Biotransformation of chalcones by the endophytic fungus aspergillus flavus isolated from paspalum maritimum trin

Article abstract of DOI:10.1590/S0103-50532011000700019

The fungus Aspergillus flavus isolated as endophytic of the plant Paspalum maritimum Trin. was evaluated for its potential application in biotransformation reactions. The compounds chalcone (1), 3,4,5-trimethoxychalcone (2) and 2,3,4,4′-tetramethoxychalco

Full text of DOI:10.1590/S0103-50532011000700019

J. Braz. Chem. Soc., Vol. 22, No. 7, 1333-1338, 2011.
Printed in Brazil - ©2011 Sociedade Brasileira de Química
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Article
A
Biotransformation of Chalcones by the Endophytic Fungus Aspergillus flavus
Isolated from Paspalum maritimum Trin.
Marivaldo J. C. Corrêa,a Fátima M. Nunes,a Heriberto R. Bitencourt,a Fábio C. Borges,a
Giselle M. S. P. Guilhon,a Mara S. P. Arruda,a Andrey M. R. Marinho,a Alberdan S. Santos,a
Cláudio N. Alves,a Davi S. B. Brasil b and Lourivaldo S. Santos*,a
aPrograma de Pós-Graduação em Química, Instituto de Ciências Exatas e Naturais and
bFaculdade de Engenharia Química, Instituto de Tecnologia, Universidade Federal do Pará,
Campus Universitário do Guamá, 66075-900 Belém-PA, Brazil
O fungo Aspergillus flavus isolado como endofítico da planta Paspalum maritimum
Trin. foi avaliado quanto ao seu potencial de aplicação em reações de biotransformações. Os
compostos chalcona (1), 3,4,5-trimetoxichalcona (2) e 2,3,4,4’-tetrametoxichalcona (3) foram
biotransformados, respectivamente, na diidrochalcona (4), 3,4,5-trimetoxidiidrochalcona (5) e
2,3,4,4’-tetrametoxidiidrochalcona (6). As estruturas destes compostos foram determinadas por
análises de RMN uni e bidimensionais e por espectrometria de massas. As diidrochalconas 5 e 6
são substâncias inéditas.
The fungus Aspergillus flavus isolated as endophytic of the plant Paspalum maritimum
Trin. was evaluated for its potential application in biotransformation reactions. The compounds
chalcone (1), 3,4,5-trimethoxychalcone (2) and 2,3,4,4’-tetramethoxychalcone (3) were
biotransformed, respectively, in dihydrochalcone (4), 3,4,5-trimethoxydihydrochalcone (5) and
2,3,4,4’-tetramethoxydihydrochalcone (6). The structures were elucidated by spectroscopic
methods including 1D and 2D NMR techniques, and MS analysis. The dihydrochalcones 5
and 6 are new compounds.
Keywords: biotransformations, Aspergillus flavus, Paspalum maritimum, chalcones,
dihydrochalcones
Introduction
Chalcones are precursors of the flavonoids and are
defined as α,ß-unsaturated ketones in which both the
carbonyl and the olefinic fragment are linked to an
aromatic ring. They possess a large spectrum of action,
such as antiviral activity,14 inhibition of NS3 protease of
dengue virus,15 activity against herpes simplex virus,16
HIV-1 replication inhibition in lymphocytes-H9 in mice,17
allelopathic activity,18 anti-inflammatory and antibacterial
properties.19 These activities have been raising the interest
in synthesizing and characterizing different chalcones,
in order to obtain the corresponding dihydrochalcones to
correlate structure versus biological activity.
Biotransformation of bioactive natural and synthetic
compounds by many microorganisms including species
of Aspergillus have been examined and investigated.20,21
Regioselective O-demethylation of flavones into their
4’-O-demethylated metabolites was performed using an
A. nigerstrain.22 Biotransformationofflavanonesbyonewild
Chemical reactions performed by microorganisms have
been used as an alternative to obtain products of chemical
and biological interest. Taking into account the modern
concepts and requirements for a safer and environmentally
friendly chemistry, the use of reactions promoted by
microorganisms tends to increase in the near future.1-3
Such reactions, usually known as biotransformation,
have certain advantages over the conventional reactions.
Biotransformation is usually performed in aqueous
systems and at neutral pH, preventing the common use of
dangerous solvents in conventional synthesis.4-9 In addition,
microorganisms are capable of performing a wide variety of
reactions, including some almost inaccessible by chemical
means.10-13
*e-mail: lss@ufpa.br

