Laccase-catalyzed phenol oxidation. Rapid assignment of ring-proton deficient polycyclic benzofuran regioisomers by experimental 1H- 13C long-range coupling constants and DFT-predicted product formation

Article abstract of DOI:10.1039/c1ob00012h

Laccase-catalyzed oxidation of substituted catechols followed by reaction with 4-hydroxy-pyrone/-benzopyrone afforded substituted benzofuran regioisomers whose structures with only two aromatic protons in total prevent a rapid structural assignment. Based

Full text of DOI:10.1039/c1ob00012h

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PAPER
Laccase-catalyzed phenol oxidation. Rapid assignment of ring-proton
deficient polycyclic benzofuran regioisomers by experimental 1H–13C
long-range coupling constants and DFT-predicted product formation†
Heiko Leutbecher,a Gerhard Greiner,a Robert Amann,a Andreas Stolz,b Uwe Beifuss*a and Ju¨rgen Conrad*a
Received 4th January 2011, Accepted 1st February 2011
DOI: 10.1039/c1ob00012h
Laccase-catalyzed oxidation of substituted catechols followed by reaction with
4-hydroxy-pyrone/-benzopyrone afforded substituted benzofuran regioisomers whose structures with
only two aromatic protons in total prevent a rapid structural assignment. Based on the evaluation of
1H–13C long-range coupling constants a rule of thumb could be deduced for an easy and unambiguous
differentiation between the possible regioisomers formed. DFT frontier orbital calculations of the
reactants offer an interesting tool to explain the regioselectivity of the key reaction.
Introduction
Natural compounds with a benzofuran skeleton are well known
for their broad range of biological activities. Coumestans with
a benzofuro[3,2-c]chromen-6-one structure are reported to ex-
hibit phytoestrogenic, antibacterial, antifungal, antihepatotoxic
and phytoalexin effects.1 Pyrano[4,3-b]benzofuran-1-ones possess
powerful antioxidant properties combined with a highly effective
UV absorbing functionality making them interesting for cosmetic
and dermatological formulations.2 Oxidation of catechols via
inorganic oxidants,3a electrochemically3b or enzymatically with
tyrosinase3c followed by reaction with substituted 4-hydroxy-2H-
pyran-2-one (1a) or 4-hydroxy-2H-chromen-2-one (1b) provides
access to this class of interesting compounds. Very recently, we have
found a simple and economical laccase-catalyzed domino reaction
for the synthesis of such benzofurans and related structures
(Scheme 1, Table 1).4
However, a major challenge comprises the rapid and unambigu-
ous determination of the regioselectivity of the aforementioned
Scheme 1 Laccase-catalyzed synthesis of regioisomers 3–6.
reactions for the following reasons: many polyphenols suffer
from a lack of aromatic protons in a way that classical NMR
1
1
1
methods such as H-NOE, H-1H-NOESY, H-1H-ROESY can
only be applied to a certain extent—or not at all—for resolving
stereochemical issues and/or regioselectivity. Even more, a limited
sample amount or the absence of single crystals very often
prevents 13C–13C correlations in NMR or X-ray analysis for
an unambiguous assignment of the carbon skeleton, especially
when looking at naturally occurring polyphenols. In those cases,
derivatization reactions followed by time and solvent consuming
work-up procedures as well as second NMR/X-ray analyses have
to be performed. Other approaches to assign carbons in such
protonless molecules rely on the prediction of 13C NMR chemical
shifts by quantum mechanical calculations and/or (semi)empirical
methods using databases and/or incremental algorithms and
subsequent comparison with experimental data.5 Much research
in this area is still being carried out with the focus on the im-
provement of the accuracy of such time-intensive computational
calculations.5 Nevertheless, the predicted chemical shifts could
suffer from inaccuracy depending on the databases6 or the applied
calculation protocols.
aBioorganische Chemie, Institut fu¨r Chemie, Universita¨t Hohenheim, Gar-
benstrasse 30, D-70599, Stuttgart, Germany. E-mail: juergen.conrad@uni-
hohenheim.de; Fax: +49 711 459 22951; Tel: +49 711 459 22944
bInstitut fu¨r Mikrobiologie, Universita¨t Stuttgart, Allmandring 31, D-70569,
Stuttgart, Germany. E-mail: andreas.stolz@imb.uni-stuttgart.de; Fax: +49
711 685 65725; Tel: +49 711 685 65489
† Electronic supplementary information (ESI) available: Results of com-
putational calculations, and 1H and 13C NMR spectra of all compounds.
See DOI: 10.1039/c1ob00012h
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Table 1 Laccase-catalyzed domino reaction of 1a or 1b, respectively, and
and solvent consuming work-up procedures along with a second
NMR analysis, we applied different computational methods for
the prediction of 13C NMR chemical shifts including quantum
mechanical DFT calculations and (semi)empirical methods using
databases and/or incremental algorithms (ACD/Labs).5,7 For
evaluation of this approach we calculated the 13C NMR chemical
shifts of the regioisomers 4a, 3d, 3f and 4f and compared
them with the experimental data. The ring A carbons of 4a
(see above), for instance, were calculated at the DFT GIAO
B3LYP/6-311+G(2d,p)//B3LYP/6-31G(d) level of theory8 to be
at dC 120.47, 149.05, 151.23 and 156.01 ppm with TMS as
reference compound. By an MSTD approach published only
recently,9 comprising benzene as a reference for sp2 carbons the
aforementioned chemical shifts were recalculated at dC 114.19,
142.77, 144.95 and 149.73 ppm which match the experimental
values much better than the calculation with TMS as reference.
