Laccase-catalysed reaction between Meldrum's acid and catechols/ hydroquinones - An investigation

Article abstract of DOI:10.1016/j.crci.2013.04.006

Enzymatic oxidation of catechols/hydroquinones in the presence of Meldrum's acid as a nucleophile in aqueous solution has been investigated in detail by applying laccase as a catalyst. Optimization of the reaction through the use of HBT/ABTS/HPI as a mediator allowed a yield of more than 65% of the isolated product. We derived some new compounds with catechol/hydroquinone ring with moderate yields based on enzymatic synthesis in environmentally benign aqueous solution. These types of reactions represent a milestone along the path to future sustainable green chemistry.

Full text of DOI:10.1016/j.crci.2013.04.006

C. R. Chimie 16 (2013) 728–735
Contents lists available at SciVerse ScienceDirect
Comptes Rendus Chimie
www.sciencedirect.com
´
Full paper/Memoire
Laccase-catalysed reaction between Meldrum’s acid and catechols/
hydroquinones – An investigation
Mazaahir Kidwai a, ,1, Arti Jain a, Abha Sharma b, Ramesh Chander Kuhad b
*
a Green Research Laboratory, Department of Chemistry, University of Delhi, North Campus, New Delhi 110007, India
b Lignocellulose Biotechnology Laboratory, Department of Microbiology, University of Delhi, South Campus, New Delhi 110021, India
A R T I C L E I N F O
A B S T R A C T
Article history:
Enzymatic oxidation of catechols/hydroquinones in the presence of Meldrum’s acid as a
nucleophile in aqueous solution has been investigated in detail by applying laccase as a
catalyst. Optimization of the reaction through the use of HBT/ABTS/HPI as a mediator
allowed a yield of more than 65% of the isolated product. We derived some new
compounds with catechol/hydroquinone ring with moderate yields based on enzymatic
synthesis in environmentally benign aqueous solution. These types of reactions represent
a milestone along the path to future sustainable green chemistry.
Received 27 June 2012
Accepted after revision 4 April 2013
Available online 2 July 2013
Keywords:
Laccase
Aqueous medium
Meldrum’s acid
Environmentally benign
Novel compounds
´
ß 2013 Academie des sciences. Published by Elsevier Masson SAS. All rights reserved.
1. Introduction
studies have investigated the use of laccase in organic
synthesis [11–23].
From the perspective of green chemistry, environmen-
tally friendly manufacturing of organic compounds has
been considerably re-examined in recent years and many
excellent methodologies have been devised. Reactions
employing natural sources as catalysts are the best
example of such type of aspects.
Laccases are copper-containing glycoprotein oxidor-
eductase enzymes found in plants and fungi [1,2]. They
catalyse the oxidation of a broad range of substrates [3–
10], such as methoxyphenols, phenols, o-, and p-diphenols,
aminophenols, polyphenols, polyamines, and lignin-
related molecules, by reducing dioxygen in a four-electron
reduction to water. Moreover, laccases frequently exhibit
high oxidative selectivity in aqueous solution and provide
Meldrum’s acid is of particular interest, since they are
known to have depressant effect on the central nervous
system and to possess a low toxicity, like barbiturates [24].
Catechol, by itself, and mono-substituted catechols are
active in part against Pseudomonas, Bacillus, but not
Penicillium species [25]. Many flavanoids and catechol
derivative turned out to be antimicrobial agents [26].
Hence, the fusion of catechol and Meldrum’s acid could
provide superior biological moiety i.e. 6,7-dihydroxy-2,2-
dimethyl-1,3,9-trioxa-fluoren-4-one. Recently, Nematol-
lahi et al. reported the one-pot reaction of Meldrum’s acid
and catechol via the oxidation of catechol by an electro-
chemical method [27]. Because enzymatic oxidation very
often parallels the chemical and electrochemical oxidation,
it was interesting to study the enzymatic oxidation of
catechols and hydroquinones in the presence of Meldrum’s
acid as CH-acidic nucleophile.
a
unique green chemistry solution for a variety of
oxidations. These properties make laccase attractive
biocatalysts in organic synthetic chemistry. Recently,
The synthesis of compounds with benzofuran moiety
or combination of benzofuran and dioxane make it
promising in the search for new pharmacologically active
substances, as part of our program aimed at developing
efficient and environmentally friendly methodologies for
*
Corresponding author.
E-mail address: kidwai.chemistry@gmail.com (M. Kidwai).
Presently vice-chancellor of Jiwaji university, Gwalior (MP), India.
1
1631-0748/$ – see front matter ß 2013 Acade´mie des sciences. Published by Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.crci.2013.04.006

M. Kidwai et al. / C. R. Chimie 16 (2013) 728–735
729
the preparation of fine chemicals [28–30]. So, herein we
are studying the enzymatic greener oxidation of catechol/
hydroquinone and their derivative in the presence of
Meldrum’s acid as a nucleophile.
from the above three organisms was harvested by adding
0.1 mol/L citrate phosphate buffer (pH 5.4) in a 1:5 ratio of
solid substrate to buffer. The mixture was homogenized for
30 min at 30 8C, 200 rpm in a New Brunswick Scientific
incubator shaker Innova = 40R (USA). The solid biomass
residues were separated from the suspensions by filtration
through muslin cloth and the filtrate obtained was
centrifuged at 10 000 g for 20 min at 4 8C in a Sigma 6 K
15 laboratory centrifugation system (UK). The supernatant
obtained was used as the source of crude enzyme
preparations.