1334
Biotransformation of Chalcones by the Endophytic Fungus Aspergillus flavus
J. Braz. Chem. Soc.
strainofA.nigerKBandthreeUVmutantsofA.nigerresulted
in the reduction of carbonyl group and dehydrogenation at
C-2 and C-3 positions; reduction of carbonyl group and
hydroxylation at C-7; and dehydrogenation at C-2 and C-3
and hydroxylation at C-3 producing flavonol.23 In the high
molecularweightfractionofthecultureextractofAspergillus
oryzae, genistein was transformed into shoyuflavone B in the
presence of ( )-transepoxysuccinic acid.24 Epigallocatechin
gallate (EGCG) has been reported to be converted into
epigallocatechin (EGC) by a hydrolase from Aspergillus
oryzae and the yield of EGC could reach at least 70%.25
Biotransformation of the flavanone by means of Aspergillus
niger MB culture led to formation of three products:
6-hydroxyflavanone, 2’,5’-dihydroxydihydrochalcone and
2’-hydroxydihydrochalcone. Their yields were 18%, 6%
and 7.5% after 9 days, respectively.26
published in literature.30 The dihydrochalcones 5 and 6
are new compounds.
The bioreduction reaction of the chalcones was
regioselective, showing only alteration in carbon-
carbon double bond of the olefin, with no reaction at the
carbonyl double bond. Several reactions of chalcones are
reported, however, none of them showed regioselectivity
for the double bond C-2 and C-3. Among the reactions
of biotransformation reported directly on chalcones,
Aspergillus alliaceus cyclized the chalcone 2’-hydroxy-
2,3-dimethoxychalcone to three flavanones and to
O-demethylated (6.5% yield) and hydroxylated (7.2% yield)
chalcones.31 The unsubstituted chalcone was converted to
2’’-hydroxychalcone and 2’’,3’’-dihydroxychalcone in
25% and 59% yields, respectively, by Escherichia coli.32
Microbial transformation of chalcone xanthohumol, using
the culture broth of Pichia membranifaciens afforded three
metabolites, one isomeric prenylflavanone in 3.3% yield,
and two modified prenylchalcones in 0.55 and 0.58%
yield.33 The yields obtained in the biotransformation
reactions of chalcones to dihydrochalcones in this work
are higher (15.6-75.5% yield) than those reported in the
literature, although no reaction can be directly compared,
because none of them performed this type of bioreduction
(direct formation of dihydrochalcones from chalcone).
The biotransformation products (dihydrochalcones 4,
5 and 6) were characterized mainly by the presence of two
Due to the potential ability of Aspergillus to
promote biotransformation reactions, we decided to
study biotransformation reactions of chalcone (1),
3,4,5-trimethoxychalcone (2) and 2,3,4,4’-tetramethoxy-
chalcone (3), using the endophytic fungus Aspergillus
flavus isolated from the plant Paspalum maritimum.
Results and Discussion
The chalcones 1 (68.5% yield),27 2 (75.0% yield)28 and
3 (60.0% yield)29 were synthesized using methodology
described in the literature18 and their structures were
determined by the analysis of 1D- and 2D-NMR, mass
spectral information and comparison with reported
physical and spectroscopic data. These chalcones
were bioreduced by Aspergillus flavus producing the
dihydrochalcones 4 (74.5% yield), 5 (36.3% yield) and
6 (15.6% yield), respectively, after 10 days of reaction
(Figure 1). The known dihydrochalcone (4) was identified
by comparison of the spectroscopic data with those
1
triplets in the H NMR spectra that indicate the reduction
of the double bond of the conjugated olefinic system. In the
13C NMR spectrum, the chemical shift of the carbonyl carbon
increased about 10 ppm due to the loss of conjugation.
Compound 5 was identified as 3,4,5-trimethoxy-
dihydrochalcone. The analysis of the LR-MS spectrum
showed that compound 5 has a molecular ion peak at m/z
300 (64%) and fragments at m/z 195 (100%), 181 (36%),
165 (42%) and 77 (36%), suggesting a molecular formula
Figure 1. Bioreduction of the chalcones 1, 2 and 3 by Aspergillus flavus.

