The laccase-catalyzed domino reaction between catechols and heterocyclic 1,3-dicarbonyls and theunambiguous structure elucidation of the products by NMR spectroscopy and X-ray crystal structure analysis

Article abstract of DOI:10.1021/jo9011915

(Chemical Equation Presented) The laccase-catalyzed reaction between catechols and heterocyclic 1,3-dicarbonyls (pyridinones, quinolinones, thiocoumarins) using aerial oxygen as the oxidant delivers benzofuropyridinones, benzofuroquinolinones, and thiocou

Full text of DOI:10.1021/jo9011915

pubs.acs.org/joc
The Laccase-Catalyzed Domino Reaction between Catechols and
Heterocyclic 1,3-Dicarbonyls and the Unambiguous Structure Elucidation of
the Products by NMR Spectroscopy and X-ray Crystal Structure Analysis
†
Szilvia Hajdok, Jurgen Conrad, Heiko Leutbecher, Sabine Strobel, Thomas Schleid,
†
†
‡
‡
€
and Uwe Beifuss*,†
†
€
Bioorganische Chemie, Institut fu€r Chemie, Universitat Hohenheim, Garbenstrasse 30, D-70599 Stuttgart, Germany,
and Institut fu€r Anorganische Chemie, Universitat Stuttgart, Pfaffenwaldring 55, D-70569 Stuttgart, Germany
‡
€
ubeifuss@uni-hohenheim.de
Received June 8, 2009
The laccase-catalyzed reaction between catechols and heterocyclic 1,3-dicarbonyls (pyridinones,
quinolinones, thiocoumarins) using aerial oxygen as the oxidant delivers benzofuropyridinones,
benzofuroquinolinones, and thiocoumestans in a simple fashion, highly regioselectively with yields
ranging from 55 to 98%. With barbituric acid derivatives the exclusive formation of dispiropyr-
imidinone derivatives takes place. The unambiguous and complete structure elucidation of all
reaction products has been achieved by means of NMR spectroscopic methods (HSQMBC and
band-selective HMBC) as well as by X-ray crystal structure analysis.
Introduction
great deal of interest in the development of efficient synthetic
approaches to these heterocyclic ring systems.1d,3,4 Nowadays
the main focus is on the development of catalytic methods.1d,3,5
Also of great interest is the use of domino reactions3,6 which
Benzofurans1 and annulated benzofurans2 are well-known
for their remarkable biological activities. This is why there is a
(1) (a) Salih, K. S. M.; Ayoub, M. T.; Saadeh, H. A.; Al-Masoudi, N. A.;
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~
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de Moraes, M. O.; Costa-Lotufo, L. V.; Lima, M. A. S.; Silveira, E. R.
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Hitzler, M.; Schmid, D.; Visser, K.; Cordes, H. P.; Csollei, J.; Seydel, J. K.;
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7230 J. Org. Chem. 2009, 74, 7230–7237
Published on Web 09/09/2009
DOI: 10.1021/jo9011915
r
2009 American Chemical Society

Hajdok et al.
JOCArticle
allow the resource-conserving and efficient construction
of benzofurans and annulated benzofurans in a single syn-
thetic operation. Enzyme-catalyzed reactions are of increasing
importance in organic synthesis7 as they allow a multitude of
selective and efficient transformations to be run in aqueous
systems at room temperature. In addition, many enzyme
catalyzed transformations fulfill a number of the principles
of green chemistry.8 A main area is the development of
enzyme-catalyzed oxidations.9 Oxidative transformations are
particularly attractive if aerial oxygen can be used as an
oxidant because atmospheric oxygen is inexhaustible and free
of cost. In addition, the only waste product formed upon
reduction of oxygen is water which is completely safe and
nontoxic. Reactions that combine several enzyme catalyzed
transformations or enzyme and chemically catalyzed transfor-
mations to make up new reaction sequences are even more
interesting than single-step enzymatic transformations.10,11
In this respect, laccases12 are particularly attractive en-
zymes as they can be used to catalyze oxidations which are
capable of triggering domino processes. Laccases (benzene-
diol: O2 oxidoreductase E.C. 1.10.3.2.) mainly occur in fungi,
but also in plants and some prokaryotes. In general, they
are easy to isolate, and some of them are even commer-
cially available. The best known laccases include the enzymes
isolated from Agaricus bisporus and Trametes versicolor. Lac-
cases are multicopper oxidases which are able to catalyze the
selective oxidation of a number of substrates under mild reac-
tion conditions with simultaneous reduction of O2 to give
H2O.13 By using mediators, the redox potential of laccases
can be changed which allows the oxidation of substrates
with a higher redox potential. Laccases have been employed
for the oxidation of aromatic methyl groups,14 benzylic,
allylic, and aliphatic alcohols,15 ethers,16 benzylamines, and
hydroxylamines.17 Relatively few studies have so far been
published on the combination of a laccase-catalyzed oxida-
tion with another enzyme or with a chemically catalyzed
transformation to make up new domino reactions.18 Recently,
we have reported on the combination of a laccase-catalyzed
oxidation of catechols with 1,4-additions of 1,3-dicarbonyls.19
We could demonstrate that 4-hydroxy-6-methyl-2H-pyran-2-
one and cyclohexane-1,3-diones, respectively, can be reacted
with catechols to efficiently produce 1H-pyrano-[4,3-b]-
benzofuran-1-ones19a and 3,4-dihydro-7,8-dihydroxy-2H-
dibenzofuran-1-ones,19b respectively. Similar reactions have
been reported by Ragauskas et al.20 They also studied the
intermolecular Diels-Alder reaction of o-and p-benzoqui-
nones, which were generated by laccase-catalyzed oxidations
of catechols and hydroquinones, respectively, with several
dienes.21
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JOCArticle
Results and Discussion
TABLE 1. Laccase (A. bisporus)-Catalyzed Domino Reaction of Pyridi-
nones 1 and Catechols 2 for the Synthesis of 6-Substituted 7,8-Dihydroxy-
benzofuro[3,2-c]pyridin-1(2H)-ones 3
To explore the scope of the domino process we studied
the laccase-catalyzed reaction of unsubstituted as well as
substituted catechols with heterocyclic 1,3-dicarbonyls.