The MSTD method with benzene and the mPW1PW91/6-
311+G(2d,p)//mPW1PW91/6-31G(d) level of theory9 resulted
in dC 113.03, 141.30, 143.30 and 147.67 ppm for the ring A
carbons, already close to the experimental data. For comparison,
the predicted chemical shifts by ACD/Labs7 were at dC 118.43,
143.73, 145.93 and 156.61 ppm. Similar consistency between the
experimental and calculated data was observed for 3d: dC(exp.)
112.83, 141.12, 146.19 and 147.77 ppm and dC(calc.) 114.51, 140.30,
146.56 and 150.46 (B3LYP/6-311+G(2d,p)//B3LYP/6-31G(d)).
When comparing the experimental 13C NMR chemical shifts
of the ring A carbons of 3f (dC 112.92, 141.26, 145.14, 147.82
ppm) and 4f (dC 113.01, 142.56, 144.29, 146.68 ppm), only small
shift differences of up to 1.3 ppm were detected. Deviations of
up to 3 ppm between the experimental and calculated chemical
shifts of these carbons, both in 3f (dC(calc.) 112.74, 138.40, 145.09,
149.01 ppm) and 4f (dC(calc.) 115.84, 138.99, 145.44, 147.37 ppm) at
the mPW1PW91/6-311+G(2d,p)//mPW1PW91/6-31G(d) level,
unfortunately did not support any assignment and thus did not
allow a differentiation of those regioisomers. A better accuracy
would probably be achieved by additional diffuse and polarization
functions and/or another basis set such as Aug-CC-pVTZ,8 but
at the expense of much higher computational costs.
2a–f
Entry
1
2
R3
Time (h)
Product(s)
Yield (%)
1
2
3
4
5
6
7
8
9
a
a
a
a
a
a
b
b
b
a
b
c
d
e
f
a
b
e
Me
3
4.5
4
6
4
6
3
5
4
4a
99
51
93
67
85
94
99
61
89
OMe
i-Pr
Cl
CO2Me
Ph
Me
4b
4c
3d
3e, 7a
3f, 4fb
6a
OMe
CO2Me
6b
5e, 8c
a 3e is accompanied by pyrano[4,3-c]isochromen-1,6-dione 7 (3e : 7 = 7 : 3)
(Fig. 1). b 3f and 4f were obtained in a 1 : 4.1 ratio. c 5e is accompanied by
isochromeno[4,3-c]chromen-6,11-dione 8 (5e : 8 = 7.1 : 2.9) (Fig. 1).
Fig. 1 Side products 7 and 8 from domino reaction of 2e and 1a or 1b,
respectively.
In this work we present (i) an experimental methodology for the
rapid differentiation of the synthesized regioisomers based on the
evaluation of experimental 1H–13C long-range coupling constants
and demonstrate that (ii) DFT frontier orbital calculations of the
reactants may offer a promising tool for the explanation of the
regioselectivity of such reactions.
Results and discussion
For our studies of the regioselectivity of the laccase-
catalyzed domino reaction we prepared in total ten pyrano[4,3-
b]benzofuran-1-ones and benzofuro[3,2-c]chromen-6-ones, four
of them (4c, 3d, 3f and 4f) being new compounds (Table 1).
The synthesis of 3e, 5e, 7 and 8 has already been published by
Leutbecher et al. elsewhere.4a
In most cases the domino reaction delivers only one of two
possible regioisomers. However, the ring-proton deficiency in the
products did not allow a rapid structural assignment by standard
NMR methods. Considering, for example, regioisomers 3 and 4,
only one proton represents aromatic ring A and another proton
In the search for more rapid and direct solutions we have found
and evaluated a methodology based on experimental 1H–13C long-
range coupling constants for the unambiguous determination
of such regioisomers independently from 13C NMR chemical
shift considerations. We used the HSQMBC pulse sequence for
1
the rapid and accurate determination of the H–13C long-range
1
coupling constants of all compounds.10 The H–13C long-range
coupling constants between HA and the ring A carbons C-9a, C-5a,
C-7 and C-8 were found to be highly consistent within each set of
regioisomers 3 and 4 despite 13C chemical shift differences for those
carbons due to different substitution patterns (Fig. 2). Since 3JCH
coupling constants in those aromatic ring systems are significantly
greater (absolute values between 4 and 12 Hz) than 2JCH and 4JCH
coupling constants, the carbons with smaller coupling constants
(e.g. 4a: absolute values of 2JCH = 2.1 and 3.1 Hz)11 were assigned
as the carbons at both ortho-positions (C-9a and C-8) and the
carbons with larger coupling constants (i.e. 4a: absolute values of
3JCH = 7.5 and 10.4 Hz) to both meta-positions (C-7 and C-5a)
with respect to HA.
ring B. In such cases, a common gHMBC optimized for JCH
=
8 Hz provides a series of JCH–4JCH long-range correlations. In
compound 4a, the aromatic proton HA (d 7.06 ppm) shows four
correlations with ring A carbons at dC 112.31, 143.36, 143.78 and
148.09 ppm. Due to missing additional aromatic protons in ring
A, those values can not be assigned to the respective positions in
the ring system. Consequently, the substitution pattern of ring A
and the connectivity with the remaining subunit B—thus fixing the
regioselectivity—can not be determined. Unfortunately, in most
NMR solvents the phenolic protons very often appear not as
2
1
sharp singlets but as broad humps which show no H–13C long-
range correlations for assignment purposes at all. To circumvent
derivatization reactions such as methylation and avoiding time
Compared to regioisomers 4, the extracted 1H–13C long-
range coupling constants between HA and the respective carbons
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Fig. 3 Products 5 and 6 from domino reaction of 1b and 2.