Vol. 22, No. 7, 2011
Corrêa et al.
1335
C18H20O4 (9 degrees of insaturation). This molecular
formula was confirmed on the basis of HRESIMS [m/z
323.1270 (M+Na)+; D –1.1 mmu] analysis and NMR data
The HETCOR spectrum showed the correlations of signals
at dC 128.6 (C2’/C6’) 128.0 (C3’/C5’) and 133.1 (C4’)
with the signals at dH 7.96 (H2’/H6’), 7.45 (H3’/H5’) and
7.56 (H4’), respectively. The HMBC spectrum showed
correlation of the signal at dH 7.45 (H3’/H5’) with signals
at dC 128.6 (C2’/C6’) and dC 136.9 (C1’) and of the signal
at dH 7.96 (H2’/H6’) with the signals at dC 128.0 (C3’/C5’),
133.1 (C4’) and 199.2 (C=O).
1
(Table 1). The H NMR spectrum of 5 (Table 1) showed
signals due to the benzylic methylene protons Hβ at dH
3.01 (2H, t, J 7.5 Hz) and to the α-carbonyl protons Hα
at dH 3.29 (2H, t, J 7.5 Hz). The coupling of the protons
Hα and Hβ was confirmed by analysis of 1H × 1H-COSY
spectrum. HETCOR spectrum (1JCH) showed correlations
of the signals of the protons at dH 3.29 (Hα) and 3.01 (Hβ)
with signals at dC 40.6 (Cα) and 30.6 (Cβ), respectively.
The triplet at dH 3.01, assigned to the signal of hydrogen
Hß, showed a correlation in the HMBC spectrum with
the signals at dC 137.1 (C1) and at dC 105.4 (C2/C6). The
singlet at dH 6.46 (2H, s, ArH) assigned to the equivalent
protons H2/H6 of ring B showed correlation with the
signal at dC 105.4 (C2/C6) in the HETCOR spectrum.
The HMBC spectrum showed the correlation of the signal
at dH 6.46 (H2/H6) with the signals at dC 153.2 (C3/C5),
136.9 (C4), 105.4 (C2/C6) and 30.6 (Cβ). The assignment
of the protons and carbons signals of ring A was proposed
from the analysis of 1H × 1H-COSY and HETCOR spectra.
Compound 6 was identified as 2,3,4,4’-tetramethoxy-
dihdrochalcone. The analysis of LR-MS spectrum of 6
showed a molecular ion peak at m/z 330 (82%) and the peaks
at m/z 195 (24%), 181 (98%), 166 (48%), 135 (100%) and
107 (16%), which are consistent with the molecular formula
C19H22O5 (9 degrees of insaturation). This molecular formula
was confirmed on the basis of HRESIMS [m/z 353.1358
(M+Na)+; D +0.7 mmu] analysis and NMR data (Table 1).
1
The H NMR spectrum of 6 showed signals attributed to
the benzylic methylene protons Hß and Hα similar to those
of compound 5. Two ortho-related doublets in the aromatic
region (1H each, J 8.4 Hz) at dH 6.87 and 6.59, were assigned
to H6 and H5, respectively. The signal at dH 6.59 correlated
with the signals of carbons at dC 127.2 and 142.3 in the
Table 1. 1H and 13C NMR chemical shifts (dH and dC in ppm) of compounds 5 and 6 in CDCl3a,b
Position
5
6
dH
dC
HMBCc
dH
dC
HMBC
5, 6, α
OMe-2
5
1
137.1d
105.4
153.2
136.9d
153.2
105.4
40.6
127.2
151.9
142.3
152.3
107.2
123.9
39.5
2
6.46 (s)
6, ß
2,OMe-3
2,6, OMe-4
6, OMe-5
2, ß
3
4
6, OMe-4
5
6.59 (d, 1H, J 8.4)
6.87 (d, 1H, J 8.4)
3.18(t, 2H, J 8.4)
2.96 (t, 2H, J 8.4)
6
6.46
3
β
α
3.29 (t, 2H, J 7.5)
3.01 (t, 2H, J 7.5)
ß
β
30.6
2, 6, α
2’, 6’
25.3
6, α
C=O
1’
199.2
136.9
128.6
128.0
133.1
128.0
128.6
198.4
129.9
130.6
113.6
163.3
113.6
130.6
60.8
α, β, 2’, 6’
3’, 5’
2’
7.96 (dd, 2H, J 7.5 and 1.5)
7.45 (t, 2H, J 7.5)
6’
7.95 (d, 2H, J 9.3)
6.92 (d, 2H, J 9.3)
3’, 6’
2’, 6’, OMe-4’
2’, 5’
3’
4’
7.56 (tt, 1H, J 13.2, 7.5 and 1.5)
7.45 (t, 2H, J 7.5)
2’, 6’
2’
5’
6.92 (d, 2H, J 9.3)
7.95 (d, 2H, J 9.3)
3.90 (s)
6’
7.96 (dd, 2H, J 1.5 and 7.5)
OMe-2
OMe-3
OMe-4
OMe-5
OMe-4’
3.84 (s)
3.82 (s)
3.84 (s)
56.1
60.8
56.1
3.88 (s)
60.6
3.83 (s)
55.3
3.85 (s)
55.9
a 1H NMR data were recorded at 300 MHz. b 13C NMR data were recorded at 75 MHz. c 1H-13C HMBC correlations are from the carbon(s) specified to
the protons indicated. dValues can be reversed.