Here, we demonstrate that catechols can be reacted with
several heterocyclic 1,3-dicarbonyls to yield a couple of
heterocyclic systems in an efficient way. In the reactions
with 4-hydroxy-6-methylpyridin-2(1H)-one, 4-hydroxy-1,6-
dimethylpyridin-2(1H)-one, 4-hydroxyquinolin-2(1H)-one,
and 4-hydroxy-1-thiocoumarin, the exclusive and highly
regioselective formation of annulated benzofuran deriva-
tives like benzofuropyridinones, benzofuroquinolinones,
and thiocoumestans was observed. Due to their distinct
biological activities, these classes of heterocyclic com-
pounds attract great interest in medicinal chemistry.22 It
has been established that 3,9-dihydroxybenzofuro[3,2-
c]quinolin-6(5H)-one exhibits unique effects of inhibiting
bone resorption and stimulating bone mineralization with-
out exhibiting estrogenic activity.22a 3,9-Bis(N,N-dimethyl-
carbamoyloxy)benzofuro[3,2-c]quinolin-6(5H)-one, a derivative
thereof, has been found to display similar pharmaceutical
activities. It might become useful in the treatment of osteo-
porosis as it is a more potent agent than ipriflavone.22b
3-Chloro-5-thiocoumestan-8,9-diacetate was shown to be
active against Aspergillus niger and Alternaria alternata.22c
Some of the compounds described here have been made
accessible by other methods, including tyrosinase-catalyzed
oxidation,23a electrochemical oxidation,23b and chemical
oxidation with potassium ferricyanide.22c In addition, we
demonstrate that the laccase-catalyzed transformations of
barbituric acid and thiobarbituric acid derivatives with
catechols do not give the expected benzofuran derivatives
but result in the exclusive formation of polycyclic dispiro-
pyrimidinones.
entry
1
R1
2
R2
time (h)
product 3
yielda (%)
1
2
3
4
5
6
a
a
a
b
b
b
H
H
H
Me
Me
Me
a
b
c
a
b
c
H
Me
OMe
H
Me
24
18
18
18
19
17
a
b
c
d
e
f
98
98
83
98
65
81
OMe
aThe reactions were performed with 156 U of laccase from A. bisporus
(AbL). The enzyme activity was determined using catechol as the
substrate.24
TABLE 2. Laccase (A. bisporus)-Catalyzed Domino Reaction of 4-Hy-
droxyquinolin-2(1H)-one (5) with Catechols 2 for the Synthesis of 10-Sub-
stituted 8,9-Dihydroxybenzofuro[3,2-c]quinolin-6(5H)-ones 6
We started with the laccase-catalyzed reaction between
4-hydroxy-6-methylpyridin-2(1H)-one (1a) and 4-hydroxy-
1,6-dimethylpyridin-2(1H)-ones (1b) with the catechols
2a-c. It was found that 6-substituted 7,8-dihydroxybenzo-
furo[3,2-c]pyridin-1(2H)-ones 3 can easily be accessed regio-
selectively using this approach (Table 1). In all cases, the
reactions (except the transformations presented in Table 4)
were performed with 156 U24 of a commercially available
laccase from A. bisporus (AbL) as a catalyst at room tem-
perature in a phosphate buffer (0.2 M) at pH 6.0. The yields
of the 6-substituted 7,8-dihydroxybenzofuro[3,2-c]pyridin-
1(2H)-ones 3a-f were in the range between 65 and 98%, and
entry
2
R2
time (h)
product 6
yielda (%)
1
2
3
a
b
c
H
Me
OMe
20
18
20
a
b
c
73
77
61
aThe reactions were performed with 156 U of laccase from A. bisporus
(AbL). The enzyme activity was determined using catechol as the
substrate.24
even the purity of the crude products was very high
(>90-95%) (Table 1).
