounds from methylated planar catechin 5 and
methylated bent epicatechin derivative 6. Methylated catechin
derivative 5 shows the B and C rings coplanar because of the
formation of a trans junction between the C ring and the newly
generated six-term cycle D, in turn condensed to ring B
according to the synthetic catechin analogue reported by
Fukuhara (Fig. 1).8 Contrarily, due to the establishment of a cis
junction between the C ring and the newly formed cycle D, in its
two more stable conformations modelled by DFT calculations
(see below), methylated epicatechin derivative 6 presents bent
geometry. The unexpected observed different chemoselectivity
Next, we focused our attention on their behaviour in air and
in the presence of Trametes villosa laccase/HBT as the catalytic
system. Based on our previous study on the oxidation of cate-
chins 3 and 4,11 we initially chose aqueous sodium acetate
buffer/tetrahydrofuran as the reaction medium. Unfortunately,
despite the retained activity of the enzyme in this medium,
methylated planar catechin 5 was not soluble. Therefore, owing
to inactivation of the enzyme when the content of water-
miscible organic solvent in the system increases14 and in an
effort to use laccase in the presence of an organic solvent, we
decided to perform the oxidation of catechin derivative 5 by the
Trametes villosa laccase/HBT system in two types of reaction
medium: (i) an aqueous sodium acetate buffer/ethyl acetate
biphasic system15 and (ii) a sodium dioctylsulfosuccinate salt
(AOT) reverse micelle system in isooctane/chloroform.16,17 In
both these reaction environments, aer 24 h, the laccase/HBT
oxidation of methylated planar catechin 5 afforded exclusively
compound 7 in 82% and 79% yields, respectively (Scheme 3;
Table 1, entries 1 and 2), deriving from oxidation at the C-2
position.
In contrast, methylated bent epicatechin 6 was soluble in
aqueous sodium acetate buffer/tetrahydrofuran, and aer
50 h it afforded the oxidation products 8 and 9 in 64% and
18% yields, respectively (Scheme 4; Table 1, entry 3).
Scheme 2 Synthesis of methylated planar catechin 5 and methylated
bent epicatechin 6.
Fig.
1 Chemical structures of natural and synthetic catechin
derivatives.
Scheme 3 Oxidation of methylated planar catechin derivative 5.
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Table 1 Experimental data of oxidation of catechin derivatives 5 and 6
Entry Substrate Reaction medium
Products (yield %)
1
2
3
5
5
6
NaOAc buffer/ethyl acetatea
Isooctane/chloroform/AOTa
NaOAc buffer/
7: 82
7: 79
8: 64; 9: 18
tetrahydrofuranb
4
5
6
6
NaOAc buffer/ethyl acetatea
Isooctane/chloroform/AOTa
8: 71; 9: 21
8: 59; 9: traces
a
b
25 ꢁC, 24 h. 25 ꢁC, 50 h.
Scheme 5 Oxidation of catechin derivatives 5 and 6 in (a) the biphasic
system and (b) the reverse micelle.
Scheme 4 Oxidation of methylated bent epicatechin derivative 6.
2. Mechanistic aspects
To shed light on such an intriguing nding, we undertook
studies to investigate more precisely the origin of this different
chemoselectivity. As described in the literature regarding the
oxidation of a benzylic alcohol or ether derivatives by the lac-
case/HBT system, the key step is the abstraction of a hydrogen
atom from the benzylic position by the oxidized form of the
mediator, the benzotriazole-N-oxyl (BTNO) radical (Scheme 6).6
a. Determination of oxidation potential. Redox features of
the substrate have no major impact upon this radical H-atom
transfer (HAT) route, where only the enthalpic balance between
the breaking and forming of bonds is relevant.6
Indeed, determination of the oxidation potential by cyclic
voltammetry in acetonitrile containing tetrabutylammonium
tetrauoroboride as supporting electrolyte indicated that
methylated catechin and epicatechin derivatives 5 and 6 were
irreversibly oxidized at a potential above 1.65 V (Table 2), thus
conrming that they are resistant to monoelectronic oxidation
Both products derive from oxidation at the C-4 position;
compound 9 is a product of over-oxidation of the aromatic
A ring.