1336
Biotransformation of Chalcones by the Endophytic Fungus Aspergillus flavus
J. Braz. Chem. Soc.
HMBC spectra, what allowed the assignment of these signals
toC1andC3, respectively. InthesameNMRspectrum, itwas
observed that the signal at dH 6.87 (H6) showed correlation
with the signals at dC 151.9 and 152.3, which indicated
that these signals can be attributed to C2 and C4 carbons,
respectively. The analysis of the HETCOR (1JCH) spectrum
of 6 confirms the assignments of the signals of the protons
H5 and H6, since they showed correlations with the signals
of carbons at dC 107.2 (C5) and 123.9 (C6). The 1H NMR
spectrum showed two ortho-related doublets at dH 7.95 and
6.92 (2H each, J 8.4 Hz), due to the protons of a system
AA’-XX’, assigned to H2’/H6’ and H3’/H5’, respectively.
This coupling was confirmed by analysis of spectrum
Oriental in Belém, Pará State, Brazil. A voucher specimen
(IAN 183337) was deposited at the herbarium of this
institution.
Microorganisms and culture
The general procedures adopted for isolation of the
microorganism followed the methodology described by
Araújo et al.34 Epiphytic hyphae and spores were eliminated
by immersion of the plant material in 70% ethanol (1 min),
followed by 5% aqueous sodium hypochlorite (4 min) and
finally with 70% aqueous ethanol (30 s), before being rinsed
in sterile distilled water. The material was then deposited
on a Petri dish containing Sabouraud (SDA), composition
meat peptone 10 g L-1 (HIMEDIA), D-glucose anhydrous
20.0 g L-1 (NUCLEAR), Agar 20.0 g L-1 (VETEC) and
chloramphenicol 1.0 g L-1 and incubated in the dark at
30 oC. Samples of the fast growing fungi were subcultured
onto other plates with the culture medium used. The
microorganisms were conserved as suspension in distilled
water. The aflpm3D1 strain was identified as Aspergillus
flavus by Dra. Maria Inês de Moura Sarquis, Fiocruz-RJ.
A voucher specimen has been deposited at “Laboratório
de Química de Microorganismos e Bioensaios-UFPA”.
1
1H × H-COSY. The analysis of HETCOR spectrum of 6,
relating to the ring A, was important to assign accurately
the signals at dC 130.3 and 113.6 to carbons C2’/C6’
and C3’/C5’, respectively, that showed 1JCH correlations with
signals at dH 7.95 and 6.92. The HMBC spectrum showed
correlations of the signals of carbons at dC 129.9 (C1’)
and 163.3 (C4’) with the signals at dH 6.92 (H3’/H5’) and
dH 7.95 (H2’/H6’).The assignment of carbonyl carbon signal
(dC 198.4) was based on the analysis of the correlations with
the signals due to Hβ and Hα.
Experimental
Synthesis of substrate:18 chalcones 1-3
General experimental procedures
In a flat-bottomed flask (125 mL), placed in an ice bath,
15 mL of EtOH, 11 mmol of acetophenone or derivatives,
15 mL of 10% NaOH solution and 12 mmol of benzaldehyde
or derivatives were added. The reaction mixture was kept
on magnetic stirring at 40 oC for 40 min. After this period,
the mixture was cooled and left in freezer for 48 h and then
filtered under vacuum. The product was crystallized in
methanol.Chalcone 1 was obtained in 68.5% yield (1.47 g),
chalcone2wasobtainedin75.0%yield(2.42g)andchalcone
3 was obtained in 60.0% yield (2.62 g).
Low-resolution mass spectra data were obtained on a
Thermo DSQII GC-MS instrument equipped with fused
silica capillary column (30 m × 0.25 mm id) with DB-5MS
stationary phase (0.25 mu thickness of the film); carrier
gas helium gas, temperature of the source and other parts
200 oC; type of injection: splitless; programmed temperature
to 100-300 oC, with a gradient of 5 oC min-1. The quadrupole
filter swept range 39-400 daltons every second. Ionization
was obtained by the technique of electron impact with
energy of 70 eV, and high resolution mass spectra were
recorded on an LC-IT-TOF mass spectrometer model
225-07100-34-SHIMADZU, by positive ionization mode of
the ESI. NMR spectra, including HMBC experiment, were
recorded on aVarian Mercury-300 spectrometer, operating
at 300 MHz for 1H and 75 MHz for 13C NMR, using solvent
signal (d-chloroform) as reference. The chemical shifts are
given in delta (d) values and the coupling constants (J) in
Hertz (Hz).
General procedure for biotransformation reactions
One Erlenmeyer flask (250 mL), containing 100 mL
SDA modified medium (meat peptone 10 g L-1 (HIMEDIA),
D-glucose anhydrous 10 g L-1 (NUCLEAR) and
chloramphenicol 1.0 g L-1) was previously autoclaved at
o
121 C for 15 min. Three small disc cut from Petri dishes
containing mycelium of A. flavus in SDA medium and the
appropriate chalcones (1-3), 0.05 g dissolved in 0.5 mL
DMSO, were added under steril conditions to the Erlenmeyer
Plant material
o
flask and shaken (160 rpm) at 30 C. After 10 days the
The leaves of Paspalum maritimum were collected in
January 2009 in the forest reserve of EMBRAPA-Amazônia
reaction mixture was extracted with EtOAc (3 × 100 mL),
and organic phases were grouped, dried (Na2SO4), filtered