The starting materials, i.e., the 4-hydroxy-6-methyl-
pyridin-2(1H)-one (1a) and the 4-hydroxy-1,6-dimethyl-
pyridin-2(1H)-one (1b), were synthesized in yields of 92
and 65%, respectively, by reacting 4-hydroxy-6-methyl-
pyran-2(2H)-one (4) with NH4OH and CH3NH2, respec-
tively25
(22) (a) Tsutsumi, N.; Kawashima, K.; Nagata, H.; Ujiie, A.; Endo, H.
Jpn. J. Pharmacol. 1995, 67, 169. (b) Yamada, T.; Saito, N.; Anraku, M.;
Imai, T.; Otagiri, M. Pharm. Dev. Technol. 2000, 5 (4), 443. (c) Reddy, B. S.;
Darbarwar, M. Indian J. Chem. 1985, 24B, 556.
(23) (a) Pandey, G.; Muralikrishna, C.; Bhalerao, U. T. Tetrahedron
We also studied the reactions between 4-hydroxyquinolin-
2(1H)-one (5) and the catechols 2a-c (Table 2). As shown in
Table 2, the 8,9-dihydroxybenzofuro[3,2-c]quinolin-6(5H)-
ones 6a-c can be isolated as single regioisomers with yields
ranging from 61 to 77% if the laccase-catalyzed reactions of
the appropriate substrates are conducted under the reaction
conditions developed in our laboratory.
ꢀ
1989, 45, 6867. (b) Tabakovic, I.; Grujic, Z.; Bejtovic, Z. J. Heterocycl.
Chem. 1983, 20, 635.
ꢀ
ꢀ
(24) Laccase activity (AbL) was determined according to a modified
procedure taken from: Felici, M.; Artemi, F.; Luna, M.; Speranza, M. J.
Chromatogr. 1985, 320, 435. Land, E. J. J. Chem. Soc., Faraday Trans. 1993,
89, 803. A 1.02 mM solution of catechol (0.45 mL) in 0.2 M phosphate buffer
(pH = 6.0) was diluted with 0.2 M phosphate buffer (2.5 mL, pH = 6.0) and
treated with a solution of laccase from A. bisporus (AbL) in the same buffer
(0.2 mL). The change in absorption was followed via UV spectroscopy (λ =
390 nm). 1 U is defined as the amount of the enzyme that catalyzes the
conversion of 1 μmol of catechol per minute by pH 6.0 and 25 °C
(Nomenclature Committee of the International Union of Biochemistry.
Eur. J. Biochem. 1979, 97, 319). The activity of laccase amounted to 3.11
U/mg.
(25) (a) Stadlbauer, W.; Fiala, W.; Fischer, M.; Hojas, G. J. Heterocycl.
ꢀ
Chem. 2000, 37, 1253. (b) Castillo, S.; Ouadahi, H.; Herault, V. Bull. Soc.
Chim. Fr. 1982, 7-8, 257.
7232 J. Org. Chem. Vol. 74, No. 19, 2009

Hajdok et al.
JOCArticle
TABLE 3. Laccase (A. bisporus)-Catalyzed Domino Reaction of 4-Hy-
droxy-1-thiocoumarin (9) with Catechols 2 for the Synthesis of 8,9-Dihy-
droxy-5-thiocoumestans 10
SCHEME 1. Formation of the Diacetates
entry
2
R2
time (h)
product 10
yielda (%)
1
2
3
a
b
c
H
Me
OMe
18
19
19
a
b
c
96
77
55
aThe reactions were performed with 156 U of laccase from A. bisporus
(AbL). The enzyme activity was determined using catechol as the
substrate.24
SCHEME 2. Possible Reaction Mechanism
TABLE 4. Model Reactions with Different Laccases
entry 1,3-dicarbonyl
2
laccase unitsa (U) product yield (%)
1
2
3
4
5
6
7
8
9
1a
1a
1a
1a
1b
9
1a
1b
9
a
a
a
a
a
a
a
a
a
a
a
a
AbLb
AbLb
AbLb
AbLb
AbLb
AbLb
TvFc
TvFc
TvFc
Lccβd
Lccβd
Lccβd
35
30
15
7
35
35
35
35
35
35
35
35
3a
3a
3a
3a
3d
10a
3a
3d
10a
3a
3d
10a
99
98
88
74
99
98
60
57
61
64
64
54
10
11
12
1a
1b
9
aThe enzyme activity was determined using ABTS as the substrate.30
bReactions were performed using a commercially available laccase from
A. bisporus (AbL) in 0.2 M phosphate buffer at pH 6.0. cReactions were
performed using a commercially available laccase from T. versicolor
(TvF) in 0.2 M acetate buffer at pH 4.38. dReactions were performed
using a recombinant β-laccase from T. versicolor produced in P. pastoris
(Lccβ) in 0.1 M acetate buffer at pH 5.0.