The oxidation reactions conducted in the biphasic system
and in the reverse micelle produced the same products 8 and 9
aer only 24 h (Scheme 4; Table 1, entries 4 and 5). As far as we
know, this is the rst description of an oxidation reaction cat-
alysed by the laccase/HBT system in either a biphasic system or
a reverse micelle environment for synthetic purposes. Of note,
the oxidation of methylated planar catechin derivative 5 and
bent epicatechin 6 in these reaction media indicates that HBT
acts as a “shuttle” connecting the organic phase, where
substrates 5 and 6 are soluble, and the aqueous phase, where
laccase retains its catalytic activity (Scheme 5). The use of these
reaction media offers the additional advantage that further
laccase addition during the oxidation is not necessary, as is
required for the aqueous sodium acetate buffer/tetrahydrofuran
medium (see Experimental section).11
The experimental data evidence that oxidation of methylated
bent epicatechin 6 by the laccase/HBT system leads to C-4
functionalization, in accordance with our previous results
obtained by oxidation of methylated catechins 3 and 4.11
Unexpectedly, methylated planar catechin 5 is oxidized on the
C-2 position. These results demonstrate the synthetic potenti-
ality of the oxidative procedure in avonoid chemistry and, in
general, medicinal chemistry in order to produce a rearranged
core structure or a ketone derivative depending on the struc-
tural features.
Scheme 6 Catalytic cycle of the laccase/HBT system in the oxidation
of a benzylic alcohol or ether derivatives.
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Table 2 Oxidation potential, rate constant kH and dissociation energies of C-2–H and C-4–H bonds of catechin derivatives 5 and 6
BDEC–H (kJ molꢀ1 d
)
Sub
E
p (V, vs. NHE)a
kH (Mꢀ1 sꢀ1 b
)
Sub.conf.c
HFe
DFT2//HFf
DFTg
DFT2//DFTh
5C-2 (1i)
5C-4 (2i)
6C-2 (1i)
6C-4 (2i)
1.75
1.69
61 ꢃ 1
56 ꢃ 1
5C-2
5C-4
6aC-2
6aC-4
6bC-2
6bC-4
259.6
291.4
244.6
289.4
268.7
281.3
355.7
380.1
350.6
370.4
360.7
385.3
354.5
378.9
358.6
375.4
359.6
381.9
355.2
373.2
a
b
[Substrate]: 2 mM, at 500 mV sꢀ1 in CH3CN containing Bu4NBF4 (0.1 M). Rate constants obtained spectrophotometrically at 25 ꢁC in CH3CN;
c
d
initial condition: [BTNO] ¼ 0.5 mM, [substrate] ¼ 5–40 mM; determination in triplicate. Calculated conformations of substrate. From the
following equation: BDEC–H ¼ DHꢁf (R) + DHꢁf (H) – DHꢁf (R–H); DHꢁf (H) ¼ 218.0 kJ molꢀ1
31G*. h B3LYP/6-311+G**//B3LYP/6-31 G*. i Number of equivalent H atoms.
.
HF/3-21G. B3LYP/6-311+G**//HF/3-21G. B3LYP/6-
e
f
g
21
and cannot be oxidized by Trametes villosa laccase (Eꢁ 0.79 V/ values (Table 2) indicated for both planar and bent catechin
NHE)18 directly.