Vol. 22, No. 7, 2011
Corrêa et al.
1337
and evaporated at reduced pressure. Experiments were
performed in triplicate and were reproducible according to
TLC analysis. Control flasks consisted of cultures containing
substrate in DMSO without fungus and cultures containing
fungus and DMSO without substrate. The purifications
were carried out from one experiment. The crude residue
(102.0 mg) obtained from the biotransformation reaction
of chalcone 1 was chromatographed on silica gel by elution
with EtOAc (300 mL) and MeOH (100 mL). Evaporation
of solvent furnished EtOAc extract (62.0 mg) and MeOH
extract (30.6 mg). TLC analysis indicated that the substances
of interest were in the extract EtOAc. This extract was
chromatographed sequentially on silica gel eluting with
hexanes and increasing polarity with EtOAc, to give eleven
fractions (A1-A11). Fractions A3-A5 (hexanes:EtOAc 9:1;
8:2 and 8:2, respectively) were grouped and recrystallized
with MeOH giving dihydrochalcone 4 (37.2 mg). The crude
residues obtained from the biotransformation reactions
of chalcones 2 and 3 after the same treatment, provided
EtOAc extract (113.0 mg) and MeOH extract (90.6 mg)
to the chalcone 2 reaction, and EtOAc extract (119.0 mg)
and MeOH extract (97.3 mg) to the chalcone 3 reaction.
The EtOAc extract obtained from the biotransformation
reactions of chalcone 2 was chromatographed sequentially
on silica gel eluting with hexanes and increasing polarity
with EtOAc, to give fourteen fractions (B1-B14). Fractions
B6-B10 (hexanes:EtOAc 8:2; 8:2; 7:3; 7:3 and 6:4,
respectively) were grouped and recrystallized with MeOH
giving dihydrochalcone 5 (18.3 mg). The EtOAc extract
obtained from the biotransformation reactions of chalcone 3
was chromatographed sequentially on silica gel eluting with
hexanesandincreasingpolaritywithEtOAc,togiveseventeen
fractions (C1-C17). Fractions C12 (hexane:EtOAc 6:4) and
C13(hexane:EtOAc6:4)weregroupedandrecrystallizedwith
MeOH giving dihydrochalcone 6 (7.8 mg).
Conclusions
Microbial transformation is an effective tool for
the structural modification of bioactive natural and
synthetic compounds, including chalcones. Microbial
factories pose advantages such as rapid growth, ease of
cultivation, convenient genetic manipulations, and high
level production on natural products biotransformation.
Moreover, microbial production increases product