Due to solubility problems, it turned out to be impossi-
ble to obtain the heterocycles 3a-f and 6a-c in analyti-
cally pure form for elemental analysis. In order to obtain
analytically pure material, the poorly soluble products
3a-f and 6a-c were transformed into their correspond-
ing diacetates28 13a-f and 14a-c (Scheme 1). For this
purpose, the substrates were dissolved in excess pyridine
and reacted with 5 equiv of acetic anhydride and 10 mol %
of DMAP at room temperature. The solid crude products
obtained after acidic workup were recrystallized to yield
analytically pure diacetates. With products 10a-c recrystal-
lization was possible and sufficient to obtain analytically
pure material.
It is assumed that the first step of the domino process is the
laccase-catalyzed oxidation of the catechol 2 with O2 to o-
benzoquinone 15, which then undergoes an intermolecular
1,4-addition with the enol of the 1,3-dione 16 as a nucleophile
to yield 17 which cannot be isolated (Scheme 2). After a
second laccase-catalyzed oxidation, a final intramolecular
1,4-addition under formation of the tricycle 19 takes place.
Altogether, this can be summarized as a domino oxidation/
1,4-addition/oxidation/1,4-addition-process.19b The regios-
electivity of these domino reactions may be rationalized in
such a way that the initial 1,4-addition exclusively occurs at
the more electrophilic carbon atom of the corresponding
o-benzoquinones 15.
The substrate 4-hydroxyquinolin-2(1H)-one (5) was pre-
pared in two synthetic steps according to known pro-
cedures.26 Upon reaction of 20-aminoacetophenone (7)
with diethyl carbonate and sodium hydride in toluene a
mixture of 5 and 4-hydroxyquinolin-2(1H)-one-3-carboxy-
lic acid ethyl ester (8) (approximately 1:1) was formed,
which was then boiled in aqueous KOH to allow for
the quantitative transformation of 8 into 4-hydroxyqui-
nolin-2(1H)-one (5).
In the reactions between 4-hydroxy-1-thiocoumarin (9)
and the catechols 2a-c the tetracyclic 8,9-dihydroxy-5-thio-
coumestans 10a-c were isolated in yields from 55 to 96% as
single regioisomers (Table 3).
4-Hydroxy-1-thiocoumarin (9) could also be prepared in
two steps.26a,27 First, thiosalicylic acid (11) was reacted with
methyllithium to yield 20-mercaptoacetophenone (12) in
90% yield. Treatment of 12 with diethyl carbonate and
sodium ethoxide in toluene resulted in the isolation of
crystalline 4-hydroxy-1-thiocoumarin (9) in 53% yield.
(26) (a) Jung, J.-C.; Jung, Y.-J.; Park, O.-S. Synth. Commun. 2001, 31 (8),
1195. (b) Koller, G. Chem. Ber. 1927, 60, 1108.
(27) Topolski, M. J. Org. Chem. 1995, 60, 5588.
€
(28) Hofle, G.; Steglich, W.; Vorbruggen, H. Angew. Chem., Int. Ed. 1978,
17, 569.
€
J. Org. Chem. Vol. 74, No. 19, 2009 7233

Hajdok et al.
JOCArticle
SCHEME 3. Reaction of Barbituric Acid Derivatives 20 with Catechols 2 for the Synthesis of 21
To explore whether other laccases could also catalyze
these transformations, some model reactions were run using
(a) a commercially available laccase from T. versicolor (TvF)
(Table 4, entries 7-9) and (b) a recombinant β-laccase from
T. versicolor produced in Pichia pastoris (Lccβ) (Table 4,
entries 10-12).29 The outcome was compared to the results
obtained with the laccase from A. bisporus (AbL) (Table 4,
entries 1, 5, and 6). For these comparative experiments, the
activities of all three enzymes were determined using 2,20-
azinobis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) as
the substrate.30 Both the commercially available laccase
from T. versicolor (TvF) and the recombinant β-laccase from
P. pastoris (Lccβ) were associated with lower yields (by 35 to
45%) and products of lower purity when compared to AbL
(Table 4). Obviously, TvF and Lccβ possess lower activity on
substrates 1a, 1b, and 9. In order to study the influence of the
amount of laccase on the yields, the transformation of 1a and
2a was also performed with 30, 15, and 7 U of AbL (Table 4,
entries 2-4). With 30 U the yield of 3a is almost identical
with the yield obtained with 35 U (Table 4, entries 1 and 2).
With 15 U the yield slightly decreased to 88%, and 7 U
yielded 74%.