As clearly stated by Sheldon, it is generally recognized
that ‘‘the best solvent is no solvent and if solvent is needed
it should preferably be water’’, hence, herein we are using
water as the reaction medium. The use of water as
the reaction medium represents a remarkable benefit,
since this green solvent is highly polar and therefore
immiscible with most of the organic compounds; moreover,
the water-soluble catalyst resides and operates in the
aqueous phase, thus, separation of the organic material is
easy [31].
2.1.3. Purification of laccase
The crude laccase enzyme extract (1000 mL) from
P. cinnabarinus was precipitated by adding ammonium
sulphate (40–80% cut off) and centrifuged at 10 000 g for
20 min at 4 8C. The precipitates were resuspended in a
50 mM citrate phosphate buffer (pH 5.4) and dialyzed
against the same buffer. This partially purified enzyme
sample was loaded to an anion exchange Q-Sepharose
column (Sigma–Aldrich, St. Louis, MO, USA), equilibrated
with a 10 mM Tris–HCl buffer (pH 7.5). The proteins were
eluted by NaCl gradient (0–0.5 M dissolved in equilibrating
buffer) at a flow rate of 1 mL/min, with each 1 mL fraction.
Fractions with laccase activity were pooled and concen-
trated using 10-kDa filter membrane (Vivaspin,
Vivascience, Sartorious group, Stone house, UK) at 4 8C
and assayed for laccase activity.
2. Experimental
2.1. Experimental procedures
Meldrum’s acid, various catechols and hydroquinones
were purchased from Aldrich. Guaiacol was purchased
from Hi Media Laboratories Pvt. Ltd. (Mumbai, India). Q-
sepharose was purchased from Sigma (St. Louis, USA). All
media components and chemicals used were of analytical
grade. All reactions and purity of products were mon-
itored by thin layer chromatography (TLC) using alumi-
nium plates coated with silica gel (Merck) employing
ethylacetate and hexane (3:7). The isolated products were
further purified by column chromatography using alu-
minium oxide (Sigma–Aldrich 24, 217-9, 70, 35-70, mesh
2.1.4. Enzyme assay
2
˚
40 A, surface area 675 m /g). IR spectra were recorded on a
Guaiacol was used as a substrate for assaying laccase
activity. One unit of laccase was defined as the change in
absorbance of 0.01 mLÀ1 minÀ1 at 470 nm [32,33].
PerkinElmer FTIR-1710 spectrophotometer using a Nujol
film and KBr pellets. 1HNMR, 13CNMR spectra were
recorded on a JEOL JNMECX 400P FT NMR system
(manufactured in Japan) using TMS as an internal
2.2. General procedure for the reaction of Meldrum’s acid and
catechols/hydroquinones
standard. The chemical shift values are recorded on
scale.
d
Catechol or hydroquinone (1 mM) and Meldrum’s acid
(2 mM) were taken in 15 mL of phosphate/citrate buffer
pH 7 solution. Then, purified laccase (400 U) and
0.01 mmol of HBT were added to the resultant solution
and stirred well at 5 8C with a magnetic stirrer. The
temperature of the reaction mixture was gradually
increased up to room temperature. The progress of the
reaction was monitored by TLC examination at intervals of
30 min. Upon completion of the reaction, approximately
after 12 h, the reaction mixture was extracted with
ethylacetate (3 Â 15, i.e. three-time extraction with 15 mL
of ethylacetate). The organic layer was dried over
anhydrous Na2SO4. The aqueous phase was further used
for another run without any pre-treatment. The product
was purified by column chromatography using aluminium
oxide as a stationary phase and hexane:ethyl acetate
(80:20) as a mobile phase. Products were unambiguously
characterized by spectral data.
2.1.1. Microorganisms
The laccase produced from Pycnoporous cinnabarinus,
were obtained from the Lignocellulose Biotechnology
laboratory, University of Delhi, South Campus, New Delhi.
The fungal cultures were grown and maintained on malt
extract agar (MEA) containing (g/L): malt extract 20.0;
KH2PO4 0.5; MgSO4Á7H2O, Ca(NO3)2Á4H2O 0.5, agar 20.0
(pH 5.4) at 30 8C. Pure cultures were stored at 4 8C and
subcultured every fortnight.
2.1.2. Isolation of laccase
Laccase
production
was
carried
out
using
P. cinnabarinus under solid-state fermentation conditions.
Then, 1000 g of air-dried wheat bran were spread in
separately sterilized enamel trays (78 Â 51 Â 8.1 cm3) to
about 1.0 cm thick and were moistened with a mineral salt
solution containing (g/L): KH2PO4 0.5; MgSO4Á7H2O,
Ca(NO3)2Á4H2O 0.5, agar 20.0 (pH 5.4) in 1:2 ratio of solid
substrate to moisture. The trays were put in autoclavable
poly bags and sterilized at 121 8C (15 psi) for 30 min. The
substrate was inoculated with 0.4% w/w of fungal pellets of
P. Cinnabarinus. The trays were incubated for 168 h at
30 Æ 1 8C in a 70% relative humidity chamber. The laccase
2.3. Procedure to recover laccase
After completion of the reaction, the product was
extracted using ethylacetate and dried over anhydrous
sodium sulphate. The solvent was evaporated under

730
M. Kidwai et al. / C. R. Chimie 16 (2013) 728–735
reduced pressure and water extract was further used for
consecutive runs.