Even more, this approach can successfully be exploited for the
fast analysis of mixtures of regioisomers e.g. 3f and 4f obtained as
a 1 : 4.1 mixture. Two big and only one small coupling constants
to oxygen bearing aromatic carbons (10.2, 7.4, 2.9 Hz) establish
structure 4f as the major whereas coupling constants of JCH = 4.2,
4.4, 5.4 and 6.5 Hz indicate 3f as the minor component (Fig. 4).
Fig. 2 1H–13C long-range couplings of HA of 4a and 3d to ring A carbons,
measured by HSQMBC.
C-5a, C-7, C-8 as well as C-9a differed considerably in size of
regioisomers 3, e.g. 3d: JC-5aH = 4.3 Hz, JC-7H = 4.3 Hz, JC-8H
=
A
A
A
6.9 Hz, JC-9aH = 5.7 Hz. However, as both coupling patterns were
A
almost constant within each set of regioisomers 3 and 4 (Table 2),
the following rule could be deduced: 1H–13C long-range coupling
constants of JCH ª 4.3, 4.3, 6.7 and 5.5 Hz to ring A carbons C-5a,
C-7, C-8 and C-9a (Scheme 1, Table 2) establish the structures of
products 3, whereas two big coupling constants of JCH ª 10.4 and
7.3 and a small one of JCH ª 3.0 Hz to the oxygen bearing carbons
C-5a, C-7 and C-8 (Scheme 1, Table 2) indicate the chemical
structures of 4 for these types of compounds.
In order to verify and broaden its applicability we tested the
rule on the determination of the regioisomers 5 and 6 obtained by
laccase-catalyzed domino reaction of catechols 2 with 4-hydroxy-
2H-chromen-2-one (1b) (Scheme 1, Fig. 3) and found, again,
JCH coupling constant patterns for the two regioisomers nearly
identical to 3 and 4 (Table 2).
Fig. 4 Expansion of the HSQMBC of a 1 : 4.1 mixture of 3f and 4f. JCH
coupling constants (absolute values) between HA and ring A carbons are
shown.
Similarly to the 13C NMR considerations we calculated the
1H–13C long-range coupling constants of 3d, 4a, 3f and 4f at
the mPW1PW91/6-311+G(2d,p)//mPW1PW91/6-31G(d) level
of theory to evaluate their suitability for assignment purposes
of the investigated molecules. Comparison with the experimental
data (Table 2, entries 1, 3, 4 and 7) reveals that the calculated values
follow the patterns observed for 3 and 4 despite deviations of up
to 1.6 Hz between the experimental and calculated data. Thus, the
calculation of 1H–13C long-range coupling constants seems to be
a much better option for the differentiation of the regioselectivity
than the 13C chemical shift considerations. However, the accurate
determination of coupling constants requires tremendous compu-
tational time (e.g. 4f: 108 h) in addition to the recording of an
HSQMBC NMR spectrum (e.g. 4f: 8 h).
Table 2 Experimental 1H–13C long-range coupling constants of HA to
ring A carbons of 3 and 4 as well as 5 and 6
Entry
Compound
C-5aa
C-7a
C-8a
C-9aa
In addition to the fast and direct NMR based determination
of the regioselectivity we have also searched for theoretical
alternatives to the time-intensive DFT GIAO 13C NMR approach
and performed DFT frontier orbital calculations of the reactants
at the B3LYP/6-31G(d) level for the prediction of the regioisomers
formed.4c–d,12 Consideration of the frontier orbital energies of 1a
and of reaction intermediate 9 revealed a smaller energy difference
between the HOMO of 1a and LUMOs of 9 than between the
LUMO of 1a and HOMOs of 9 (Table 3). Thus, a nucleophilic
attack of 2H-pyran-2-one 1a on quinones 9 could be expected.
The softer nucleophilic center, e.g. the carbon atom of the enol of
1a (atomic orbital coefficients of the enol: oxygen -0.285, carbon
0.553), should react with the most electrophilic center of quinones
1
2
3
4
5
6
7
3db
3e
4.3 (5.5)
4.4
4.4 (5.2)
10.4 (9.0)
10.4
4.3 (4.5)
4.4
4.2 (5.4)
7.5 (6.7)
7.4
6.9 (6.2)
6.5
6.5 (6.3)
3.1 (3.3)
2.9
5.7 (4.7)
5.7
5.4 (4.8)
2.1 (2.2)
1.9
3fb
4ab
4b
4c
10.3
7.4
2.8
1.8
4fb
10.2 (9.5)
C-10aa
4.5
10.3
10.2
7.4 (7.0)
2.9 (4.5)
1.9 (1.4)
C-6ba
5.8
2.5
2.0
C-9a
C-8a
8
9
10
5e
6a
6b
4.2
7.4
7.5
6.6
3.0
2.8
a JCH in Hz (absolute values). b DFT-calculated JCH of 3d, 4a, 3f and 4f in
parentheses (mPW1PW91/6-311+G(2d,p)//mPW1PW91/6-31G(d)).