SCHEME 4. Possible Reaction Mechanism
Apart from the reactions with 4-hydroxymethylpyri-
din-2(1H)-ones, 4-hydroxyquinolin-2(1H)-ones, and 4-hy-
droxy-1-thiocoumarins we also studied the domino reactions
with barbituric acid derivatives as substrates using AbL as
a catalyst. We found that the reaction of N,N-dimethylbar-
bituric acid (20a) with catechol (2a) did not yield the expec-
ted benzofuran derivative but resulted in the exclusive
formation of the polycyclic dispiropyrimidinone 21a in
90% yield (Scheme 3). The exclusive formation of com-
pounds with this skeleton (21b and 21c) was also observed
in the reactions of 20a with the substituted catechols 2b
and 2c. Similarly, laccase-catalyzed domino reaction of 2a
with thiobarbituric acid (20b) exclusively afforded 21d
which precipitated from the reaction mixture in 77%. The
spirocyclic compounds 21a-d can also be synthesized in
comparable yields by using an excess of up to 4 equiv of
potassium ferricyanide as an oxidant31a or by electrochemi-
cal oxidation.31b-e
(29) Koschorreck, K.; Richter, S. M.; Swierczek, A.; Beifuss, U.; Schmid,
R. D.; Urlacher, V. B. Arch. Biochem. Biophys. 2008, 474, 213.
(30) Laccase activity was determined according to a modified proce-
dure taken from: Nicotra, S.; Intra, A.; Ottolina, G.; Riva, S.; Danieli, B.
Tetrahedron: Asymmetry 2004, 15, 2927. A 0.91 mM solution of ABTS
(0.3 mL) in 0.2 M phosphate buffer (pH = 6.0) was diluted with 0.2 M
phosphate buffer (2.6 mL, pH = 6.0) and treated with a solution of laccase
from A. bisporus (AbL) in the same buffer (0.1 mL). The change in absorption
was followed via UV spectroscopy (λ = 414 nm). For AbL 1 U is defined as
the amount of the enzyme that catalyzes the conversion of 1 μmol of ABTS
per minute by pH 6.0 and 25 °C. The activity of AbL with ABTS as the
substrate amounted to 0.61 U/mg. For TvF 1 U is defined as the amount of
the enzyme that catalyzes the conversion of 1 μmol of ABTS per minute by
pH 4.38 and 25 °C. For Lccβ 1 U is defined as the amount of the enzyme
that catalyzes the conversion of 1 μmol of ABTS per minute by pH 5.0 and
25 °C.
Presumably, the compounds 21 are formed according to
the mechanism depicted in Scheme 4 using the reaction of 2a
(31) (a) Alizadeh, A.; Nematollahi, D.; Hbibi, D.; Hesari, M. Synthesis
2007, 10, 1513. (b) Nematollahi, D.; Goodarzi, H. J. Org. Chem. 2002, 67,
5036. (c) Nematollahi, D.; Goodarzi, H.; Tammari, E. J. Chem. Soc., Perkin
Trans. 2 2002, 829. (d) Szanto, D.; Trinidad, P.; Walsh, F. J. Appl. Electro-
chem. 1998, 28, 251. (e) Azzem, A. M.; Zahran, M.; Hagagg, E. Bull. Chem.
Soc. Jpn. 1994, 67, 1390.
7234 J. Org. Chem. Vol. 74, No. 19, 2009

Hajdok et al.
JOCArticle
FIGURE 3. Observed NOEs in products 3c.
FIGURE 1. Product numbering of 3 and important HMBC
correlations (HfC) of 3f (blue arrow: 2J coupling; red arrow:
3J coupling; green arrow: 4J coupling).
cases, e.g., 3a, where the two phenolic OH groups of ring A
showed strong and sharp resonances. In compounds with
broad/overlapping OH signals, failure to determine the
substitution pattern was due to the absence of impor-
tant HMBC correlations (Figure 1). Preliminary studies
at various temperatures and pH values to obtain correla-
tion signals failed. By using another NMR approach focus-
ing on the evaluation of long-range 1H-13C coupling
constants, we were able to determine the substitution
pattern of ring A. The pulse sequences for the accurate
determination of 1H-13C coupling constants as well as
their application in the structure elucidation process were
published elsewhere.32 Due to its good sensitivity and ex-
cellent applicability for proton singlets, the HSQMBC32a,33
pulse sequence was implemented to provide the required
long-range heteronuclear coupling constants. From the
HSQMBC spectrum of 3a (Figure 4), the following coupl-
ing constants were extracted: H-9 (7.31 ppm) to C-8 (143.9
ppm), 3.1 Hz (2J), to C-7 (145.4 ppm), 7.3 Hz (3J) and to
C-5a (148.9 ppm), 10.3 Hz (3J). H-6 (7.03 ppm) to C-5a
(148.9 ppm), 4.3 Hz (2J), to C-7 (145.4 ppm), 4.4 Hz (2J),
and to C-8 (143.9 ppm), 6.3 Hz (3J). The coupling cons-
tants between H-9 (7.31 ppm) and C-9b (108.8 ppm), 2.9 Hz
(3J), as well as C-9a (115.4 ppm), 2.8 Hz (2J). From H-6
(7.03 ppm) to C-9a (115.4 ppm), 5.6 Hz (3J). Due to a lack
of reference data in the literature, we assigned the quater-
nary carbons of ring A on the basis of the size of the ob-
FIGURE 2. Product numbering of 6 and 10 and important
HMBC correlations (HfC) of 10a (blue arrow: 2J coupling; red
arrow: 3J coupling; pink arrow: ambiguous assignment).
with 20a as an example.31 Accordingly, the first step of the
domino process is the laccase catalyzed oxidation of catechol
(2a) with oxygen to o-quinone (15) which then reacts with
two molecules of N,N-dimethylbarbituric acid (20a) as a
nucleophile to yield the intermediate 22 (not isolable)
which;after oxidation to 23a;undergoes cyclization with
a second molecule of o-quinone (15). Oxidation of 24a finally
affords the dispiropyrimidinone 21a.
