Laccase-generated quinones in naphthoquinone synthesis via Diels-Alder reaction

Article abstract of DOI:10.1016/j.tetlet.2007.03.013

The tandem synthesis of naphthoquinones was conducted from the reaction of laccase-generated quinones and acyclic dienes via Diels-Alder reaction. This reaction was carried out under mild condition in aqueous medium and yielded naphthoquinones up to 80%. In addition, the effect of solvent was also investigated and water was shown to be optimal for this reaction.

Full text of DOI:10.1016/j.tetlet.2007.03.013

Tetrahedron Letters 48 (2007) 2983–2987
Laccase-generated quinones in naphthoquinone synthesis via
Diels–Alder reaction
Suteera Witayakran,a Abdullah Zettilib and Arthur J. Ragauskasa,*
aSchool of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332, USA
bDepartment of Physical and Earth Sciences, School of Chemistry, Jacksonville State University, Jacksonville, AL 36265, USA
Received 20 November 2006; revised 27 February 2007; accepted 1 March 2007
Available online 4 March 2007
Abstract—The tandem synthesis of naphthoquinones was conducted from the reaction of laccase-generated quinones and acyclic
dienes via Diels–Alder reaction. This reaction was carried out under mild condition in aqueous medium and yielded naphtho-
quinones up to 80%. In addition, the effect of solvent was also investigated and water was shown to be optimal for this reaction.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Laccases (benzenediol:oxygen oxidoreductase, EC
1.10.3.2) are copper-containing glycoprotein oxidore-
ductase enzymes found in plants and fungi.1 They cata-
lyze the oxidation of a broad range substrates,2 such as
methoxyphenols, phenols, o-, and p-diphenols, amino-
phenols, polyphenols, polyamines, and lignin-related
molecules, by reducing dioxygen in a four-electron
reduction to water. Moreover, laccases frequently exhi-
bit high oxidative selectivity in an aqueous solution
and provide a unique green chemistry solution for a
variety of oxidations. These properties make laccase
an attractive biocatalysts in organic synthetic chemistry.
Recently, studies have investigated the use of laccase in
organic synthesis.2a,3 For example, utilizing their well
known propensity to oxidize phenolics, Lalk and co-
workers reported laccase catalyzed a nuclear animation
tandem reaction.3a These studies have demonstrated the
synthetic research capabilities of this oxidative enzyme.
The combination of enzymatic with nonenzymatic trans-
formations for tandem reactions was first reported by
Waldmann and co-workers in 1998.8 They reported
the synthesis of highly functionalized bicycle[2.2.2]oct-
enes by a tyrosinase-initiated hydroxylation–oxidation
of phenols followed by a Diels–Alder (DA) reaction
with electron rich dienophiles (see Scheme 1). These
studies, conducted in chloroform, provided a unique
three-step one-pot reaction of bicyclic DA products in
high yields with the key intermediate being reactive
ortho-quinones. The applicability of enzyme catalyzed
domino reactions in green chemistry has only recently
been fully appreciated.
OH
R1
OH
R1
tyrosinase
CHCl3, O2
OH
This study summarizes our interests in the use of laccase
for the synthesis of substituted napthoquinones. Naphtho-
quinones are naturally occurring compounds which
have attracted interest in total synthesis because of their
wide range of biological activity including antitumor,4
wound healing,5 anti-inflammatory,5 and antimicrobial6
and antiparasitic activities.7
tyrosinase
CHCl3, O2
O
O
O
O
O
R2
O
+
R1
R2
R1
R2
R1
Keywords: Laccase; Diels–Alder reaction; Naphthoquinone; Quinone;
Green chemistry; Tandem reaction.
*
Scheme 1. The example of enzyme-initiated reaction cascade reported
by Waldmann and co-workers.8
Corresponding author. Tel.: +1 404 894 9701; fax: +1 404 894
4778; e-mail: arthur.ragauskas@chemistry.gatech.edu
0040-4039/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2007.03.013