tetramethyl ethers 5 and 6 a more advantageous H-abstraction
b. H-abstraction reactivity. We focused our efforts on from carbon C-2 (359.6 and 355.2 kJ molꢀ1, respectively) than
investigating the HAT reactivity (kH) of BTNO from methylated from C-4 (381.9 and 373.2 kJ molꢀ1, respectively). Moreover, the
catechin derivatives 5 and 6 by using UV-Vis spectroscopy and values attributed to the C-2 radical formation were found to be
monitoring the disappearance of the OD at l ¼ 474 nm (OD474
)
even smaller than those calculated elsewhere for 3 and 4 (377.4
of BTNO under reported conditions in CH3CN solution at 25 and 367.4 kJ molꢀ1, respectively).11 However, such ndings can
ꢁC.11,19 Briey, BTNO was generated in the spectrophotometric only explain the C-2 functionalization observed in the oxidation
cuvette by quickly mixing a 0.5 mM solution of monoelectronic of the planar catechin 5, but do not justify the reactivity man-
oxidant (NH4)2Ce(NO3)6 (CAN) with a 0.5 mM HBT solution, ifested by compound 6 that led to the formation of 8 and 9 (i.e.
both in CH3CN at 25 ꢁC. The reading of the initial absorption at the reactivity at carbon C-4). Again, taking inspiration from our
l ¼ 474 nm conrmed quantitative generation of BTNO. Rapid previous study involving, amongst others, catechins 3 and 4,11 we
addition of a solution of the C–H-bearing compound marked theoretically evaluated the possibility that the observed C-4
the beginning of the kinetic experiment; 3–5 initial concentra- reactivity of 6, assessed as thermodynamically disadvantageous,
tions of the substrate were typically investigated at concentra- may originate from kinetic factors. For this purpose, four rele-
tions exceeding that of BTNO by 10–80-fold. The time-dependent vant transition states (TS) simulating H-abstraction from either
depletion of BTNO at OD474 was monitored by stopped-ow the C-2 or C-4 position of 5 and 6a were also modelled (i.e. the
spectrophotometry because the reaction times were generally in structures hereaer denoted as TS_5C-2-BTNO, TS_5C-4-BTNO,
the few seconds range. Under these pseudo rst-order condi- TS_6aC-2-BTNO and TS_6aC-4-BTNO; Fig. S1;† details are given in
tions, the H-abstraction rate from the substrate was higher than the Experimental section and ESI†). From inspection of the
the spontaneous decay of BTNO (kdecay ¼ 6.3 ꢂ 10ꢀ3 sꢀ1).19 The achieved energetic results (Table S1 of ESI†), it is clear that
pseudo rst-order rate constants k0, determined from at least methylated catechin and epicatechin derivatives 5 and 6 (in its
duplicated experiments, were converted into second-order H- more stable and greatly dominant 6a conformation) display a
abstraction rate constants (kH) by determining the slope of a k0 very different reactive behaviour towards oxidation. Indeed,
versus [subst]0 linear plot (Table 2). As expected, methylated catechin 5 shows an energetically more favourable and then
5
catechin derivative 5 is about twice more reactive than epi- faster H-abstraction from the C-2 carbon atom (DDEC-2–C-4
¼
catechin derivative 6 (kH/number of equivalent H atoms ¼ ETS_5C-2ꢀBTNO ꢀ ETS_5C-4-BTNO ¼ ꢀ12.30 kJ molꢀ1 from M06-2x
61 Mꢀ1 sꢀ1 and 28 Mꢀ1 sꢀ1, respectively) due to the presence, in calculations), whereas this occurs from the C-4 position for
6
the rst one of the two, of the lone-pair of the oxygen atom O-1 epicatechin 6 (DDEC-2–C-4 ¼ ETS_6aC-2-BTNO ꢀ ETS_6aC-4-BTNO
¼
that weakens the adjacent C-2–H bond and stabilizes the 10.85 kJ molꢀ1 from M06-2x calculations). This means that, at