In all of the products (3, 6, and 10), full assignment of the
1H and 13C chemical shifts of ring C of 3 as well as rings C and
D of 6 and 10 could be achieved by evaluating their gHSQC,
gHMBC, and ROESY spectra (Figures 1 and 2).
3
served coupling constants [values for aromatic J (C, H)
are between 4 and 12 Hz34] as shown in Figure 5. For all
other compounds, corresponding HSQMBC correlations
and heteronuclear long-range coupling constants were
observed.
The connectivity between the substructures (rings C and
ring A) of 3, which establishes the regioselectivity of the
domino process, was deduced as follows: in the HMBC
spectrum of 3 (optimized for 8 Hz) the aromatic proton
To confirm the above assignment of ring A, the trimethoxy
derivative of 3f (= 25) was prepared (Scheme 5) and its
structure completely elucidated by common HSQC, HMBC,
ROESY, and NOE NMR experiments (Figure 6). Subse-
quent comparison of the nJ (C, H) data of 25 with the data
of 3f revealed similar coupling constants in both compounds
verifying the assignment on the sole basis of the analysis of
heteronuclear long-range coupling constants.
3
H-4 showed a strong JH/C correlation to carbon C-9b. A
3J-HMBC correlation from the latter carbon to proton
H-9 unambiguously fixed the regioselectivity of the reac-
tion (Figure 1). Additionally, selective 1D-NOE measure-
ments independently supported the assignments above.
For instance, irradiation of H-4 at δ 6.53 of 3c caused
1
an H,1H NOE enhancement of the signal of the methoxy
In conclusion, nJ(C,H) data may offer a promising
approach toward the assignment of quaternary aromatic
group at C-6 (instead of C-9) at δ 61.2 showing the spatial
proximity between these protons and confirming the ob-
served regioselectivity (Figure 3). In a similar way, the
structures of all products were elucidated, and it was estab-
lished that only one regioisomer was obtained in all the
reactions performed.
A prerequisite for the determination of the aforemen-
tioned regioselectivity is the complete and unambiguous
assignment of all quaternary carbons in all compounds,
especially those of rings A (C-8, C-7, C-5a, C-9a). Direct
assignment, however, could only be achieved in a few
(32) (a) Lacerda, V. Jr; da Silva, G. V. J.; Tormena, C. F.; Williamson, R.
T.; Marquez, B. L. Magn. Reson. Chem. 2007, 45, 82. (b) Williamson, R. T.;
Boulanger, A.; Vulpanovici, A.; Roberts, M. A.; Gerwick, W. H. J. Org.
Chem. 2002, 67, 7927. (c) Marquez, B. L.; Gerwick, W. H.; Williamson, R. T.
Magn. Reson. Chem. 2001, 39, 499. (d) Meissner, A.; Sørensen, O. W. Magn.
Reson. Chem. 2001, 39, 49. (e) Matsumori, N.; Kaneno, D.; Murata, M.;
Nakamura, H.; Tachibana, K. J. Org. Chem. 1999, 64, 866.
€ ꢀ
(33) (a) Kover, K. E.; Batta, G.; Feher, K. J. Magn. Reson. 2006, 181, 89.
(b) Williamson, R. T.; Marquez, B. L.; Gerwick, W. H.; Kover, K. E. Magn.
ꢀ
€ ꢀ
Reson. Chem. 2000, 38, 265.
(34) Hansen, P. E. Prog. Nucl. Magn. Reson. Spectrosc. 1981, 14, 175.
J. Org. Chem. Vol. 74, No. 19, 2009 7235

Hajdok et al.
JOCArticle
FIGURE 4. Part of the HSQMBC contour plot (500 MHz) of 3a and 1D antiphase HSQC slices from the contour plot.
FIGURE 5. Important HSQMBC correlations and assignments
of 3a.
FIGURE 6. Important HMBC, ROESY, and NOE correlations
of 25.
SCHEME 5. Preparation of the Trimethoxy Derivative of 3f
correlation between H-7 and C-6a unambiguously estab-
lished the connectivity between ring A and C as shown in
Figure 2.
Unequivocal evidence for the structure of the products
3a-f was produced by X-ray structure analysis. For this
purpose, crystals of the diacetate 13f which was derived from
3f were studied by X-ray crystal structure analysis.36 The
result confirmed the structure assignments made by NMR
experiments.
carbons in proton-poor NMR spectra of heterocycles
without any need for derivatization, solvent- and time-
consuming purification steps, as well as additional NMR
time.