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Table 3 Frontier orbital energies of 9a–f and 1a
the nucleophile and the quinones, other effects, e.g. steric factors,
must be involved and influence the reaction kinetics.12,13
Entry
Compound
R
HOMO (eV)
LUMO (eV)
Conclusions
1
2
3
4
5
6
7
9a
9b
9c
9d
9e
9f
Me
-6.69
-6.29
-6.67
-7.16
-7.05
-6.44
-6.15
-3.46
-3.43
-3.42
-3.86
-4.03
-3.49
-1.18
OMe
i-Pr
Cl
CO2Me
Ph
In conclusion, we have synthesized and analyzed the regioisomers
formed by the laccase-catalyzed domino reaction of catechols
and substituted 4-hydroxy-2H-pyran-2-one (1a) or 4-hydroxy-
2H-chromen-2-one (1b). During these studies we have found a
rapid and elegant methodology for the unequivocal determination
of the regioselectivity of those reactions yielding ring-proton
deficient products based on experimental 1H–13C long-range
coupling constants which were partially corroborated by time-
intensive DFT GIAO calculations. Furthermore, frontier orbital
consideration of the intermediates seems to be a valuable tool
for the explanation of the regioselectivity of those reactions.
Overall, we are convinced that both strategies—(i) experimental
1H–13C long-range coupling constants and (ii) frontier orbital
consideration—can be easily and successfully exploited for a much
wider range of even more complicated molecules avoiding quite
often painstaking and time consuming identification procedures.
1a
Experimental
Computational studies
All the calculations reported in this paper were performed within
Density Functional Theory, using the Gaussian 03 package.8
Frontier orbital energies were calculated at the B3LYP/6-31G(d)
level of theory. Atomic orbital coefficients of frontier orbitals
were obtained using the STO-3G basis set. 13C NMR chemical
shifts of selected compounds 3d, 4a, 3f and 4f were calculated
as follows: The rigid structures were optimized with the MM2
force field implemented in Chem3D Pro.14 In the second step,
the optimized structures were subsequently reoptimized at the
AM1 level followed by the RHF/3-21G level and finally by
the B3LYP/6-31G(d) level of theory within the Gaussian 03
package. In the final step, the 13C NMR chemical shifts of the
reoptimized geometries were computed once at the B3LYP/6-
311+G(2d,p)//B3LYP/6-31G(d) level of theory and twice at
the mPW1PW91/6-311+G(2d,p)//mPW1PW91/6-31G(d) level
of theory using the IEFPCM model of solvation and DMSO as
solvent in both calculations.8 The references TMS and benzene
for the MSTD approach according to Sarotti and Pellegrinet9
were computed in the same manner as for 3d, 4a, 3f and 4f. For
comparison the 13C NMR chemical shifts were predicted using
Scheme
reaction.
2 Proposed mechanism of the laccase-catalyzed domino
9 (Scheme 2). That is the carbon atom with the largest atomic
orbital coefficient, C-4 or C-5, respectively.
As shown in Fig. 5, quinones 9a, 9b and 9c have their largest
atomic orbital coefficients at C-5. According to expectations,
regioisomers 4a, 4b and 4c are the exclusive products of the domino
reaction between 1a and 2a, 2b and 2c (Table 1). C-4 of 9d and
9e should be the preferred position for the nucleophilic attack of
1a. The experimental results are also in excellent agreement with
the DFT-calculations (Table 1). Only the reaction of 1a with 2f
leads to a mixture of regioisomers 3f and 4f (Table 1), which is
probably due to the steric hindrance caused by the bulky phenyl
substituent during the first step of the reaction.13 A preferred
attack of 1a onto the less hindered carbon atom C-5 of 9f can be
observed. Presumably, in addition to the electronic properties of
1
ACD/CNMR Predictor.7 The H–13C long-range coupling con-
stants between HA and the ring A carbons of 3d, 4a, 3f and 4f were
calculated at the mPW1PW91/6-311+G(2d,p)//mPW1PW91/6-
31G(d) level of theory using the IEFPCM model and DMSO as
solvent. An HP Compaq with a 2.39 GHz processor and 2 GB
RAM was used for the calculations.
Determination of laccase activity
The activity of commercially available laccase (EC 1.10.3.2) from
Trametes versicolor (Fluka, Buchs) was determined following a
procedure taken from Nicotra et al.15 A solution of ABTS (0.3 mL;
51.61 mg ABTS in 10 mL of 0.2 M acetate buffer, pH = 4.4)
was diluted with 0.2 M acetate buffer (2.67 mL, pH = 4.4) and
Fig. 5 Atomic orbital coefficients of LUMOs of 9a–f.
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treated with a solution of laccase in the same buffer (0.03 mL). The
change in absorption was followed via UV-spectroscopy (l = 414
nm). One unit was defined as the amount of laccase that converts
1 mmol of ABTS per minute at pH = 4.4 at room temperature.
3-CH3), 3.97 (3 H, s, OCH3), 6.92 (1 H, br s, 4-H), 6.93 (1 H, s, 9-H),
8.96 (1 H, br s, OH), 9.50 (1 H, br s, OH); dC(125 MHz; DMSO-
d6) 19.81 (3-CH3), 60.52 (OCH3), 95.88 (C-4), 99.17 (C-9), 102.92
(C-9b), 113.58 (C-9a), 133.54 (C-6), 137.42 (C-7), 140.82 (C-5a),
145.46 (C-8), 158.88 (C-1), 161.54 (C-3), 163.16 (C-4a); m/z(EI,
70 eV) 262 (M+, 100%), 247 (M+ - CH3, 44), 233 (2), 219 (5), 205
(3), 191 (3), 163 (2), 146 (2), 131 (M2+, 5), 43 (31).