selectivity and reduces the usage of toxic chemicals while
conserving energy usage. The use of microorganisms
in bioreduction reactions of chalcones has led to the
formation of several products in mixture, formed by
cyclization, reduction, O-demethylation, hydroxilation
reactions, among others. Generally, these reactions have
shown low yield and stereoselectivity. In this work we
performed the biotransformation reaction of chalcones
to dihydrochalcones in excellent yields, compared to
other biotransformation reactions of flavonoids. The
bioreduction reaction was regioselective, showing only
alteration in carbon-carbon double bond of the olefin, not
occurring any alteration in the carbonyl double bond of
the α,β-unsaturated ketone system. In addition, to the best
of our knowledge this is the first report of the obtention of
dihydrochalcones directly from biotransformation reaction
of chalcones.
Supplementary Information
Supplementary Information (Figure S1-S19) is
available free of charge at http://jbcs.org.br as PDF file.
Acknowledgments
The authors would like to thank CAPES, CNPq
and FAPESPA for financial support; Dra. Maria Inês
de Moura Sarquis (Fiocruz-RJ) by identification of the
fungus; Laboratory of Mass Spectrometry of Northeast
(LEMANOR) by acquisition of mass spectra (LCMS-IT-
TOF: quadrupole ion trap-time of flight mass spectrometer).
3,4,5-Trimethoxydihydrochalcone (5)
o
1
Crystalline solid; mp 59-61 C (MeOH); H NMR
(300 MHz, CDCl3): see Table 1; 13C NMR (75 MHz,
CDCl3): see Table 1. LR-MS: m/z (rel. int.): 300 (64), 195
(100), 181 (36), 165 (42), 77 (36). HRESIMS [M+Na]+;
Found: 323.1270 [M +Na]+ ; Calc. for C18H20O4Na:
323.1259.
References
1. Demain, A. L.; Appl. Microbiol. Biotechnol. 1999, 52, 455.
2. Schmid, A.; Dordick, J. S.; Hauer, B.; Wubbolts, M.; Witholt,
B.; Nature 2001, 409, 258.
2,3,4,4’-Tetramethoxydihydrochalcone (6)
o
1
Crystalline solid; mp 75-77 C (MeOH); H NMR
(300 MHz, CDCl3): see Table 1; 13C NMR (75 MHz,
CDCl3): see Table 1. LR-MS: m/z (rel. int.): 330 (82), 195
(24), 181 (98), 166 (48), 135 (100), 107 (16). HRESIMS
[M+Na]+ Found: 353.1358 [M+Na]+; Calc. for C19H22O5Na:
353.1365.
3. Faber, K.; Biotransformations in Organic Chemistry, 4th ed.;
Springer: Berlim, 2000.
4. Davis, B. G.; Boyer, V.; Nat. Prod. Rep. 2001, 18, 618.
5. Comasseto, J. V.; Omori, A. T.; Porto, A. L. M.; Andrade, L.
H.; Tetrahedron Lett. 2004, 45, 473.