intervening C-2-radical generated by H-abstraction.20
25 ꢁC and referring both the C-2 and C-4 HAT transition states of
each catechin 5 or 6 to a relevant and common ground state
adduct (hereaer denoted as 5:BTNO and 6a:BTNO, respec-
tively), for compound 5 the generation of radical C-2 is assessed
3. Rationalization of the observed chemoselectivity
Estimations of BDEC–H values for H-abstraction from the C-2 or about 143 times faster than that of radical C-4, whereas for
C-4 position of each compound 5 and 6 (one representative compound 6 it is estimated that radical C-2 is generated about 80
conformation for 5 and two for 6, hereaer denoted as 6a and times slower than radical C-4. Interestingly, stable ground states
6b) were performed by quantum chemical calculations based on of complexes 5:BTNO and 6a:BTNO displaying the BTNO oxygen
the hybrid Hartree-Fock (HF)/Density Functional Theory (DFT) virtually equidistant from both hydrogen atoms linked to
method (for details see ESI†). Similar to what was previously carbons C-2 and C-4 (i.e. just geometries eligible to be considered
observed for the parent structures 3 and 4,11 the achieved BDEC–H as the common starting point leading to both transition states
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of lignin or manganese peroxidases. 1H NMR and 13C NMR
spectra were recorded on a spectrometer 200 and 300 MHz
using CDCl3 as solvent. All chemical shis are expressed in
parts per million (d scale) and coupling constants are in Hertz
(Hz). HPLC analysis was performed with a C18 column (150 mm
ꢂ 4.6 mm ꢂ 5 mm) and a UV detector at l ¼ 280 nm. Samples
were eluted at a ow rate of 1.0 mL minꢀ1 with the following
method: acetonitrile/water ¼ 10/90 (0–10 min); linear gradient
until a ratio of acetonitrile/water ¼ 60/40 (10–25 min); aceto-
nitrile/water ¼ 60/40 (5 min); linear gradient until a ratio of
acetonitrile/water ¼ 80/20 (30–35 min) and then acetonitrile/
water ¼ 80/20 (5 min). High-resolution mass spectra (HRMS)
were performed with an Electrospray Ionization Time of Flight
Micromass spectrometer. Kinetic studies were performed with a
stopped ow instrument interfaced to a diode array spec-
trophometer having a thermostated cuvette holder.
Fig. 2 Calculated structures of methylated planar catechin 5 and
methylated bent epicatechin 6 in their free states (top position);
ground state adducts with BTNO (middle position); and transition state
adducts with BTNO (bottom position).
Purication of laccase
Laccase from a strain of Trametes villosa (viz. Poliporus pinsitus)
(Novo Nordisk Biotech) was employed. It was puried by ion-
exchange chromatography on a Q-Sepharose column (pre-
equilibrated with 10 mM Tris–HCl buffer at pH 7.5, 1 mS) by
elution with a mixture of 10 mM Tris–HCl and 0.2 M NaCl
(linear gradient 1–70 mM NaCl); laccase fractions having an
absorption ratio A280/A610 of 20–30 were considered sufficiently
pure.22 The collected fractions were concentrated by dialysis in
cellulose membrane tubing (Sigma) against poly(ethylene
glycol) to a nal activity of 5277 U mLꢀ1, as determined spec-
trophotometrically by the standard assay with ABTS (where 1 U
is the number of mmol of ABTS oxidized by 1 mL of laccase
solution per min).23
TS_XC-2-BTNO and TS_XC-4-BTNO, with X being equal to 5 or 6;
Fig. 2 and ES1†) have indeed been obtained by suitable
optimization at equilibrium geometry level of the structures
TS_5C-2-BTNO and TS_6aC-4-BTNO.