2984
S. Witayakran et al. / Tetrahedron Letters 48 (2007) 2983–2987
OH
O
R2
R5
R2
R5
O
O
OH
O
R3
R4
R1
R1
R1
R2
R3
Laccase
[O ]
DA
or
+
or
or
0.1 M Acetate Buffer pH4.5
R4
R5
OH
O
O
O
R3
R4
R1
R1
R1
OH
O
Scheme 2. Laccase-initiated cascade synthesis of substitute naphthoquinones via aqueous Diels–Alder reaction.
In this study, a series of substituted naphthoquinones
were synthesized via an aqueous cascade reaction
between acyclic dienes and in situ-generated quinones.
The ortho- and para-quinones were synthesized in situ
by the oxidation of the corresponding o- or p-benzo-
hydroquinone by laccase. The initial Diels–Alder adduct
was shown to undergo further oxidization by laccase
and/or quinone to yield the desired naphthoquinones
(see Scheme 2).
ature and medium were shown to have an effect on the
reaction outcome. For example, if the reaction was pre-
formed at room temperature or 60 ꢁC the yield of 3
was diminished to only 10% and 0%, respectively. This
result was attributed an increase in the rate of decompo-
sition and polymerization of the in situ-generated o-qui-
none. Therefore, we retarded the rate of decomposition
and polymerization by maintaining the initial reaction
temperature to 3 ꢁC for the first 2 h and then allowing
the reaction mixture warm to room temperature. This
cascade reaction system provided 47% of 3. The reaction
was performed in an aqueous acetate buffer at pH 4.5,
generally known to be the optimum pH for laccase activ-
ity in the formation of quinone,13 these conditions pro-
vided the best result (see Table 2). The lower percent
yield in other solvent systems was due to a decrease of
laccase activity in organic and aqueous–organic mixed
solvent.14 Moreover, the Diels–Alder reaction has shown
to exhibit higher reactivity and selectivity in aqueous
medium than in organic solvent.15 Interestingly, the use
of a 1:1 acetate buffer/chloroform medium, provided
the aromatized DA adduct (5,8-dihydro-6,7-dimethyl-
1,2-naphthoquinone) instead of fully oxidized product
(3).
Initially, we focused our attention on the reaction of
laccase with 1,2-catechols yielding the corresponding o-
quinones which have an interesting reactivity profile in
cycloaddition reactions,9 and have been used in o-naph-
thoquinone synthesis.
In a preliminary study, the reaction of catechol (1) and
2,3-dimethyl-1,3-butadiene (2) in the presence of laccase
was investigated. As summarized in Table 1, optimal
yields of 6,7-dimethyl-1,2-naphthoquinone (3) was
achieved when the reaction was conducted with 1 equiv
of 1 and 10 equiv of 2 in the presence of laccase10 in
0.1 M acetate buffer pH 4.5 at 3 ꢁC for the first 2 h of
the reaction. The reaction mixture was then warmed to
room temperature and stirred for another 22 h.11
After developing the optimum reaction conditions, the
reaction of a variety of catechol substrates with diene
2 were examined11 and these results are summarized in
An excess of the diene was required to overcome the
intrinsic instability of the o-benzoquinone as it will
undergo competing decomposition, dimerization, and
polymerization if insufficient diene is present for the
Diels–Alder reaction.12 In addition, the reaction temper-
Table 2. Solvent effect on the reaction of 1 and 2a
Entry Solvent
Yieldb of 3 (%)
1
2
3
4
5
6
0.1 M Acetate buffer pH 4.5
Water
47
18
25
0
8
15
Table 1. Preliminary study of the reaction of catechol (1) and 2,3-
dimethyl-1,3-butadiene (2)
5% Aqueous PEG 2000
p-Dioxane
1:1 p-Dioxane/acetate buffer
1:1 Ethylene glycol/
acetate buffer
O
OH
O
OH
Laccase
0.1M acetate buffer pH 4.5
24 hours
7
8
1:1 MeOH/acetate buffer
1:1 Chloroform/acetate buffer 0% of 3
18
1
3
2
OH
Entry
1:2 (equiv)
Temperature
Yielda of 3 (%)
HO
27% of
1
2
3
4
5
1:10
1:10
1:10
1:5
3 ꢁC (2 h), rt
rt
60 ꢁC
3 ꢁC (2 h), rt
3 ꢁC (2 h), rt
47
10
No product formed
8
32
a Reaction conditions: 1 (1 equiv) and 2 (10 equiv) was stirred with
laccase (4000 U/1g substrate) in solvent at 3 ꢁC for 2 h and then
stirred at room temperature for another 22 h.
b Isolated yield.
1:15
a Isolated yield.