1338
Biotransformation of Chalcones by the Endophytic Fungus Aspergillus flavus
J. Braz. Chem. Soc.
6. Comasseto, J. V.; Andrade L. H.; Omori, A. T.; Assis, L. F.;
21. Das, S.: Rosazza, J. P. N.; J. Nat. Prod. 2006, 69, 499.
Porto, A. L. M.; J. Mol. Catal. B: Enzym. 2004, 29, 55.
7. Kamerbeek, N. M.; Moonen, M. J. H.; van der Vem, J. G. M.;
van Berkel, W. J. H.; Fraaije, M. W.; Janssen, D. B.; Eur. J.
Biochem. 2001, 268, 2547.
22. Buisson, D.; Quintin, J.; Lewin, G.; J. Nat. Prod. 2007, 70,
1035.
23. Kostrzewa-Suslow, E.; Dmochowska-Gladysz, L.; Bialonska,
A.; Ciunik, Z.; Rymowicz, W.; J. Mol. Catal. B: Enzym. 2006,
39, 18.
8. Andrade, L. H.; Keppler,A. F.; Crusius-Schoenlein, I. H.; Porto,
A. L. M.; Comasseto, J. V.; J. Mol. Catal. B: Enzym. 2004, 31,
129.
24. Kinoshita, E.; Murakami, S.;Aishima, T.; J. Agric. Food Chem.
2000, 48, 2149.
9. Gotor-Fernandez, V.; Brieva, R.; Gotor, V.; J. Mol. Catal. B:
Enzym. 2006, 40, 111.
25. Zhong, K.; Shao, Z.; Hong, F.; Biotechnol. Prog. 2008, 24, 583.
26. Kostrzewa-Suslowa, E.; Dmochowska-Gladysz, J.; Bialonska,
A.; Ciunik, Z.; J. Mol. Catal. B: Enzym. 2008, 52, 34.
27. Annapoorna, S. R.; Rao, M. P.; Sethuram, B.; Indian J. Chem.,
Sect. A: Inorg., Bio-inorg., Phys., Theor. Anal. Chem. 2002, 41,
1341.
10. Pinheiro, S.; Ferreira, V. F.; Quim. Nova 1998, 21, 312.
11. Ogawa, J.; Shimizu, S.; Curr. Opin. Biotechnol. 2002, 13, 367.
12. Sahoo, S.; Kumar, P.; Lefebvre, F,; Halligudi, F. B.; Appl. Catal.,
A 2009, 354, 17.
13. Wang, P.-Y.; Tsai, S.-W.; J. Mol. Catal. B: Enzym. 2009, 59,
70.
28. Edwards, M. L.; Stemerick, D. M.; Sunkara, P. S.; J. Med. Chem.
1990, 33, 1948.
14. Ishitsuka, H.; Ninomiya, Y. T.; Ohsawa, C.; Fujiu, M.; Suhara,
Y.; Antimicrob. Agents Chemother. 1982, 22, 617.
15. Kiat, T. S.; Pippen, R,; Yusof, R.; Ibrahim, H.; Khalid, N.;
Rahman, N. A.; Bioorg. Med. Chem. Lett. 2006, 12, 3337.
16. Phrutivorapongkul,A.; Lipipun,V.; Ruangrungsi, N.; Kirtikara,
K.; Nishikawa, K.; Maruyama, S.; Watanabe, T.; Ishikawa, T.;
Chem. Pharm. Bull. 2003, 51, 187.
29. Russell, A.; J. Chem. Soc. 1934, 218.
30. Sushma, D.; Asian J. Chem. 2007, 19, 4957.
31. Sanchez-Gonzalez, M.; Rosazza, J. P. N.; J. Nat. Prod. 2004,
67, 553
32. Shindo, K.; Kagiyama,Y.; Nakamura, R.; Hara,A.; Ikenaga, H.;
Furukawa, K.; Misawa, N.; J. Mol. Catal. B: Enzymol. 2003,
23, 9.
17. Wu, J.-H.; Wang, X.-H.; Yi, Y.-H.; Lee, K.-H. Bioorg. Med.
Chem. Lett. 2003, 13, 1813.
33. Herath, W. H. M. W.; Ferreira, D.; Khan, I. A.; Phytochemistry
2003, 62, 673.
18. Bitencourt, H. R.; Santos, L. S.; Souza Filho, A. P. S.; Planta
Daninha 2007, 25, 747.
34. Araújo,W. L.; Lima,A. O. S.;Azevedo, J. L.; Marcon, J.; Sobral,
J. K.; Lacava, P. T.; Manual: Isolamento de Microrganismos
Endofíticos, CALQ: Piracicaba, Brazil, 2002.
19. Herencia, F.; López-Garcia, M. P.; Ubeda, A.; Ferrándiz, M.
L.; Biol. Chem. 2002, 6, 242.
20. Wang,A.; Zhang, F.; Huang, L.;Yin, X.; Li, H.;Wang, Q.; Zeng,
Z.; Xie, T.; J. Med. Plant. Res. 2010, 4, 847.
Submitted: July 8, 2010
Published online: March 22, 2011