Structure elucidation of the dispiropyrimidinones 21a-d
was achieved by NMR spectroscopic methods including
1
HSQC and HMBC. Both H and 13C NMR spectra are
Structure elucidation of compounds 6 and 10 (Figure 2)
was performed in a similar manner as described for 3, but
here was hampered by an additional problem: as the carbon
signals of C-6a and C-6b are very close to each other or even
overlap (e.g., δ 114.36 and 114.49 ppm, 10a), the HMBC
correlations with the protons H-7 and H-10 cannot be
resolved by indirect detected standard HMBC with 256 or
512 increments in F1 (Figure 7a). Hence, the connectivity
between rings A and C could not be determined. This is why
we used a recently published band-selective adiabatic
gHMBC pulse sequence35 combined with a modified
gHMBC for super long-range connectivities (4J (C, H)).
The 13C band-selection offers the possibility to record adia-
batic gHMBCs with a high resolution in the indirect dimen-
distinguished by the fact that they contain fewer reso-
nance signals than expected for the corresponding annu-
lated benzofurans. For example, the 1H NMR of 21a exhibits
only three resonance signals, namely three singlets: at 3.29,
6.61, and 9.34 ppm. In the 13C NMR spectrum of 21a
only seven resonance signals appear (29.7, 58.2, 113.8,
124.0, 146.9, 151.7, 169.9 ppm). These results indicate
a highly symmetrical structure. Together with the mass
spectrometry results suggesting that each of the compounds
21a-d is made up of two catechol and two barbituric
acid units it was deduced that 21a-d are polycyclic dis-
piropyrimidinones.
(36) (a) CCDC-714093 (13f) contains the supplementary crystallographic
data for this paper. These data can be obtained free of charge from the Cambridge
Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif. (b)
Otwinowski, Z.; Minor, W. Methods Enzymol. 1997, 276, 307. (c) Sheldrick,
G. M. SHELXS-97, Programs for X-ray Crystal Structure Solution; University
4
sion and without spectral aliasing (Figure 7b). A J (C, H)
3
correlation between H-1 and C-6a along with a J (C, H)
€ €
of Gottingen: Gottingen, Germany, 1997. (d) Sheldrick, G. M. SHELXL-97,
(35) (a) Bradley, S. A.; Krishnamurthy, K. Magn. Reson. Chem. 2005, 43,
117. (b) Crouch, R.; Boyer, R. D.; Johnson, R.; Krishnamurthy, K. Magn.
Reson. Chem. 2004, 42, 301.
€
Programs for X-ray Crystal Structure Refinement; University of Gottingen:
€
Gottingen, Germany, 1997.
7236 J. Org. Chem. Vol. 74, No. 19, 2009

Hajdok et al.
JOCArticle
FIGURE 7. Part of HMBC spectra of 10a displaying long-range correlations form H-7 and H-10 to C-6a and C-6b, respectively. (a) Part of
adiabatic gHMBC with ni = 256 and sw1 = 30165 Hz. (b) Band-selective gHMBC with ni = 256 and sw1 = 1887 Hz.
Conclusions
7,8-Acetyloxy-6-methoxy-2,3-dimethylbenzofuro[3,2-c]pyri-
din-1(2H)-one (13f). Mp: 180-181 °C. Rf = 0.24 (EtOAc). IR
(ATR): ν~ 1768 cm-1, 1670, 1572, 1375, 1174, 1088, 1037, 1016,
883, 792. UV (CH3CN): λmax (lg ε) 314 nm (4.27), 287 nm (3.86),
272 nm (3.92), 221 nm (4.40). 1H NMR (300 MHz, DMSO-d6): δ
2.33 ppm (s, 3H, CH3), 2.36 (s, 3H, CH3), 2.53 (s, 3H, CH3), 3.57
(s, 3H, NCH3), 4.14 (s, 3H, OCH3), 6.83 (s, 1H, 4-H), 7.50 (s, 1H,
9-H). 13C NMR (75 MHz, DMSO-d6): δ 20.7 ppm, 21.1, 22.0,
31.0, 61.5, 95.3, 107.1, 108.2, 123.6, 132.4, 138.5, 141.0, 143.2,
149.8, 159.7, 162.9, 168.9, 169.5. MS (70 eV, EI): m/z 359.1 (20)
[Mþ], 317.0 (67), 275.0 (100), 260 (25). Anal. Calcd for
C18H17NO7 (359.1): C, 60.17; H, 4.77; N, 3.90. Found: C,
60.44; H, 4.67; N, 4.12.
Here we presented a simple and efficient method for the
synthesis of several types of annulated benzofurans by
employing a laccase-catalyzed domino reaction between
catechols and heterocyclic 1,3-dicarbonyls. The reactions
can be performed under mild conditions using aerial oxy-
gen as an oxidant. When pyridinones, quinolinones, and
thiocoumarins are used as reactants, the corresponding
benzofuropyridinones, benzofuroquinolinones, and thio-
coumestans are formed with yields ranging from 55 to
98%. With barbituric acid derivatives as reactants polycyclic
dispiropyrimidinones are formed exclusively. The structure
of all products was elucidated unambiguously by NMR
spectroscopy. The results were supported by X-ray crystal
structure analysis of one product (13f).