General
Commercial reagents were used withoutfurther purification. Thin-
layer chromatography (TLC) was performed on silica gel SIL
G/UV254 (Macherey-Nagel); compounds were visualized with UV
light (l = 254 nm) and/or immersion in vanillin/H2SO4 solution
followed by heating. Melting points were determined on a Kofler
melting point apparatus (Reichert) and are uncorrected. UV/Vis
spectra were measured using a CARY 4E (Varian). IR (atr) spectra
were taken on a Spectrum One (Perkin Elmer). NMR spectra were
recorded on a Varian UnityINOVA spectrometer (500/125 MHz)
7,8-Dihydroxy-6-isopropyl-3-methyl-1H-pyrano[4,3-b]benzofur-
an-1-one (4c). Brown solid, 93% yield; mp 259–261 ◦C (from
ethyl acetate, dec.); lmax(MeOH)/nm 245 (lg e 4.23), 333 (4.17);
˜
n
max(atr)/cm-1 3553 and 2961 (OH), 1736 (C O), 1611 and 1569
(C C), 1382 (CH3), 1294 (OH), 1213 and 1041 (C–O), 854 ( C–
3
H); dH(500 MHz; DMSO-d6) 1.35 (6 H, d, J(CH
7.1,
)
,(CH
)
CH
3 2
3 2
(CH3)2CH), 2.34 (3 H, br s, 3-CH3), 3.56 (1 H, sept, 3J(CH
)
CH,(CH )
3 2
3 2
7.1, (CH3)2CH), 6.93 (1 H, br s, 4-H), 7.08 (1 H, s, 9-H), 8.60 (1 H,
br s, OH), 9.76 (1 H, br s, OH); dC(125 MHz; DMSO-d6) 19.81 (3-
CH3), 21.08 [(CH3)2], 24.85 [CH(CH3)2], 95.96 (C-4), 101.74 (C-9),
102.82 (C-9b), 112.97 (C-9a), 118.76 (C-6), 142.57 (C-7), 143.86
(C-8), 147.47 (C-5a), 158.98 (C-1), 161.14 (C-3), 162.74 (C-4a);
m/z(EI, 70 eV) 274.0818 (M+, 100%. C15H14O5 requires 274.0842),
259 (M+ - CH3, 95), 231 (5), 213 (5), 203 (4), 185 (3), 137 (M2+, 2),
129 (5), 99 (3), 43 (9).
1
in DMSO-d6; the H and 13C chemical shifts were referenced to
residual solvent signals at dH = 2.49 and dC = 39.5 (DMSO) relative
to TMS. J values are given in Hz. All 1D (1H, 13C) and 2D NMR
(COSY, ROESY, gHSQCAD, gHMBCAD) measurements were
performed using standard pulse sequences. HSQMBC parameters:
sw = 4000–6000 Hz, sw1 = 8000–12 000 Hz, np = 8192, fn = 16 384,
jnxh = 8, ni = 128–512, nt = 16–64, d1 = 1. Linear Prediction in F1.
Mass spectra (EI) were recorded on a MAT 95 (Finnigan MAT)
with 70 eV ionization energy.
9-Chloro-7,8-dihydroxy-3-methyl-1H-pyrano[4,3-b]benzofuran-
1-one (3d). White solid, 67% yield; mp >300 ◦C (dec.);
˜
l
max(MeCN)/nm 227 (lg e 4.28), 328 (4.22); nmax(atr)/cm-1 3575
General procedure for the synthesis of
and 3209 (OH), 1733 (C O), 1618, 1564 and 1490 (C C),
1380 (OH), 1221 (C–O), 1037 (C–O or C–Cl), 849 ( C–H);
dH(500 MHz; DMSO-d6) 2.32 (3 H, s, 3-CH3), 6.84 (1 H, s, 4-
H), 7.06 (1 H, s, 6-H), 9.18 (1 H, br s, OH), 10.29 (1 H, br s,
OH); dC(125 MHz; DMSO-d6) 19.68 (3-CH3), 95.33 (C-4), 97.24
(C-6), 102.29 (C-9b), 111.69 (C-9), 112.83 (C-9a), 141.12 (C-8),
146.19 (C-7), 147.77 (C-5a), 156.99 (C-1), 162.30 (C-3), 163.77
(C-4a); m/z(EI, 70 eV) 265.9941 (M+, 85%. C12H7ClO5 requires
265.9982), 251 (M+ - CH3, 25), 237 (8), 233 (4), 196 (8), 133 (3),
43 (23).
7,8-dihydroxy-1H-pyrano[4,3-b]benzofuran-1-ones 3 and 4, and
8,9-dihydroxy-6H-benzofuro[3,2-c]chromen-6-ones 5 and 6
4-Hydroxy-6-methyl-2H-pyran-2-one (1a) (378 mg, 3.0 mmol)
or 4-hydroxy-2H-chromen-2-one (1b) (491 mg, 3.0 mmol), re-
spectively, and catechol 2a, 2b and 2e (3.3 mmol), respectively,
were dissolved in acetate buffer (200 mL, pH = 4.4, 0.2 M) and
vigorously stirred under air at r.t. in the presence of 475 U laccase
of T. versicolor until the substrates had been fully consumed, as
judged by TLC. The reaction mixture was saturated with NaCl
and filtered on a Buchner funnel. The filter cake was washed
with a solution of 15% NaCl (200 mL) and water (10 mL). The
crude products obtained after drying exhibited a purity of 90–
95% (NMR). Analytically pure products could be obtained by
recrystallization.