J. Braz. Chem. Soc., Vol. 22, No. 7, S1-S10, 2011.
Printed in Brazil - ©2011 Sociedade Brasileira de Química
0103 - 5053 $6.00+0.00
Supplementary Information
SI
Biotransformation of Chalcones by the Endophytic Fungus Aspergillus flavus
Isolated from Paspalum maritimum Trin.
Marivaldo J. C. Corrêa,a Fátima M. Nunes,a Heriberto R. Bitencourt,a Fábio C. Borges,a
Giselle M. S. P. Guilhon,a Mara S. P. Arruda,a Andrey M. R. Marinho,a Alberdan S. Santos,a
Cláudio N. Alves,a Davi S. B. Brasil b and Lourivaldo S. Santos*,a
aPrograma de Pós-Graduação em Química, Instituto de Ciências Exatas e Naturais and
bFaculdade de Engenharia Química, Instituto de Tecnologia, Universidade Federal do Pará,
Campus Universitário do Guamá, 66075-900 Belém-PA, Brazil
Figure S1. 1H NMR spectrum (300 MHz, CDCl3) of compound 5.
*e-mail: lss@ufpa.br

S2
Biotransformation of Chalcones by the Endophytic Fungus Aspergillus flavus
J. Braz. Chem. Soc.
Figure S2. 13C NMR spectrum (75 MHz, CDCl3) of compound 5.
Figure S3. DEPT spectrum (75 MHz, CDCl3) of compound 5.

Vol. 22, No. 7, 2011
Corrêa et al.
S3
Figure S4. HETCOR spectrum (CDCl3) of compound 5.
Figure S5. HMBC spectrum of compound 5 (expansion).

S4
Biotransformation of Chalcones by the Endophytic Fungus Aspergillus flavus
J. Braz. Chem. Soc.
Figure S6. HMBC spectrum of compound 5 (expansion).
Figure S7. HMBC spectrum of compound 5 (expansion).

Vol. 22, No. 7, 2011
Corrêa et al.
S5
Figure S8. HMBC spectrum of compound 5 (expansion).
Figure S9. Low resolution mass spectrum of compound 5.

S6
Biotransformation of Chalcones by the Endophytic Fungus Aspergillus flavus
J. Braz. Chem. Soc.
Figure S10. High resolution mass spectrum of compound 5.
Figure S11. 1H NMR spectrum (300 MHz, CDCl3) of compound 6.

Vol. 22, No. 7, 2011
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S7
Figure S12. 13C NMR spectrum (75 MHz, CDCl3) of compound 6.
Figure S13. DEPT spectrum (75 MHz, CDCl3) of compound 6.

S8
Biotransformation of Chalcones by the Endophytic Fungus Aspergillus flavus
J. Braz. Chem. Soc.
Figure S14. HETCOR spectrum (75 MHz, CDCl3) of compound 6.
Figure S15. HMBC spectrum of compound 6 (expansion).

Vol. 22, No. 7, 2011
Corrêa et al.
S9
Figure S16. HMBC spectrum of compound 6 (expansion).
Figure S17. HMBC spectrum of compound 6 (expansion).

S10
Biotransformation of Chalcones by the Endophytic Fungus Aspergillus flavus
J. Braz. Chem. Soc.
Figure S18. Mass spectrum of compound 6 (CG-MS).
Figure S19. High resolution mass spectrum of compound 6.