S. Witayakran et al. / Tetrahedron Letters 48 (2007) 2983–2987
2985
Table 3. The results show that the reaction depended on
the reactivity of the in situ-generated o-quinones. The
very high reactivity quinones, such as 3-methoxy-1,2-
benzoquinone and 4-chloro-1,2-benzoquinone, which
have rich electron donating group (OMe) and strong
electron withdrawing group (Cl), respectively, did not
provide good yields of the o-naphthoquinone product
(entries 4 and 5). These quinones preferently underwent
dimerization and polymerization. For example, in situ
synthesis 3-methoxy-1,2-benzoquinone by laccase from
the corresponding hydroquinone yielded 32% of the
undesired product, which was converted from the dimer-
ization intermediate, and only 11% of naphthoquinone
product. Besides the reactivity of the in situ-generated
quinones, steric factor also affected the formation of
the product. Quinones with bulky groups provided very
low yield of the products such as 4-tert-butyl-1,2-benzo-
quinone yielded only 14% product for a 4 day reaction,
and 3,5-di-tert-butyl-1,2-benzoquinone gave no product
but 97% of it remaining in the reaction solution (entries
6 and 7). From Table 3, the in situ-generated o-quinones
with moderate reactivity clearly exhibited higher yields
of the o-naphthoquinone adduct (entries 1–3), and 4-
methyl-1,2-benzoquinone provided the highest yield
(57%) in this reaction system (entry 2).
The versatility of this system for a variety of dienes was
investigated by using 4-methylcatechol as starting
material to generate 4-methyl-1,2-benzoquinone in situ.
Table 4 demonstrates that many dienes can be used
to react with 4-methyl-1,2-benzoquinone to generate
o-naphthoquinone products in moderate to high
yield. Optimal results were achieved when 1-methoxy-
1,3-butadiene and 1-acetoxy-1,3-butadiene were used
as diene reagent (entries 4 and 5). Both dienes provided
very high yields of the product, and only 2 equiv of 1-
acetoxy-1,3-butadiene was needed. This high yielding
reaction can be attributed to the elimination of the
methoxy or acetoxy group that ‘pushed’ the reaction
forward to the product.
Table 3. Reaction of 2 with a variety of catechol substrates
OH
O
OH
O
Laccase
R1
0.1M acetate buffer pH 4. 5
3 oC - RT, 24 hours
R1
2
In this study, we also conducted p-naphthoquinone
synthesis by using a variety of 1,4-benzohydroquinones
as a substrate for laccase to generate 1,4-benzoquinone
in situ. As the result of o-quinone reaction above, the
reactive 1-acetoxy-1,3-butadiene was chosen for this
study. However, we found that the reaction of these less
10
1
:
Entry
Catechol
Yielda (%)
47
OH
OH
1
Table 4. Reaction of 4-methylcatechol with a variety of dienes
OH
OH
O
R2
OH
R2
2
57
O
R3
R4
OH
R3
R4
Laccase
CH3
OH
0.1M acetate buffer pH 4.5
3 oC - RT, 24 hours
CH3 R5
CH3
R5
OH
:
1
10
3
4
5
28 (R1 = H)
11 and 32 of
Entry
1
Diene
Yielda (%)
57
O
CH
O
OCH3
OH
O
H3CO
OH
OH
2
3
71
10
H3CO
OCH3
OH
No product formed
OCH3
OCH3
Cl
4
77 (R2 = H)
76 (R2 = H)
OH
O
OH
OH
6b
14 and 15 of
O
O
O
CH3
5b
OH
O
7
No product formed
97 of quinone
O
6
No product formed
a Isolated yield.
b 96 h reaction.
a Isolated yield.
b Only 2 equiv of 1-acetoxy-1,3-butadiene was used.