1.79 (s, 3H), 1.80 (s, 3H), 3.81 (s, 3H), 4.40 (s, 1H), 6.75 (s, 1H),
6.87 (s,1H), 7.90 (s, br), 8.12 (s, br); 13CNMR,
(100 MHz,
d
DMSO-d6) 24.1, 24.8, 28.11, 53.55, 107.19, 116.30, 120.61,
2.4. Characterization of the products
124.19, 127.29, 145.19, 147.11, 169.34. Anal. calcd. for
C13H14O7; C, 55.32; H, 5.00; found C, 55.39, H, 4.98; ESI–
2.4.1. Compound 5a: 6,7-dihydroxy-2,2-dimethyl-1,3,9-
trioxa-fluoren-4-one
MS: 283.02 (M + 1).
Mp > 300 8C; IR (KBr) 3430, 3448,1715, 1620,
2.4.7. Compound 4g: 5-(2-(tert-butyl)-4, 5-
1618 cmÀ1
;
1HNMR,
d
(400 MHz, DMSO-d6) 1.75 (s, 3H),
1.78 (s, 3H), 6.87 (s, 1H), 6.91 (s, 1H), 7.90 (s, br), 8.01 (s,
br); 13CNMR,
(100 MHz, DMSO-d6) 25.1, 25.5, 85.01,
Dihydroxyphenyl)-2,2-dimethyl-1,3-dioxane-4,6-dione
Mp 215–217 8C; IR (KBr) 3430, 3009,1761, 1727,1696,
d
1613,1518, 1512 cmÀ1 1HNMR,
; d(400 MHz, DMSO-d6)
101.23, 105.01, 110.10, 125.11, 127.98, 144.56,151.24,
162.34, 170.20. Anal. calcd. for C12H10O6; C, 57.60; H, 4.03;
found C, 57.89, H, 3.98; ESI–MS: 251.00 (M + 1).
1.35 (s, 9H), 1.70 (s, 3H), 1.72 (s, 3H), 4.38 (s, 1H), 6.72 (s,
1H), 6.86 (s,1H), 7.78 (s, br), 8.10 (s, br); 13CNMR,
d
(100 MHz, DMSO-d6) 27.1, 27.3, 31.13, 34.25, 53.55,
107.19, 116.30, 120.61, 124.19, 127.29, 145.19, 147.11,
169.34. Anal. calcd. for C16H20O6; C, 62.33; H, 6.54;
found C, 62.99, H, 6.98; ESI–MS: 309.11 (M + 1).
2.4.2. Compound 5b: 6,7-dihydroxy-2,2,8-trimethyl-1,3,9-
trioxa-fluoren-4-one
Mp > 300 8C; IR (KBr) 3427, 3440,1711, 1621,
1610 cmÀ1
;
1HNMR,
d
(400 MHz, DMSO-d6) 1.73 (s, 3H),
1.74 (s, 3H), 2.15 (s, 3H), 6.85 (s, 1H), 7.87 (s, br), 8.00 (s,
br); 13CNMR,
(100 MHz, DMSO-d6) 19.01, 24.9, 25.3,
85.00, 104.91, 109.35,112.01, 124.37, 141.14,
2.4.8. Compound 9a: 5,8-Dihydroxy-2,2-dimethyl-1,3,9-
trioxa-fluoren-4-one
d
Mp > 300 8C; IR (KBr) 3433, 3449, 1712, 1620,
1618 cmÀ1
;
1HNMR,
d
(400 MHz, DMSO-d6) 1.70 (s, 3H),
1.77 (s, 3H), 6.84 (d, 1H), 6.89 (d, 1H), 8.00 (s, br),
8.11 (s, br); 13CNMR,
(100 MHz, DMSO-d6) 24.4, 25.5,
145.38,162.70, 170.00. Anal. calcd. for C13H12O6; C,
59.09; H, 4.58; found C, 59.34, H, 4.38; ESI–MS: 265.12
(M + 1).
d
80.01, 100.23, 104.11, 110.19, 124.11, 128.00, 144.60,
150.94, 162.14, 170.00. Anal. calcd. for C12H10O6; C,
57.60; H, 4.03; found C, 57.67, H, 4.17; ESI–MS: 251.34
(M + 1).
2.4.3. Compound 5d: 6,7-dihydroxy-2,2-dimethyl-8-
methoxy-1,3,9-trioxa-fluoren-4-one
Mp > 300 8C; IR (KBr) 3426, 3442,1710, 1619, 1617,
1225 cmÀ1
;
1HNMR,
d
(400 MHz, DMSO-d6) 1.75 (s, 3H),
1.76 (s, 3H), 3.81 (s, 3H), 6.80 (s, 1H), 7.81 (s, br), 8.00 (s,
br); 13CNMR,
(100 MHz, DMSO-d6) 24.7, 25.0, 61.50,
2.4.9. Compound 9b: 5,8-Dihydroxy-2,2,7-trimethyl-1,3,9-
trioxa-fluoren-4-one
d
Mp > 300 8C; IR (KBr) 3425, 3442,1710, 1619,
86.23, 105.97, 110.15, 114.78, 125.11, 142.38, 144.14,
163.25, 171.45. Anal. calcd. for C13H12O7; C, 55.72; H, 4.32;
found C, 55.82, H, 4.98; ESI–MS: 281.09 (M + 1).