7,8-Dihydroxy-3-methyl-1-oxo-1H-pyrano[4,3-b]benzofuran-9-
carboxylic acid methyl ester (3e). White solid, 60% yield; tR/min
8 (HPLC on Phenomenex Aqua RP-18 250 ¥ 10 mm column,
5 mm, 30% MeCN (0.01% TFA), flow rate of 4 mL min-1); mp
237–239 ◦C (dec.); lmax(MeCN)/nm 233 (lg e 4.28), 335 (4.18);
˜
Transformations of 1a (0.5 mmol) and 2c or 2d (0.55 mmol),
respectively, were performed in acetate buffer (70 mL) on a smaller
scale. Reaction of 1a (0.45 mmol) and 2f (0.5 mmol) was performed
in a buffer/acetone mixture (85 mL, v/v = 16 : 1).
n
max(atr)/cm-1 3405 and 3119 (OH), 1703 (C O), 1440 (CH3),
1296 (OH), 1220 and 1050 (C–O), 839 ( C–H); dH(500 MHz;
DMSO-d6) 2.33 (3 H, s, 3-CH3), 3.81 (3 H, s, OCH3), 6.91 (1
H, s, 4-H), 7.17 (1 H, s, 6-H); dC(125 MHz; DMSO-d6) 19.75
(3-CH3), 51.68 (OCH3), 95.59 (C-4), 99.29 (C-6), 102.26 (C-9b),
110.19 (C-9a), 113.69 (C-9), 141.27 (C-8), 145.64, 147.68 (C-5a or
C-7), 157.96 (C-1), 162.00 (C-3), 163.55 (C-4a), 165.97 (CO2CH3);
m/z(EI, 70 eV) 290.0425 (M+, 20%. C14H10O7 requires 290.0427),
258 (M+ - CH4O, 100), 229 (4), 202 (16), 174 (11), 69 (7), 43 (19).
7,8 -Dihydroxy-3,6-dimethyl-1H-pyrano[4,3-b]benzofuran-1-one
◦
(4a). Brown solid, 99% yield; mp >300 C (dec.) (lit.,3b 257–
259 ◦C, dec.); dH(500 MHz; DMSO-d6) 2.28 (3 H, s, 6-CH3), 2.32
(3 H, br s, 3-CH3), 6.86 (1 H, br s, 4-H), 7.06 (1 H, s, 9-H), 8.74 (1
H, br s, OH), 9.63 (1 H, br s, OH); dC(125 MHz; DMSO-d6) 8.93
(6-CH3), 19.81 (3-CH3), 95.91 (C-4), 101.69 (C-9), 103.11 (C-9b),
108.37 (C-6), 112.31 (C-9a), 143.36 (C-7), 143.78 (C-8), 148.09
(C-5a), 158.99 (C-1), 161.06 (C-3), 162.91 (C-4a); m/z(EI, 70 eV)
246 (M+, 100%), 231 (M+ - CH3, 11), 217 (13), 203 (5), 190 (3),
176 (7), 147 (2), 123 (5), 43 (24).
7,8-Dihydroxy-3-methyl-6-phenyl-1H-pyrano[4,3-b]benzofuran-
1-one (4f). Brown solid, 76% yield; mp 262–264 ◦C (from CHCl3,
dec.); lmax(MeOH)/nm 251 (lg e 4.44), 335 (4.19); nmax(atr)/cm-1
˜
3495 and 3057 (OH), 1736 (C O), 1616, 1570 and 1493 (C C),
1308 (OH), 1238 and 1043 (C–O), 885, 764 and 695 ( C–H);
dH(500 MHz; DMSO-d6) 2.32 (3 H, s, 3-CH3), 6.89 (1 H, br s,
7,8-Dihydroxy-6-methoxy-3-methyl-1H-pyrano[4,3-b]benzofur-
an-1-one (4b). ◦ Brown solid, 51% yield; mp 230–234 ◦C (dec.)
(lit.,3b 225–227 C, dec.); dH(500 MHz; DMSO-d6) 2.33 (3 H, br s,
3
3
4-H), 7.24 (1 H, s, 9-H), 7.38 (1 H, t, J4¢-H,3¢-H = J4¢-H,5¢-H 7.4,
3
3
3
4¢-H), 7.47 (2 H, t, 3J3¢-H,2¢-H = J3¢-H,4¢-H = J5¢-H,4¢-H = J5¢-H,6¢-H 7.6, 3¢-
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3
H, 5¢-H), 7.59 (2 H, d, 3J2¢-H,3¢-H = J6¢-H,5¢-H 7.7, 2¢-H, 6¢-H), 8.83 (1
(C-11b), 114.05 (C-7), 116.95 (C-4), 121.38 (C-1), 124.93 (C-2),
131.73 (C-3), 141.69 (C-8), 146.45 (C-9), 148.76 (C-10a), 152.45
(C-4a), 156.57 (C-6), 158.44 (C-11a), 165.97 (CO2CH3); m/z(EI,
70 eV) 326.04335 (M+, 16%. C17H10O7 requires 326.04266), 294
(M+ - CH4O, 100), 265 (8), 228 (11), 210 (56), 126 (16), 92 (24),
69 (24), 39 (18).
H, s, 7-OH), 9.99 (1 H, s, 8-OH); dC(125 MHz; DMSO-d6) 19.83
(3-CH3), 95.95 (C-4), 102.93 (C-9b), 103.28 (C-9), 113.01 (C-9a),
113.42 (C-6), 127.42 (C-4¢), 128.01 (C-3¢, C-5¢), 130.25 (C-2¢, C-
6¢), 132.09 (C-1¢), 142.56 (C-7), 144.29 (C-8), 146.68 (C-5a), 158.93
(C-1), 161.30 (C-3), 163.12 (C-4a); m/z(EI, 70 eV) 308.0687 (M+,
100%. C18H12O5 requires 308.0685), 293 (M+ - CH3, 5), 279 (7),
237 (9), 181 (3), 152 (4), 43 (4).