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S. Witayakran et al. / Tetrahedron Letters 48 (2007) 2983–2987
Table 5. Reaction of 1-acetoxy-1,3-butadiene with a variety of 1,4-
benzohydroquinone
2004, 17, 973–977; (h) d’Acunzo, F.; Galli, C.; Gentili, P.;
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Mol. Catal. B: Enzym. 2005, 35, 86–92; (i) Leutbecher, H.;
Conrad, J.; Klaiber, I.; Beifuss, U. Synlett 2005, 3126–
3130.
O
O
CH3
OH
O
O
R1
R1
Laccase
0.1M acetate buffer pH 4.5
55 oC, 24 hours
OH
2
1
:
Entry
R1
Yielda (%)
1
2
3
4
5
H
67
75
81
67
69
CH3
OCH3
Br
Cl
a Isolated yield.
4. (a) Subramanian, S.; Ferreira, M. M. C.; Trsic, M. Struct.
Chem. 1998, 9, 47–57; (b) Powis, G. Free Radical. Biol.
Med. 1989, 6, 63–101; (c) Afrasiabi, Z.; Sinn, E.; Chen, J.;
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reactive p-benzoquinones gave very low yield of the
desired product at low temperature. Therefore, the
reaction was conducted at 55 ꢁC for p-naphthoquinone
synthesis, and 1 equiv of 1,4-benzohydroquinone and
2 equiv of diene were used (Table 5).16 The results in
Table 5 show that this reaction system can be used for
a one-pot synthesis of p-naphthoquinones in excellent
overall yield.
In summary, an efficient green chemistry synthesis of
naphthoquinone using laccase as an oxidant in aqueous
medium was developed. In this reaction, laccase was
used to oxidized o- and p-diphenols to generate o- and
p-quinones in situ which further underwent Diels–Alder
reaction and oxidation to form napthoquinone product.
This reaction system can yield naphthoquinones up to
80% depending on the exact structure of the starting
hydroquinone and diene.
8. Muller, G. H.; Lang, A.; Seithel, D. R.; Waldmann, H.
¨
Chem. Eur. J. 1998, 4, 2513–2522.
9. Patai, S. The Chemistry of the Quinonoid Compounds;
Interscience: London, New York, 1974.
10. Enzyme assay: Laccase from Trametes Villosa (EC
1.10.3.2) was obtained from Novozymes, North Carloina.
Laccase activity was determined by oxidation of 2,20-
azinobis-(3-ethylbenzyl thiozoline-6-sulfonate) (ABTS).17
The assay mixture contained 25 lM ABTS, 0.1 M sodium
acetate (pH 5.0), and a suitable amount of enzyme. The
oxidation of ABTS was followed by an absorbance
increase at 420 nm (e420 = 3.6 · 104 Mꢀ1 cmꢀ1). Enzyme
activity was expressed in units (U = lmol of ABTS
oxidized per minute).
Acknowledgements
The authors acknowledge financial support from the
IPST@GT student fellowship, NSF Research Experi-
ence for Undergraduate in Chemistry and Biochemistry
(award #: 0552722) and the Royal Thai Government
Scholarship.
References and notes
11. Typical experimental procedure for o-naphthoquinone syn-
thesis: In a 250-mL round-bottom flask, 20 ml of cold
0.10 M acetate buffer pH 4.5 and 2,3-dimethyl-1,3-buta-
diene (2) (0.7 g, 9 mmol) were mixed together. The flask
was then placed in an ice bath over a stirring plate. Next,
0.1 g (0.9 mmol) of catechol (1) dissolved in 20 mL of
0.10 M acetate buffer, and laccase (100 U) were added to
the flask drop-wise. In the next 3 h of the reaction, 100 U
of laccase was added each per hour.The reaction was then
stirred under room temperature. After 24 h of the
reaction, the reaction mixture was extracted by EtOAc.
The organic phase was combined, dried over MgSO4, and
evaporated. The resulting crude products were purified by
silica column chromatography, using ethyl acetate and
petroleum ether as an eluent to obtain the products 3
(47%). Most compounds have been previously reported
and characterized except the two compounds below. 4,7,8-
Trimethyl-1,2-naphthoquinone: orange solid; mp 118 ꢁC
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S. Witayakran et al. / Tetrahedron Letters 48 (2007) 2983–2987
2987
(decomp.); 1H NMR (CDCl3, 400 MHz) d 2.36 (s, 3H,
CH3), 2.37 (s, 3H, CH3) 2.59 (s, 3H, CH3), 6.32 (s, 1H,
CH), 7.29 (d, J = 8 Hz, 1H, Ar), 7.41 (d, J = 8 Hz, 1H,
Ar); 13C NMR (CDCl3, 100 MHz) d 17.6, 20.9, 21.2,
124.5, 126.3, 129.8, 134.8, 135.6, 141.8, 144.1, 154.9, 181.6,
183.5; MS (EI) m/z 200 (M+, 31%), 172 (100), 157 (22),
141(11), 129 (38), 115 (12), 102 (4), 77 (7), 63 (7), 51 (8), 44
(27); HRMS (EI) calcd for C13H12O2 requires 200.0837,
found: 200.0840. 4-Methyl-6,7-dimethoxy-1,2-naphtho-
quinone: red solid; mp 124 ꢁC (decomp.); 1H NMR
(CDCl3, 400 MHz) d 2.37 (s, 3H, CH3), 3.97 (s, 3H,
OCH3), 4.03 (s, 3H, OCH3), 6.25 (s, 1H, CH), 6.92 (s, 1H,
Ar), 7.61 (s, 1H, Ar); 13C NMR (CDCl3, 100 MHz) d 20.7,
56.3, 108.7, 112.0, 125.2, 125.9, 130.8, 150.5, 153.2, 154.6,
178.3, 181.0; MS (EI) m/z 232 (M+, 54%), 204 (100), 189
(37), 175 (4), 161 (9), 133 (9), 118 (8), 105 (12), 77 (5), 63
(7), 39 (6); HRMS (EI) calcd for C13H12O4 requires
232.0735, found: 232.0734.
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16. Typical experimental procedure for p-naphthoquinone syn-
thesis: p-Hydroquinone (0.1 g, 0.9 mmol), 1-acetoxy-1,3-
butadiene (0.2 g, 1.8 mmol), and laccase (100 U) were
stirred in 40 ml of 0.1 M acetate buffer pH 4.5 under air at
55 ꢁC. In the next 3 h of the reaction, 100 U of laccase was
added each per hour. After 24 h of the reaction, the
reaction mixture was extracted by EtOAc. The organic
phase was combined, dried over MgSO4, and evaporated.
The resulting crude products were purified by silica
column chromatography, using ethyl acetate and petro-
leum ether as an eluent to obtain the products.
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