1605 cmÀ1
;
1HNMR,
d
(400 MHz, DMSO-d6) 1.73 (s, 3H),
1.76 (s, 3H), 2.10 (s, 3H), 6.90 (s, 1H), 7.80 (s, br), 8.04
(s, br); 13CNMR,
(100 MHz, DMSO-d6) 19.00, 25.02,
d
25.37, 86.12, 104.91, 109.40, 112.34, 125.17, 141.10,
145.38,161.99, 171.14. Anal. calcd. for C13H12O6; C,
59.09; H, 4.58; found C, 59.24, H, 4.40; ESI–MS: 265.54
(M + 1).
2.4.4. Compound 5f: 8-(tert-butyl)-6,7-dihydroxy-2,2-
dimethyl-1,3,9-trioxa-fluoren-4-on
Mp > 300 8C; IR (KBr) 3421, 3423,1709, 1610,
1611 cmÀ1
;
1HNMR,
d
(400 MHz, DMSO-d6) 1.30 (s, 9H),
1.66 (s, 3H), 1.68 (s, 3H), 6.54 (s, 1H), 7.92 (s, br), 8.11 (s,
br); 13CNMR,
(100 MHz, DMSO-d6) 22.7, 22.9, 24.7, 31.6,
2.4.10. Compound 9d: 5,8-Dihydroxy-2,2-dimethyl-7-
methoxy-1,3,9-trioxa-fluoren-4-one
d
84.10, 103.27, 109.11, 111.77, 123.97, 141.45, 142.78,
161.05, 169.35. Anal. calcd. for C15H16O6; C, 62.74; H, 5.92;
found C, 61.89, H, 5.58; ESI–MS: 307.10 (M + 1).
Mp > 300 8C; IR (KBr) 3424, 3440,1710, 1620, 1617,
1224 cmÀ1
;
1HNMR,
d
(400 MHz, DMSO-d6) 1.73 (s, 3H),
1.75 (s, 3H), 3.80 (s, 3H), 6.79 (s, 1H), 7.92 (s, br), 7.99
(s, br); 13CNMR,
(100 MHz, DMSO-d6) 24.74, 25.50,
d
2.4.5. Compound 4c: 5-(4, 5-Dihydroxy-2-methyl-phenyl)-2,
2-dimethyl-[1,3]-dioxane-4,6-dione
63.00, 88.13, 104.17, 108.34, 114.78, 125.71, 142.89,
146.24, 165.05, 172.95. Anal. calcd. for C13H12O7; C,
55.72; H, 4.32; found C, 56.02, H, 4.18; ESI–MS: 280.99
(M + 1).
Mp 245–247 8C; IR (KBr) 3430, 3001,1763, 1724,1698,
1610,1526, 1510 cmÀ1
; d(400 MHz, DMSO-d6)
1HNMR,
1.70 (s, 3H), 1.78 (s, 3H), 2.10 (s, 3H), 4.41 (s, 1H), 6.55
(s, 1H), 6.80 (s,1H), 7.87 (s, br), 8.00 (s, br); 13CNMR,
2.4.11. Compound 9f: 7-(tert-butyl)-5,8-dihydroxy-2,2-
dimethyl-1,3,9-trioxa-fluoren-4-one
d(100 MHz, DMSO-d6) 18.01, 23.9, 24.3, 53.40, 106.11,
115.35, 119.70, 125.17, 127.28, 144.14, 144.10, 168.01.
Anal. calcd. for C13H14O6; C, 58.64; H, 5.30; found C, 58.66,
H, 5.38; ESI–MS: 267.01 (M + 1).
Mp > 300 8C; IR (KBr) 3423, 3422,1706, 1615,
1618 cmÀ1
;
1HNMR,
d
(400 MHz, DMSO-d6) 1.28 (s, 9H),
1.64 (s, 3H), 1.66 (s, 3H), 6.50 (s, 1H), 8.02 (s, br), 8.16 (s,
br); 13CNMR,
(100 MHz, DMSO-d6) 21.90, 22.84, 23.17,
d
2.4.6. Compound 4e: 5-(4, 5-Dihydroxy-2-methoxy-phenyl)-
2, 2-dimethyl-[1,3]-dioxane-4,6-dione:
31.65, 85.18, 102.17, 108.00, 110.79, 124.40, 143.25,
144.78, 163.25, 170.16. Anal. calcd. for C15H16O6; C,
62.74; H, 5.92; found C, 62.80, H, 5.98; ESI–MS: 307.28
(M + 1).
Mp 240–242 8C; IR (KBr) 3433, 3010,1766, 1729,1700,
1619,1530, 1519, 1220 cmÀ1;1H NMR,
d(400 MHz, DMSO-d6)

M. Kidwai et al. / C. R. Chimie 16 (2013) 728–735
731
Scheme 1. Synthesis of 6,7-dihydroxy-2,2-dimethyl-1,3,9-trioxa-fluoren-4-one using catechol and Meldrum’s acid at room temperature, using laccase as a
catalyst in phosphate/citrate buffer at pH 7.