Therefore, in the case of epicatechin 6, the whole of the
quoted ndings resolutely agrees with the interpretation of a
kinetic control of the process prevailing on the thermodynamic
one, providing in this way a theoretical persuasive ration-
alization of the differential chemoselective oxidizability dis-
played by compounds 5 and 6. By visual analysis of the
optimized structures obtained for TS_5C-2-BTNO, TS_5C-4
-
BTNO, TS_6aC-2-BTNO and TS_6aC-4-BTNO, a more direct rela-
tionship due to steric effects can be established between the
observed chemoselectivity and the planar or bent structure of
the considered catechins, as described in detail in the ESI.†
Synthesis of catechin and epicatechin derivatives
(a). Synthesis of tetramethylated catechin 3 and epi-
catechin 4. (+)-Catechin 1 or epicatechin 2 (290 mg, 1.0 mmol)
was solubilized in acetone (10 mL) at 25 ꢁC. Potassium
carbonate (828 mg, 6.0 mmol) and dimethyl sulfate (3.15 g, 25
mmol) were then added. Aer 24 h, the substrate disappeared
and the solvent was evaporated under reduced pressure. The
mixture was neutralized with 1 M HCl; it was then extracted
with ethyl acetate. The organic phases were recombined and
dried over Na2SO4. Following evaporation of the solvent and
chromatographic purication of the product on silica gel
(eluent: ethyl acetate/hexane ¼ 1 : 1), methylated catechin 3 and
4 were isolated and characterized by spectroscopic analysis.
Methylated catechin 3. Yellow solid (339 mg, 98%), mp 142–
144 ꢁC (from acetone/hexane). Spectroscopic data were identical
with those in the literature.24
Conclusions
Unreported methylated planar catechin and bent epicatechin
derivatives 5 and 6 were synthesized via an oxa-Pictet-Spengler
reaction and then oxidized in air using Trametes villosa laccase/
1-hydroxybenzotriazole as the catalytic system. The oxidations
were performed in a biphasic system and in a reverse micelle
and were determined to be chemoselective and efficient. To the
best of our knowledge, this is the rst time an exploitation of
laccase/mediator in these reaction media for synthetic purposes
has been reported. The different chemoselective oxidations of
methylated planar catechin 5 (at the C-2 position) and bent
epicatechin derivative 6 (at the C-4 position) were rationalized
using a molecular modelling approach.
Methylated epicatechin 4. Yellow solid (329 mg, 95%), mp 144–
146 ꢁC (from acetone/hexane). Spectroscopic data were identical
with those in the literature.24
(b). Synthesis of methylated planar catechin 5 and meth-
ylated bent epicatechin 6. The synthesis was performed
Experimental
Materials and instruments
All chemicals were of the highest commercially available quality according to Fukuhara procedure.13 Briey, 2.56 mL of BF3–Et2O
and were used as received. Crude laccase from Trametes villosa solution (2.87 g, d ¼ 1.12 g mLꢀ1) was added at 0 ꢁC to a solution
(Novozym 51002) was a gi from Novo Nordisk Co. and was free of 1 g (2.9 mmol) of compound 3 or 4 in 120 mL di acetone. The
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reaction mixture was stirred for 3 h at room temperature then of 0.05 sodium acetate buffer pH 4.7) was added and thus
diluted with diethyl ether. The organic solution was washed reverse micelle solution was prepared (Wo ¼ 15).17 Aer the
with brine, dried over Na2SO4, ltered and concentrated in addition of HBT (5.4 mg, 40 mmol) the reaction was stirred at
vacuum. The residue was puried by silica gel (Merck, silica gel 25 ꢁC for 24 h. Reactions were monitored by TLC analysis. At the
60, 230–400 mesh) column chromatography using hexane/ethyl end of the experiment, the internal standard (1-methyl-6-methoxy-
acetate ¼ 5/1 as eluent. Products 5 and 6 were characterized by 3,4-dihydro-1H-isochromen-7-ol, 7.8 mg, 40 mmol)11 was added
spectroscopic analysis.
and the reaction vessel was dipped into a hot water bath (70–80
Methylated planar catechin 5. White power (817 mg, 73%), ꢁC) to denature the enzyme. The crude was diluted with acetoni-
mp: 136–138 ꢁC (from acetone/hexane). Anal. Found: C, 68.2; H, trile. The yields of the oxidation reactions were determined by
1
6.8; O: 25.0. Calcd. for C22H26O6: C, 68.4; H, 6.8; O, 24.8%. H- HPLC analysis with the internal standard method; suitable
NMR (CDCl3): d 1.57 (6H, s, 2 ꢂ CH3), 2.56 (1H, dd, J1 ¼ 16 Hz, response factors were determined from authentic products.
