Biotransformation of Chalcones by the Endophytic Fungus Aspergillus flavus Isolated from Paspalum maritimum Trin .

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.


Introduction
Chemical reactions performed by microorganisms have been used as an alternative to obtain products of chemical and biological interest.][3] Such reactions, usually known as biotransformation, have certain advantages over the conventional reactions.[12][13] 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. 19These 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. 20,21egioselective O-demethylation of flavones into their 4'-O-demethylated metabolites was performed using an A. niger strain. 22Biotransformation of flavanones by one wild strain of A. niger KB and three UV mutants of A. niger resulted 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. 23In the high molecular weight fraction of the culture extract of Aspergillus oryzae, genistein was transformed into shoyuflavone B in the presence of (±)-transepoxysuccinic acid. 24Epigallocatechin 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%. 25iotransformation 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. 26ue 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'-tetramethoxychalcone (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 literature 18 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 published in literature. 30The dihydrochalcones 5 and 6 are new compounds.
The bioreduction reaction of the chalcones was regioselective, showing only alteration in carboncarbon 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. 31The unsubstituted chalcone was converted to 2''-hydroxychalcone and 2'',3''-dihydroxychalcone in 25% and 59% yields, respectively, by Escherichia coli. 32icrobial 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. 33The 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 triplets in the 1 H NMR spectra that indicate the reduction of the double bond of the conjugated olefinic system.In the 13 C 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-trimethoxydihydrochalcone.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 C 18 H 20 O 4 (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 (Table 1).The 1 H NMR spectrum of 5 (Table 1) showed signals due to the benzylic methylene protons Hβ at d H 3.01 (2H, t, J 7.5 Hz) and to the α-carbonyl protons Hα at d H 3.29 (2H, t, J 7.5 Hz).The coupling of the protons Hα and Hβ was confirmed by analysis of 1  HMBC spectra, what allowed the assignment of these signals to C1 and C3, respectively.In the same NMR spectrum, it was observed that the signal at d H 6.87 (H6) showed correlation with the signals at d C 151.9 and 152.3, which indicated that these signals can be attributed to C2 and C4 carbons, respectively.The analysis of the HETCOR ( 1 J CH ) 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 d C 107.2 (C5) and 123.9 (C6).The 1 H NMR spectrum showed two ortho-related doublets at d H 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

General experimental procedures
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 o C; type of injection: splitless; programmed temperature to 100-300 o C, with a gradient of 5 o C 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 a Varian Mercury-300 spectrometer, operating at 300 MHz for 1 H and 75 MHz for 13 C 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).

Plant material
The leaves of Paspalum maritimum were collected in January 2009 in the forest reserve of EMBRAPA-Amazônia 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 o C. 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".

Synthesis of substrate: 18 chalcones 1-3
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 o C 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), chalcone 2 was obtained in 75.0%yield (2.42 g) and chalcone 3 was obtained in 60.0% yield (2.62 g).

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 121 o 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 flask and shaken (160 rpm) at 30 o C.After 10 days the reaction mixture was extracted with EtOAc (3 × 100 mL), and organic phases were grouped, dried (Na 2 SO 4 ), filtered Vol.22, No. 7, 2011   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 hexanes and increasing polarity with EtOAc, to give seventeen fractions (C1-C17).Fractions C12 (hexane:EtOAc 6:4) and C13 (hexane:EtOAc 6:4) were grouped and recrystallized with 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.

Table 1 .
H × 1 H-COSY spectrum.HETCOR spectrum ( 1 J CH ) showed correlations of the signals of the protons at d H 3.29 (Hα) and 3.01 (Hβ) with signals at d C 40.6 (Cα) and 30.6 (Cβ), respectively.The triplet at d H 3.01, assigned to the signal of hydrogen Hß, showed a correlation in the HMBC spectrum with the signals at d C 137.1 (C1) and at d C 105.4 (C2/C6).The singlet at d H 6.46 (2H, s, ArH) assigned to the equivalent protons H2/H6 of ring B showed correlation with the signal at d C 105.4 (C2/C6) in the HETCOR spectrum.The HMBC spectrum showed the correlation of the signal at d H 6.46 (H2/H6) with the signals at d C 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 1 H × 1 H-COSY and HETCOR spectra.+0.7 mmu] analysis and NMR data (Table 1).The 1 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 d H 6.87 and 6.59, were assigned to H6 and H5, respectively.The signal at d H 6.59 correlated with the signals of carbons at d C 127.2 and 142.3 in the 1 H and 13 C NMR chemical shifts (d H and d C in ppm) of compounds 5 and 6 in CDCl 3 13 D H NMR data were recorded at 300 MHz.b 13 C NMR data were recorded at 75 MHz.c 1 H-13C HMBC correlations are from the carbon(s) specified to the protons indicated.d Values can be reversed.

1
H × 1 H-COSY.The analysis of HETCOR spectrum of 6, relating to the ring A, was important to assign accurately the signals at d C 130.3 and 113.6 to carbons C2'/C6' and C3'/C5', respectively, that showed 1 J CH correlations with signals at d H 7.95 and 6.92.The HMBC spectrum showed correlations of the signals of carbons at d C 129.9 (C1') and 163.3 (C4') with the signals at d H 6.92 (H3'/H5') and d H 7.95 (H2'/H6').The assignment of carbonyl carbon signal (d C 198.4) was based on the analysis of the correlations with the signals due to Hβ and Hα.