HO
2.4.12. Compound 8c: 5-(3,6-Dihydroxy-2,4-dimethyl-
phenyl)-2,2-dimethyl-[1,3]-dioxane-4,6-dione
OH
O
Mp > 300 8C; IR (KBr) 3427, 3006, 1766, 1728,1690,
O
1609,1528 cmÀ1
;
1HNMR,
d
(400 MHz, DMSO-d6) 1.69 (s,
3H), 1.76 (s, 3H), 2.09 (s, 3H), 2.15 (s, 3H), 4.38 (s, 1H), 6.77
(s,1H), 7.80 (s, br), 8.07 (s, br); 13CNMR,
(100 MHz, DMSO-
O
laccase
0.1M phosphate buffer
pH 7
+
O
OH
O
3
O
d
6
OH
d6) 17.91, 23.98, 24.97, 55.20, 59.24, 106.11, 115.35,
119.70, 125.17, 127.28, 140.14, 143.17, 167.00. Anal. calcd.
for C13H14O6; C, 59.99; H, 5.75; found C, 59.66, H, 5.78; ESI–
MS: 280.22 (M + 1).
O
O
9
Scheme 2. Synthesis of 5,8-dihydroxy-2,2-dimethyl-1,3,9-trioxa-
fluoren-4-one using hydroquinone and Meldrum’s acid, using laccase
as a catalyst in phosphate/citrate buffer at pH 7.
2.4.13. Compound 8e: 5-(3,6-Dihydroxy-2,2-dimethoxy-
phenyl)-2,2-dimethyl-[1,3]-dioxane-4,6-dione
In addition, the reaction temperature was shown to
have an effect on the reaction outcome. For example, if the
reaction was performed at room temperature or 60 8C, the
yield of the product was diminished by only 0–10%. This
result was attributed to an increase in the rate of
decomposition and polymerization of the in situ generated
o-quinone and of in situ decomposition of Meldrum’s acid–
Mp > 300 8C; IR (KBr) 3430, 3009,1772, 1730,1704,
1610,1533 cmÀ1
;
1HNMR,
d
(400 MHz, DMSO-d6) 1.76 (s,
3H), 1.78 (s, 3H), 3.80 (s, 3H), 4.40 (s, 1H), 6.87 (s,1H), 7.89
(s, br), 8.10 (s, br); 13CNMR,
(100 MHz, DMSO-d6) 24.1,
d
24.8, 28.11, 53.55, 107.19, 116.30, 120.61, 124.19, 127.29,
145.19, 147.11, 169.34. Anal. calcd. for C13H14O7; C, 55.32;
H, 5.00; found C, 55.39, H, 4.98; ESI–MS: 283.02 (M + 1).
catechol derivative to benzofuran as
a side-product
because of the prone tendency of Meldrum’s acid to
decompose into CO2 and acetone. Therefore, we retarded
the rate of decomposition and polymerization by main-
taining the initial temperature to 5 8C for first 2 h and then,
allowing the reaction mixture to come up to room
temperature.
The enzymes have been immobilized using several
methodologies for their biocatalytic applicability affecting
their activity, effectiveness of utilization, deactivation,
regeneration kinetics and cost [36], without considering
the toxicity of the immobilized reagents and the waste
disposal problems. Hence, in this study, the laccase-
catalysed synthesis was carried out in aqueous medium for
three consecutive runs and monitored by TLC. Table 1
depicts the yield of the product from the reaction catalysed
by laccase in three consecutive runs. The yield was
observed to decrease slightly after each run.
3. Results and discussion
To explore the catalytic activity of laccase for the
coupling of catechol and Meldrum’s acid, a trial reaction
was performed using 1.0 mM catechol in aqueous solution
containing 0.1 M acetate buffer, (pH = 5.0), 2.0 mM Mel-
drum’s acid and 400 U of laccase. 6,7-Dihydroxy-2,2-
dimethyl-1,3,9-trioxa-fluoren-4-one was obtained in 45%
yield when the reaction was stirred for 10 h. The same
reaction was performed in the absence of laccase; no yield
of product was formed, as catechol is unable to be
converted into o-quinone in the absence of laccase. An
excess of Meldrum’s acid was required to overcome the
intrinsic instability of the o-benzoquinones as it will
undergo competing decomposition, dimerization and
polymerization if insufficient amounts of dione are present
for the Michael addition. The low pKa value of Meldrum’s
acid [34,35] allowed the use of neutral conditions (acetate
buffer pH 7) for the enzymatic study of catechols in the
presence of Meldrum’s acid. Hence, the same reaction was
tried at pH 7. This resulted in an increase in the yield
of the coupling product (55%). To further explore the
catalyst divergence, the same reaction was carried out
with 1,4-benzoquinone in the place of catechol. It was
found that 1,4-benzoquinone is as reactive as catechol and
gives the desired product in quantitative yield (Schemes 1
and 2).
Table 1
Catalyst recycling studiesa.
Catalyst recycle
Time (h)
Yieldb (%)
1
2
3
12
12
12
55
52
49
a
Reaction conditions: 1 mmol of 1 (Meldrum’s acid) and 2 mmol of 3
(catechol) were taken in 15 mL of buffer solution and 400 U of laccase and
stirred well for 12 h.
b
Isolated yield.

732
M. Kidwai et al. / C. R. Chimie 16 (2013) 728–735
Table 2
Solvent optimization for 1,3,9-trioxa-fluoren-4-one derivativesa.