20,30,60,70-Tetrahydroxy-1,100,3,300-tetramethyldispiro[pyrimidine-
5(2H),90(100H)-anthracene-100,500(200H)-pyrimidine]-2,200,4,400,6,600-
(1H,100H,3H,300H)-hexone (21a). Mp: >410 °C (lit.31e mp
>300 °C). Rf = 0.69 (CH2Cl2/MeOH = 6:4). IR (ATR):
3300 cm-1 (OH), 1706 and 1648 (CdO), 1537, 1454, 1416,
1
1380, 1350, 1251, 1206, 1060, 837, 758. H NMR (300 MHz,
Experimental Section
DMSO-d6): δ 3.28 ppm (s, 12H, N-CH3), 6.61 (s, 4H, arom H),
9.33 (s, 4H, OH). 13C NMR (75 MHz, DMSO-d6): δ 29.7 ppm
(CH3), 58.2 (spiro C), 113.8 (arom CH), 124.0 (arom C), 146.9
(COH), 151.7 (NCON), 169.9 (CdO). MS (FAB, matrix:
glycerin): 523.9 [Mþ]. C24H20N4O10 (524.12).
General Procedure for the Laccase-Initiated Domino Reaction.
A solution of 1.5 mmol of 1,3-dione and 1.7 mmol of catechol
in 100 mL of 0.2 M phosphate buffer (pH 6.0) was placed in
a 250 mL flask. A 50 mg portion of laccase from A. bisporus
(AbL) [3.11 U/mg; the enzyme activity was determined with
catechol as the substrate]24 was added and the mixture vigor-
ously stirred in air at room temperature until the substrates
had been fully consumed, as judged by TLC. The reaction
mixture was acidified with 2 M HCl to pH ∼4, saturated with
NaCl, and filtered with suction on a Buchner funnel. The filter
cake was washed with a solution of 50 mL of 15% NaCl
and 5 mL of H2O. The crude products obtained after drying
exhibited a purity of 90-95% (NMR). Analytically pure pro-
ducts could be obtained by recrystallization of the correspond-
ing diacetates.
7,8-Dihydroxy-6-methoxy-2,3-dimethylbenzofuro[3,2-c]pyri-
din-1(2H)-one (3f). Mp: 276-282 °C dec. Rf = 0.12 (EtOAc). IR
(ATR): ν~ 3458 cm-1 (OH), 3087 (OH), 1658, 1621, 1559, 1432,
1343, 1321, 1195, 1100, 1075, 1037, 1018, 996, 893, 786, 682. 1H
NMR (500 MHz, DMSO-d6): δ 2.49 ppm (s, 3H, 3-CH3), 3.54 (s,
3H, N-CH3), 3.99 (s, 3H, OCH3), 6.72 (s, 1H, 4-H), 7.14 (s, 1H,
9-H), 8.77 (s, 1H, 7-OH), 9.29 (s, 1H, 8-OH). 13C NMR (75
MHz, DMSO-d6): δ 21.8 ppm (3-CH3), 30.8 (NCH3), 61.2
(OCH3), 95.4 (C-4), 100.7 (C-9), 108.2 (C-9b), 115.9 (C-9a),
134.0 (C-6), 137.4 (C-7), 141.1 (C-5a), 145.1 (C-8), 146.4 (C-3),
159.9 (C-1), 161.0 (C-4a). MS (70 eV, EI): m/z 275 (100) [Mþ],
260 (63), 232 (20).
Acknowledgment. We thank Ms. Sabine Mika for record-
ing the NMR spectra and Dr. Heiko Leutbecher, Ms.
€
Fadime Mert-Balci, and Ms. Katrin Wohlbold (Institut fur
Organische Chemie der Universitat Stuttgart) for recording
€
the mass spectra. We thank Mr. Frank Hambloch (Institut
€
€
fur Organische und Biomolekulare Chemie der Universitat
Gottingen) for combustion elemental analysis. We thank Dr.
€
Katja Koschorreck and PD Dr. Vlada B. Urlacher (Institut
€
€
fur Technische Biochemie der Universitat Stuttgart) for the
recombinant laccase (Lccβ: β-laccase from T. versicolor
produced in P. pastoris). This work was performed within
the Collaborative Research Centre SFB 706 (Selective Cat-
alytic Oxidations Using Molecular Oxygen; Stuttgart) and
funded by the German Research Foundation.
Supporting Information Available: Full characterizations of
all new compounds, HSQMBC data for compounds 3a-f,
6a-c, 10a-c, and 25, selected NMR data for the spiro com-
pounds 21a-d, crystallographic data, and the X-ray structure of
the diacetate 13f. This material is available free of charge via the
Internet at http://pubs.acs.org.
J. Org. Chem. Vol. 74, No. 19, 2009 7237