J2 ¼ 10 Hz, 4-H), 3.07 (1H, dd, J1 ¼ 16 Hz, J2 ¼ 6.0 Hz, 4-H), 3.80
(c). Oxidation in sodium acetate buffer/tetrahydrofuran.
ꢁ
(7H, bs, 2 ꢂ OCH3, 3-H), 3.89 (3H, s, OCH3), 3.96 (3H, s, OCH3), Reaction was performed in air at 25 C in magnetically stirred
4.63 (1H, d, J ¼ 8.0 Hz, 2-H), 6.12 (1H, s, 8-H), 6.20 (1H, s, 6-H), 1 : 1 (v/v) buffered water (pH 4.7, 0.05 M sodium acetate)/THF.
6.56 (1H, s, 50-H), 7.17 (1H, s, 20-H). 13C-NMR (CDCl3): d 26.9, Compound 6 (14 mg, 40 mmol) was solubilized in 2.0 mL of
28.1, 31.6, 55.4, 55.5, 56.0, 56.1, 66.3, 73.3, 75.8, 91.9, 93.2, reaction medium, followed by the addition of HBT (5.4 mg,
102.5, 107.7, 108.0, 125.0, 135.1, 147.9, 148.5, 155.3, 158.9, 40 mmol) and laccase (40 U). Additional aliquots of laccase
159.6.
(40 U) were added every 3 h up until 280 U. Reaction was
Methylated bent epicatechin 6. White power (459 mg, 41%), monitored by TLC analysis. At the end of the experiment (50 h),
mp 146–148 ꢁC (from diethyl ether/hexane). Anal. Found: C, the internal standard (1-methyl-6-methoxy-3,4-dihydro-1H-iso-
68.1; H, 7.0; O, 24.9. Calcd. for C22H26O6: C, 68.4; H, 6.8: O, chromen-7-ol, 7.8 mg, 40 mmol)11 was added. The mixture was
1
24.8%. H-NMR (CDCl3): d 1.51 (3H, s, CH3), 1.53 (3H, s, CH3), extracted with ethyl acetate; the organic phases were washed
2.89 (2H, d, J¼ 4.0 Hz, 4-H), 3.70 (3H, s, OCH3), 3.73 (3H, s, with a saturated solution of NaCl and dried over Na2SO4. The
OCH3), 3.87 (3H, s, OCH3), 3.90 (3H, s, OCH3), 4.27 (1H, m, 3-H), yield of the oxidation reaction was determined by HPLC analysis
4.56 (1H, bs, 2-H), 6.05 (2H, m, 6, 8-H), 6.62 (1H, s, 50-H), 6.86 with the internal standard method; suitable response factors
(1H, s, 20-H). 13C-NMR (CDCl3): d 25.6, 27.6, 31.6, 55.3, 55.4, were determined from authentic products.
56.0, 56.1, 63.3, 70.8, 75.5, 91.6, 93.2, 100.5, 108.3, 112.3, 123.9,
136.0, 147.8, 149.8, 155.0, 158.8, 159.3.