S. No.
Solvent
Yieldb (%)
1
1 M acetate buffer pH 4
23
28
31
34
45
55
–
2
Water
3
THF
4
Ethanol
5
1 M phosphate/citrate buffer pH 4.5
6
1 M phosphate/citrate buffer pH 7
7
DMSO
8
MeCN
–
9
DMF
–
10
11
Methanol
1,4-Dioxane
30
18
a
Reaction conditions: 1 mmol 1 (Meldrum’s acid) and 2 mmol of 3
(catechol) were taken in 15 mL of solvent and 400 U of laccase
(P. cinnabarinus) were added and the resultant solution was stirred well
for 12 h.
b
Isolated yield.
Fig. 1. Various mediators used.
Table 2 shows the effect of different solvent systems on
the product yield. In DMSO, DMF, and MeCN, no reaction
was observed due to the denaturation of laccase. The
reaction in aqueous buffer proceeded in higher yield using
buffer tablets of NaH2PO4, Na2HPO4, and potassium
phthalate (pH 7). The lower percentage yields in other
solvent systems were due to a decrease in laccase activity
in non-intermiscible organic and aqueous phases. More-
over, the domino reaction was shown to exhibit higher
reactivity and selectivity in aqueous medium rather than
in organic solvents.
3.2. Effect of redox mediator on product formation
When laccase was used as the sole catalyst, the yield of
the product was very less. Hence, synthetic redox
mediators, 1-hydroxybenzotriazole (HBT), N-hydro-
xyphthalimide (HPI) and 2,2-azinobis (3-ethylbenzthiazo-
line-6-sulfonate) (ABTS) were employed along with
laccase as laccase mediator systems (LMSs) for improvisa-
tion of the product (Fig. 1).
The mediator needs to be easily oxidised by laccase to
the Medox status (Scheme 3).
1-Hydroxybenzotriazole [37] (HBT) and N-hydro-
xyphthalimide [38] (HPI) are two N–OH compounds that
are known to mediate the activity of laccase, driving it
towards non-phenolic substrates, and the relative effi-
ciency of these mediators has been recently evaluated [39].
A consensus has been reached upon the preliminary mono-
electronic oxidation of these N–OH mediators by laccase to
the corresponding N-oxyl radical (N–Oꢀ) forms [40]
through the fleeting intervention of their radical cations,
HPTꢀ+ or HPIꢀ+, respectively. Two different hypotheses
have been proposed to explain the subsequent oxidation of
the substrate. Either these N–Oꢀ radicals perform a one-
electron oxidation (ET) of the substrate to a radical cation
[41], or they abstract the H-atom from the substrate (HAT),
converting it into a radical [42]; this supports the ET route
3.1. Effect of enzyme concentration
The effect of enzyme concentration on the reaction was
studied to determine the minimum amount of enzyme
required for a maximum formation of desired product. It is
essential to optimize the concentration of the enzyme in
the reaction mixture because it greatly influences the
reaction rate. In the present investigation, it was observed
that the rate of laccase-catalysed product formation
increased with increasing enzyme concentration and
decreased after reaching optimal enzyme concentration.
A similar trend was observed for all the derivatives. About
400 U of laccase is used as a standard concentration for all
the reactions (Table 3).
with laccase/ABTS through the likely formation of ABTS++
.
All the mediators used enhanced the rate of reaction
appreciably but not enough to make the reaction economic-
ally viable; HBT was used as a mediator. (Table 4).
Table 3
Synthesis of 1,3,9-trioxa-fluoren-4-one derivatives using catechol and
Meldrum’s acid, with different concentration of laccase (P. cinnabarinus)
in phosphate/citrate buffer at pH 7a
After developing the optimum reaction conditions, the
reaction of
a variety of catechol and hydroquinone
S. No.
Concentration of enzyme (U)
Yieldb (%)
1
2
3
4
100
200
400
500
21
30
55
57
a
Reaction conditions: 1 mmol 1 (Meldrum’s acid) and 2 mmol of 3
(catechol) were taken in 15 mL of buffer solution. Different units of
laccase were added and the resultant solution was stirred well for 12 h.
Scheme 3. General mechanism for the reaction between different
b
Isolated yield.
catechols and Meldrum’s acid in the presence of laccase.

M. Kidwai et al. / C. R. Chimie 16 (2013) 728–735
733
Table 4
Besides 3-substituted catechols, 4-substituted cate-
chols, such as 4-methyl/4-chlorocatechol or 3,4-dihydro-
xybenzoic acid were also used for the reaction, but the
presence of substituted groups in the C-4 position of the
catechol ring, which is a reactive site of o-benzoquinones,
causes a decrease in the rate of reaction and stops the
reaction at a particular stage and does not lead to the final
desired product. It was found that the reaction stopped
with the formation of product 4 in which the catechol ring
is already occupied with a substituent and does not permit
the formation of the desired cyclised product. A general
reaction mechanism is demonstrated in Scheme 4 and
shows the formation of products 4 and 5 under different
circumstances.
Synthesis of 1,3,9-trioxa-fluoren-4-one derivatives using different
catechol and Meldrum’s acid in laccase (P. cinnabarinus) using different
mediators in phosphate/citrate buffer at pH 7a.
S. No.
Different mediators used
Yieldb (%)
1
2
3
HPI
59
65
65
ABTS
HBT
a
Reaction conditions: 1 mmol 1 (Meldrum’s acid) and 2 mmol of 3
(catechol) were taken in 15 mL of buffer solution and 100 U of laccase
(P. cinnabarinus) with different mediators was added and the resultant
solution was stirred well for 12 h.
b
Isolated yield.
derivatives were examined and these results are explained
comprehensively (Tables 5 and 6).