Acknowledgements
7,8-Dihydroxy-3-methyl-9-phenyl-1H-pyrano[4,3-b]benzofuran-
1-one (3f). NMR-spectroscopic data of 3f were deduced from
the mixture of 3f and 4f. Only 4f could be isolated in pure form by
recrystallization from CHCl3.
We thank Ms. Sabine Mika for the 13C NMR chemical shift
predictions by ACD/Labs and Mr. Matthew Witherspoon for
careful consideration of the manuscript.
dH(500 MHz; DMSO-d6) 2.26 (3 H, s, 3-CH3), 6.82 (1 H, br s, 4-
H), 7.12 (1 H, s, 6-H), 7.23 (2 H, m, 2¢-H, 6¢-H), 7.29 (3 H, m, 3¢-H,
4¢-H, 5¢-H), 8.14 (1 H, s, 8-OH), 10.17 (1 H, s, 7-OH); dC(125 MHz;
DMSO-d6) 19.61 (3-CH3), 95.25 (C-4), 97.22 (C-6), 102.49 (C-9b),
112.92 (C-9a), 126.52 (C-4¢), 126.65 (C-3¢, C-5¢), 130.61 (C-2¢, C-
6¢), 136.04 (C-1¢), 141.26 (C-8), 145.14 (C-7), 147.82 (C-5a), 156.60
(C-1), 161.28 (C-3), 163.47 (C-4a).
Notes and references
1 (a) A. J. M. da Silva, P. A. Melo, N. M. V. Silva, F. V. Brito, C. D.
Buarque, D. V. de Souza, V. P. Rodrigues, E. S. C. Poc¸as, F. Noe¨l, E. X.
Albuquerque and P. R. R. Costa, Bioorg. Med. Chem. Lett., 2001, 11,
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Planta Med., 1986, 52, 370–373.
2 PCT Int. Appl., WO 2008/110465 A1 20080918.
8,9-Dihydroxy-10-methyl-6H -benzofuro◦[3,2-c]chromen-6-one
(6a). Brown solid, 99% yield; mp >300 C (dec.) (lit.,16 304–
306 ◦C, dec.); dH(500 MHz; DMSO-d6) 2.36 (3 H, s, 10-CH3), 7.15
3 (a) H.-W. Wanzlick, R. Gritzky and H. Heidepriem, Chem. Ber., 1963,
96, 305–307; (b) D. Nematollahi and Z. Forooghi, Tetrahedron, 2002,
58, 4949–4953; (c) G. Pandey, C. Muralikrishna and U. T. Bhalerao,
Tetrahedron, 1989, 45, 6867–6874.
3
3
4
(1 H, s, 7-H), 7.42 (1 H, ddd, J2-H,1-H = J2-H,3-H 7.5, J2-H,4-H 1.0,
2-H), 7.50 (1 H, dd, 3J4-H,3-H 8.1, 4J4-H,2-H 0.9, 4-H), 7.61 (1 H, ddd,
3J3-H,2-H 7.2, 3J3-H,4-H 8.4, 4J3-H,1-H 1.6, 3-H), 7.94 (1 H, dd, 3J1-H,2-H 7.7,
4J1-H,3-H 1.6, 1-H); dC(125 MHz; DMSO-d6) 8.98 (10-CH3), 101.86
(C-7), 105.64 (C-6a), 108.53 (C-10), 112.41 (C-11b), 112.95 (C-
6b), 116.98 (C-4), 121.10 (C-1), 124.81 (C-2), 131.14 (C-3), 144.17
(C-8), 144.22 (C-9), 149.03 (C-10a), 152.25 (C-4a), 157.52 (C-6),
157.58 (C-11a); m/z(EI, 70 eV) 282 (M+, 100%), 265 (<1), 253 (1),
208 (2), 141 (M2+, 7), 115 (2).
4 (a) H. Leutbecher, J. Conrad, I. Klaiber and U. Beifuss, Synlett,
2005, 3126–3130; (b) H. Leutbecher, S. Hajdok, C. Braunberger,
M. Neumann, S. Mika, J. Conrad and U. Beifuss, Green Chem.,
2009, 11, 676–679; (c) H. Leutbecher, Ph.D. Thesis, Universita¨t
Hohenheim, Stuttgart, Germany, 2007; (d) S. Hajdok, H. Leutbecher,
G. Greiner, J. Conrad and U. Beifuss, Tetrahedron Lett., 2007, 48, 5073–
5076.
5 (a) M. E. Elyashberg, K. A. Blinov, Y. D. Smurnyy, T. S. Churanova
and A. J. Williams, Magn. Reson. Chem., 2010, 48, 219–229; (b) K. A.
Blinov, Y. D. Smurnyy, T. S. Churanova, M. E. Elyashberg and A. J.
Williams, Chemom. Intell. Lab. Syst., 2009, 97, 91–97; (c) S. Thomas,
I. Bru¨hl, D. Heilmann and E. Kleinpeter, J. Chem. Inf. Comput. Sci.,
1997, 37, 726–730.
8,9-Dihydroxy-10-methoxy-6H-benzofuro[3,2-c]chromen-6-one
(6b). Brown solid, 61% yield; mp 236–238 ◦C (from
ethanol/water, dec.) (lit.,16 260–261 ◦C, dec.); dH(500 MHz;
DMSO-d6) 4.08 (3 H, s, OCH3), 7.05 (1 H, s, 7-H), 7.46 (1 H,
6 (a) D. Nematollahi and M. Rafiee, J. Electroanal. Chem., 2004, 566,
31–37; (b) S. S. H. Davarani, N. M. Najafi, S. Ramyar, L. Masoumi and
M. Shamsipur, Chem. Pharm. Bull., 2006, 54, 959–962.
7 ACD/NMR Predictors, V10, Advanced Chemistry Development,
Toronto, Canada, 2006.