(S)-3-(2-Hydroxy-4,6-dimethoxybenzyl)-6,7-dimethoxy-1,1-dime-
thylisochroman-4-one 7. Yellow oil. 1H-NMR (CDCl3): d 1.49 (6H,
s, 2xCH3), 2.73 (1H, dd, J1 ¼ 15 Hz, J2 ¼ 9.0 Hz, CH2), 3.64 (1H,
dd, J1 ¼ 15 Hz, J2 ¼ 3.0 Hz, CH2), 3.71 (3H, s, OCH3), 3.74 (3H, s,
OCH3), 3.86 (3H, s, OCH3), 3.89 (3H, s, OCH3), 4.44 (1H, dd, J1 ¼
9.0 Hz, J2 ¼ 3.0 Hz, 3-H), 6.02 (1H, d, J ¼ 2.0 Hz, 50-H), 6.11 (1H,
Oxidation of methylated planar catechin (5) and methylated
bent epicatechin (6)
(a). Oxidations in sodium acetate buffer/ethyl acetate d, J ¼ 2.0 Hz, 30-H), 6.52 (1H, s, 8-H), 7.41 (1H, s, 5-H), 8.33 (1H,
(biphasic system). Reactions were performed in air at 25 C in s, OH). 13C-NMR (CDCl3): d 24.9, 26.3, 29.7, 55.2, 55.7, 56.1, 56.2,
ꢁ
magnetically stirred 1 : 1 (v/v) buffered water (pH 4.7, 0.05 M 75.6, 76.4, 91.3, 94.5, 101.9, 105.8, 108.2, 131.9, 143.9, 148.4,
NaOAc)/ethyl acetate. Compound 5 (or 6, 14 mg, 40 mmol) was 154.3, 157.6, 159.0, 160.1, 193.8. HRMS (ESI-TOF) m/z: [M + Na]+
solubilized in 2.0 mL of reaction medium, followed by the Found 425.1576. Calcd. for C22H26O7Na 425.1590.
addition of 1-hydroxybenzotriazole HBT (5.4 mg, 40 mmol) and
(6aS,12aR)-2,3,8,10-Tetramethoxy-5,5-dimethyl-6a,12a-dihydro-
laccase (40 U). Reactions were monitored by TLC analysis. At the isochromeno[4,3-b]chromen-7(5H)-one 8. Yellow oil. 1H-NMR
end of the experiment (24 h), the internal standard (1-methyl-6- (CDCl3): d 1.58 (6H, s, 2xCH3), 3.81 (3H, s, OCH3), 3.89 (3H, s,
methoxy-3,4-dihydro-1H-isochromen-7-ol, 7.8 mg, 40 mmol)11 OCH3), 3.91 (3H, s, OCH3), 3.94 (3H, s, OCH3), 4.15 (1H, d, J ¼
was added. The organic phase was separated and the water 2.0 Hz, 6a-H), 4.99 (1H, d, J ¼ 2.0 Hz, 12a-H), 6.08 (1H, d, J ¼ 3.0
phase was further extracted with ethyl acetate; the combined Hz, 11-H), 6.12 (1H, d, J ¼ 3.0 Hz, 9-H), 6.64 (1H, s, 4-H), 6.89
organic phases were washed with a saturated solution of NaCl (1H, s, 1-H). 13C-NMR (CDCl3): d 27.8, 31.3, 55.9, 56.3, 56.4, 56.5,
and dried over Na2SO4. The yields of the oxidation reactions 71.5, 73.9, 76.4, 93.5, 93.6, 105.0, 108.3, 112.4, 120.7, 136.6,
were determined by HPLC analysis with the internal standard 148.4, 150.7, 163.3, 165.0, 166.7, 186.0. HRMS (ESI-TOF) m/z: [M
method; suitable response factors were determined from + Na]+ Found 423.1420. Calcd. for C22H24O7Na 423.1457.
authentic products. Pure samples of compound 7 (or 8 and 9)
(6aR,12aR)-7-Hydroxy-2,3,10-trimethoxy-5,5-dimethyl-6a,7-di-
were obtained aer chromatographic purications using hydroisochromeno[4,3-b]chromene-8,11(5H,12aH)-dione 9. Yellow
1
mixtures of hexane/ethyl acetate ¼ 1/1 (v/v) and dichloro- oil. H-NMR (CDCl3): d 1.54 (3H, s, CH3), 1.58 (3H, s, CH3), 3.76
methane/methanol ¼ 98/2 (v/v) and characterized by spectro- (3H, s, OCH3), 3.88 (3H, s, OCH3), 3.89 (3H, s, OCH3), 4.17 (1H, d,
scopic and analytical data.