It was found that when 3-substituted catechols were
used with Meldrum’s acid, then approximately 65% of
desired product (5) was separated out. Side-products may
contain partially reacted product (4), but in too small
amounts to be separated. When 4-substituted catechols
were used with Meldrum’s acid, then, there is no scope for
the formation of the desired product (5). The only
separated product was (4).
The rate of reaction and the nature of the products are
dependent on the nature and position of the substituted
group on the catechol ring. The presence of bulky groups,
such as tert-butyl, on the catechol ring causes a decrease in
rate of the reaction. Furthermore, 3-fluoro-1,2-benzoqui-
none, which has a strong electron withdrawing group (F),
did not provide any desired product. This reactivity pattern
may be caused by side reactions of highly reactive quinone.
1H-NMR of catechol gave a multiplet for aromatic
hydrogens in the 6.82–6.94 ppm region; Meldrum’s acid
Table 5
Synthesis of 1,3,9-trioxa-fluoren-4-one derivatives using different
catechol and Meldrum’s acid employing 400 U of laccase
(P. cinnabarinus) in phosphate/citrate buffer at pH 7a.
S. No.
Product
R1
R2
Yield (%)b
Time(h)
1
2
3
4
5
6
7
8
9
5a
5b
4c
5d
4e
5f
H
H
65
61
35
51
40
39
39
–
12
12
12
12
12
12
12
12
12
–CH3
H
H
–CH3
–OCH3
H
H
–OCH3
–C(CH3)3
H
4g
5h
4i
H
H
F
–C(CH3)3
F
H
–
a
Reaction conditions: 1 mmol 1(a–i) and 2 mmol of 3 (Meldrum’s acid)
were taken in 15 mL of solvent and 400 U of laccase and 0.01 mmol of HBT
were added and the resultant solution was stirred well for 12 h.
b
Isolated yield.
Table 6
Synthesis of 1,3,9-trioxa-fluoren-4-one derivatives using different
hydroquinones and Meldrum’s acid employing 400 U of laccase
(P. cinnabarinus) phosphate/citrate buffer of pH 7a.
S. No.
Product
R1
R2
Yield (%)b
Time (h)
1
2
3
4
5
6
7
8
9a
9b
8c
9d
8e
9f
H
H
68
61
40
59
41
43
–
12
12
12
12
12
12
12
12
–CH3
–CH3
–OCH3
–OCH3
–C(CH3)3
–C(CH3)3
F
H
–CH3
H
–OCH3
H
8g
9h
–C(CH3)3
H
–
a
Reaction conditions: 1 mmol 6(a–h) and 2 mmol of 3 (Meldrum’s
acid) were taken in 15 mL of solvent and 400 U of laccase and 0.01 mmol
of HBT were added and the resultant solution was stirred well for 12 h.
Scheme 4. General mechanism for the reaction between different
b
Isolated yield.
catechol and Meldrum’s acid in the presence of laccase.

734
M. Kidwai et al. / C. R. Chimie 16 (2013) 728–735
gave a singlet in the 3.10–2.30 ppm region for methylene
protons and two singlets at around 1.59 and 1.61 for
methyl group protons, respectively. On the other hand, our
product (5) displays no multiplet in the aromatic region,
which confirms that two of the aromatic protons are
removed during the coupling with Meldrum’s acid. The
peak for the methylene protons of Meldrum’s acid has also
disappeared. Moreover, the ppm values for the methyl
proton of Meldrum’s acid are changed as it is coupled with
the catechol ring. In the case of product (4), it was observed
that the peak at approximately 4.4 is due to the methine
proton left on Meldrum’s acid, as it does not participate in
the reaction due to the occupancy of the C-4 position of
catechol.
When substituted hydroquinone was used in place of
substituted catechol, the same pattern was observed. But
when 2,6-disubstituted hydroquinone was used, then a
decrease in the rate of reaction was observed and the
reaction stops at a particular stage, and does not lead to the
desired final product (Scheme 5).
Scheme 5. General mechanism for the reaction between different hydroquinones and Meldrum’s acid in the presence of laccase.

M. Kidwai et al. / C. R. Chimie 16 (2013) 728–735
735
[12] S. Hajdok, J. Conrad, H. Leutbecher, S. Strobel, T. Schleid, U. Beifuss, J.
Org. Chem. 74 (2009) 7230.
4. Conclusion
[13] S. Witayakran, A. Zettilib, A.J. Ragauskas, Tetrahedron Lett. 48 (2007)
2983.
[14] S. Witayakran, L. Gelbaum, A.J. Ragauskas, Tetrahedron 63 (2007)
10958.
[15] M. Hosny, J. Rosazza, J. Agric. Food Chem. 50 (2002) 5539.
[16] M. Kurisawa, J.E. Chung, H. Uyama, S. Kobayashi, Biomacromolecules 4
(2003) 1394.
[17] H. Leutbecher, J. Conrad, I. Klaiber, U. Beifuss, Synlett (2005) 3126.
[18] S. Ciecholewski, E. Hammer, K. Manda, G. Bose, V.T.H. Nguyen, P.
Langer, F. Schauer, Tetrahedron 61 (2005) 4615.