3
3
3
pt, J2-H,1-H = J2-H,3-H 7.7, 2-H), 7.55 (1 H, d, J4-H,3-H 8.4, 4-H),
7.64 (1 H, ddd, 3J3-H,2-H 7.4, 3J3-H,4-H 8.6, 4J3-H,1-H 1.7, 3-H), 8.03 (1
8 M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb,
J. R. Cheeseman, J. A. Montgomery, Jr., T. Vreven, K. N. Kudin, J. C.
Burant, J. M. Millam, S. S. Iyengar, J. Tomasi, V. Barone, B. Mennucci,
M. Cossi, G. Scalmani, N. Rega, G. A. Petersson, H. Nakatsuji, M.
Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T.
Nakajima, Y. Honda, O. Kitao, H. Nakai, M. Klene, X. Li, J. E.
Knox, H. P. Hratchian, J. B. Cross, V. Bakken, C. Adamo, J. Jaramillo,
R. Gomperts, R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi,
C. Pomelli, J. Ochterski, P. Y. Ayala, K. Morokuma, G. A. Voth, P.
Salvador, J. J. Dannenberg, V. G. Zakrzewski, S. Dapprich, A. D.
Daniels, M. C. Strain, O. Farkas, D. K. Malick, A. D. Rabuck, K.
Raghavachari, J. B. Foresman, J. V. Ortiz, Q. Cui, A. G. Baboul, S.
Clifford, J. Cioslowski, B. B. Stefanov, G. Liu, A. Liashenko, P. Piskorz,
I. Komaromi, R. L. Martin, D. J. Fox, T. Keith, M. A. Al-Laham, C. Y.
Peng, A. Nanayakkara, M. Challacombe, P. M. W. Gill, B. G. Johnson,
W. Chen, M. W. Wong, C. Gonzalez and J. A. Pople, GAUSSIAN 03
(Revision E.01), Gaussian, Inc., Wallingford, CT, 2004.
3
4
H, dd, J1-H,2-H 7.9, J1-H,3-H 1.7, 1-H), 9.13 (1 H, br s, OH), 9.64
(1 H, br s, OH); dC(125 MHz; DMSO-d6) 61.34 (OCH3), 100.04
(C-7), 106.24 (C-6a), 113.02 (C-11b), 114.95 (C-6b), 117.78 (C-4),
122.04 (C-1), 125.63 (C-2), 132.18 (C-3), 134.36 (C-10), 138.83 (C-
9), 142.44 (C-10a), 146.58 (C-8), 153.09 (C-4a), 158.19 (C-11a),
158.68 (C-6); m/z(EI, 70 eV) 298 (M+, 100%), 283 (M+ - CH3,
52), 255 (20), 237 (2), 171 (3), 149 (M2+, 6), 115 (5).
8,9-Dihydroxy-6-oxo-6H-benzofuro[3,2-c]chromen-7-carboxylic
acid methyl ester (5e). White solid, 63% yield; tR/min 15 (HPLC
on Phenomenex Aqua RP-18 250 ¥ 10 mm column, 5 mm, 42%
MeCN (0.01% TFA), flow rate of 4 mL min-1); mp 244–246 ◦C
(dec.); lmax(MeCN)/nm 210 (lg e 4.62), 239 (4.25), 289 (3.84), 353
(4.32); nmax(atr)/cm-1 3372 and 2957 (OH), 1756 (C O), 1679,
˜
9 A. M. Sarotti and S. C. Pellegrinet, J. Org. Chem., 2009, 74, 7254–
7260.
1623 and 1606 (C C), 1445 (CH3), 1330 (OH), 1272 and 1079
10 (a) E. K. Ko¨ve´r, G. Batta and K. Fehe´r, J. Magn. Reson., 2006, 181,
89–97; (b) R. T. Williamson, B. L. Ma´rquez, W. H. Gerwick and E. K.
Ko¨ve´r, Magn. Reson. Chem., 2000, 38, 265–273; (c) V. Lacerda Jr,
G. V. J. da Silva, C. F. Tormena, R. T. Williamson and B. L. Marquez,
Magn. Reson. Chem., 2007, 45, 82–86.
11 (a) S. Hajdok, J. Conrad, H. Leutbecher, S. Strobel, T. Schleid and U.
Beifuss, J. Org. Chem., 2009, 74, 7230–7237; (b) P. E. Hansen, Prog.
Nucl. Magn. Reson. Spectrosc., 1981, 14, 175–295.
(C–O), 892 and 754 ( C–H); dH(500 MHz; DMSO-d6) 3.85 (3
3
H, s, OCH3), 7.31 (1 H, s, 10-H), 7.46 (1 H, ddd, J2-H,1-H 7.4,
3
3J2-H,3-H 7.4,4J2-H,4-H 1.0, 2-H), 7.54 (1 H, br d, J4-H,3-H 8.4, 4-H),
7.65 (1 H, ddd, 3J3-H,2-H 7.3, 3J3-H,4-H 8.3, 4J3-H,1-H 1.6, 3-H), 7.99 (1
3
4
H, dd, J1-H,2-H 7.7, J1-H,3-H 1.4, 1-H); dC(125 MHz; DMSO-d6)
51.87 (OCH3), 99.35 (C-10), 104.93 (C-6a), 110.76 (C-6b), 112.01
2672 | Org. Biomol. Chem., 2011, 9, 2667–2673
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©

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This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 2667–2673 | 2673
©