J ¼ 2.0 Hz, 12a-H), 4.32 (1H, dd, J1 ¼ 2.0 Hz, J2 ¼ 1.0 Hz, 6a-H),
(b). Oxidations in isooctane/chloroform/sodium dio- 4.43 (1H, d, J ¼ 1.0 Hz, 7-H), 5.72 (1H, s, 9-H), 6.57 (1H, s, 4-H),
ctylsulfosuccinate salt (AOT) (reverse micelle). Compound 5 (or 6.75 (1H, s, 1-H). 13C-NMR (CDCl3): d 27.8, 31.3, 56.3, 56.4, 57.0,
6, 14 mg, 40 mmol) was solubilized in 2.1 mL of sodium dioctyl 61.4, 62.8, 64.1, 76.4, 92.5, 107.3, 107.9, 112.9, 121.8, 127.5, 128.2,
sulfosuccinate sodium salt (0.2 M)/isooctane/chloroform 5/1 136.1, 148.5, 150.2, 167.4, 193.6. HRMS (ESI-TOF) m/z: [M + Na]+
(v/v) mixture; an aqueous solution of laccase (40 U in 0.113 mL Found 425.1212. Calcd. for C21H22O8Na 425.1226.
8188 | RSC Adv., 2014, 4, 8183–8190
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318–332; (c) F. Xu, T. Damhus, S. Danielsen and
L. H. Ostergaard, in Modern Biooxidation: Enzymes,
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Kinetic procedure
BTNO radical was generated in acetonitrile in a thermostated
quartz cuvette (optical path ¼ 1 cm) by adding a 0.5 mM solu-
tion of CAN to a 0.5 mM solution of HBT. A broad absorption
band developed immediately (8 ms) in the l ¼ 350–600 nm
region (lmax at 474 nm, 3 ¼ 1840 Mꢀ1 cmꢀ1). The rate of decay
was unaffected by the use of degassed acetonitrile. Rate
constants of H-abstraction from catechin derivatives 5 and 6
were determined at 25 ꢁC in acetonitrile by following the
decrease of the absorbance at l ¼ 474 nm of BTNO. The initial
concentrations of the substrate were in the 5 ꢂ 10ꢀ3 to 40 ꢂ
10ꢀ3 M range to enable a pseudo-rst-order treatment of the
kinetic data. A rst order exponential was a good t for plots of
(At ꢀ Aꢁ) vs. time over more than three half-lives; the rate
constants (k0) were obtained. From a plot of four to ve k0 vs.
[substrate]0 data pairs, the second-order rate constant of H-
abstraction (kH) was obtained.
˜
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Cyclic voltammetry
The electrochemical equipment consisted of a computer-
controlled in-house potentiostat with a Vernier Soware Multi
Purpose Laboratory Interface (MPLI) program for Windows. The
three electrodes consisted of a glassy-carbon disk (diameter of 1
mm) working electrode, an Ag/AgCl/KCl 3 M reference electrode
(Eꢁ vs. NHE ¼ Eꢁ(vs. Ag/AgCl) + 0.221), and a Pt auxiliary elec-
trode (1 cm2). All scans were obtained at room temperature. The
cyclic voltammetry scans of 2.0 mM of compounds 5 or 6 in
acetonitrile solution containing 0.1 M Bu4NBF4 as supporting
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electrolyte, were run at a rate of 0.5 V sꢀ1
.
Computational methods
All calculations were performed with soware packages running
on a PC equipped with Quad-Core AMD Opteron(tm) Processor
2376, 2.30-GHz (2 processors), 16 GB of RAM and OS 64-bit
Windows 7 Ultimate. All optimizations of geometries of ground
states, radicals, and transition states were performed by the
computer program SPARTAN 08 (Wavefunction, Inc., Irvine, CA,
USA). All details regarding the performed calculations are
reported in ESI.†25
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8190 | RSC Adv., 2014, 4, 8183–8190
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