[19] K. Manda, E. Hammer, A. Mikolasch, T. Niedermeyer, J. Dec, A.D. Jones,
A.J. Benesi, F. Schauer, J.-M. Bollag, J Mol. Catal. B: Enzym. 35 (2005) 86.
[20] T.H. Niedermeyer, A. Mikolasch, M. Lalk, J. Org. Chem. 70 (2005) 2002.
[21] A. Mikolasch, E. Hammer, U. Jonas, K. Popowski, A. Stielow, F. Schauer,
Tetrahedron 58 (2002) 7589.
The laccase-catalysed reaction for the coupling of
catechols/hydroquinones and Meldrum’s acid has been
scrutinized with a view to the synthesis of new series of
compounds. This investigation is tunable for catalyst and
solvent to facilitate the reaction and the reduction
of waste, with the use of more benign solvents. Hence,
in addition to the simple reaction conditions, the easy
work-up procedure makes our methodology a valid
contribution to the existing processes in the field of
enzymatic catalysis.
[22] S. Riva, Trends Biotechnol. 24 (2006) 219.
[23] A. Schfer, M. Specht, A. Hetzheim, W. Francke, F. Schauer, Tetrahedron
57 (2001) 7693.
Acknowledgements
[24] A.N. Kost, I.N. Khaimov, Y. Dzhuravakulov, K. Kh Khaidurov, L.D. Lebe-
deva, A.C. Kotov, Khim. Heterostsiki. Soedin. 11 (1975) 1482.
[25] D. Nematollahi, M. Alimordi, S.W. Husain, Electrochim. Acta 51 (2006)
2620.
[26] A. Pauli, Third world congress on allelopathy, Tsukuba, 2002, 26–30.
[27] D. Nematollahi, H. Ghayani-Jam, J. Org. Chem. 7 (2008) 3428.
[28] M. Kidwai, A. Jain, A. Sharma, R.C. Kuhad, Water Sci. Technol. 66 (2012)
385.
The authors (M. Kidwai and A. Jain) are thankful to UGC
New Delhi for providing financial assistance. The authors
(A. Sharma and R.C. Kuhad) acknowledge the financial
support from DBT.
References
[29] M. Kidwai, A. Jain, A. Sharma, R.C. Kuhad, J. Mol.Catal. B: Enzym. 74
(2012) 236.
[30] M. Kidwai, A. Jain, S. Bhardwaj, Catal. Lett. 141 (2011) 183.
[31] R.A. Sheldon, J. Mol. Catal. A 107 (1996) 75.
[1] C.F. Thurston, Microbiology 140 (1994) 19.
[2] A.I.O. Yaropolov, V.S.S. Skorobogat’ko, S.D. Vartanov, Appl. Biochem.
Biotechnol. 49 (1994) 257.
[3] S.G. Burton, Curr. Org. Chem. 7 (2003) 1317.
[4] H. Claus, Micron 35 (2004) 93.
[5] S. Nicotra, A. Intra, G. Ottolina, S. Riva, B. Danieli, Tetrahedron: Asym-
metry 15 (2004) 2927.
[32] S. Dhawan, R.C. Kuhad, Bioresource Tecnol. 84 (1) (2002) 35.
[33] K. Vasdev, R.C. Kuhad, Microbiol. 39 (1994) 61.
[34] E.M. Arnett, S.G. Maroldo, S.L. Schilling, J.A. Harrelson, Am. Chem. Soc.
106 (1984) 6759.
[6] S. Nicotra, M.R. Cram arossa, A. Mucci, U.M. Pagnoni, S. Riva, L. Forti,
Tetrahedron 60 (2004) 595.
[7] X. Zhang, G. Eigendorf, D.W. Stebbing, S.D. Mansfield, J.N. Saddler, Arch.
Biochem. Biophys. 405 (2002) 44.
[35] E.M. Arnett, J.A. Harrelson, J. Am. Chem. Soc. 109 (1987) 809.
[36] N. Fadnavis, W.A. Deshpande, Curr. Org. Chem. 6 (2002) 393.
[37] R. Bourbonnais, M.G. Paice, B. Freiermuth, E. Bodie, S. Borneman, Appl.
Environ. Microb. 63 (1997) 4627.
[8] F. d’Acunzo, C. Galli, P. Gentili, F. Sergi, New J. Chem. 30 (2006) 583.
[9] A. Leonowicz, N.S. Cho, J. Luterek, A. Wilkolazka, M. Wojtas- Wasi-
lewska, A. Matuszewska, M. Hofrichter, D. Wesenberg, J. Basic Micro-
biol. 41 (2001) 185.
[10] C. Galli, P. Gentili, J. Phys. Org. Chem. 17 (2004) 973.
[11] S. Hajdok, J.U. Conrad, J. Org. Chem. 77 (2012) 445.
[38] E. Fritz-Langhals, B. Kunath, Tetrahedron Lett. 39 (1998) 5955.
[39] M. Fabbrini, C. Galli, P. Gentili, J. Mol. Catal. B: Enzym. 16 (2002) 231.
[40] C. Crestini, D.S. Argyropoulos, Bioorg. Med. Chem. 6 (1998) 2161.
[41] F. Xu, H.-J.W. Deussen, B. Lopez, L. Lam, K. Li, Eur. J. Biochem. 268 (2001)
4169.
[42] K. Li,R.F. Helm,K.-E.L. Eriksson, Biotechnol.Appl. Biochem. 27(1998)239.