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Coumarin metabolic routes in Aspergillus spp.
f u n g a l b i o l o g y 1 1 5 ( 2 0 1 1 ) 2 4 5 e2 5 2
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Coumarin metabolic routes in Aspergillus spp. Celeste AGUIRRE-PRANZONI, Alejandro A. ORDEN, Fabricio R. BISOGNO, Carlos E. ARDANAZ, Carlos E. TONN, Marcela KURINA-SANZ* INTEQUI-CONICET, Facultad de Quımica, Bioquımica y Farmacia, Universidad Nacional de San Luis, Chacabuco y Pedernera, CP 5700 San Luis, Argentina
article info
abstract
Article history:
Coumarin metabolism by several Aspergillus strains was studied. Aspergillus ochraceus and
Received 29 June 2010
Aspergillus niger carried out the reduction of the C3eC4 double bond to yield dihydrocou-
Received in revised form
marin in 24 h. Meanwhile, the first strain did not transform dihydrocoumarin after 7 d,
16 November 2010
A. niger demonstrated to have two divergent catabolic pathways: (a) the lactone
Accepted 19 December 2010
moiety opening and further reduction of the carboxylic acid furnishing the primary alcohol
Available online 29 December 2010
2-(3-hydroxypropyl)phenol and, (b) the hydroxylation of the aromatic ring of dihydrocou-
Corresponding Editor: Steven Harris
marin at a specific position to give 6-hydroxy-3,4-dihydrochromen-2-one. Aspergillus flavus did not perform double bond reductions, and only produced oxygenated metabolites,
Keywords:
mainly 5-hydroxycoumarin. Enzyme-specific inhibitors and a coumarin analogous were
Aspergillus
useful to confirm the A. niger catabolic route.
Coumarin
ª 2010 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Dihydrocoumarin Hydroxycoumarin Metabolism
Introduction In nature, coumarin (1) and its derivatives occur in a huge number of living organisms. A possible explanation for such a vast distribution among plants is the ability of 1 to dissuade herbivores from eating them (Adfa et al. 2010). It has been proposed that toxicity and bitter taste are the main reasons for deterrence. In terms of coevolution, herbivores had to evolve in order to circumvent this situation by expressing several metabolic pathways to detoxify xenobiotic substances (Li et al. 2003). On the other hand, a similar scenario becomes evident between plants and fungi. It has been reported that when plants are attacked by different fungal strains, their metabolite profile is drastically perturbed, displaying the induction of defense metabolic routes. Likewise, fungi express several detoxification strategies, being the cytochrome P450 usually involved (Subramanian & Yadav 2008).
Concerning 1 metabolism, several studies have been carried out in mammals such as mice, rats and humans. Cytochrome P450 monooxygenases (P450s) are the enzymes responsible for the two main oxygenations reported: the epoxidation of the double bond C3eC4 to yield coumarin 3,4-epoxide and the hydroxylations at the aromatic ring, with preference in C7. The epoxide may be further conjugated with glutathione or be rearranged to o-hydroxyphenylacetaldehyde which is subsequently oxidized to o-hydroxyphenylacetic acid or reduced to o-hydroxyphenylethanol by a NADþ aldehyde dehydrogenase (Vassallo et al. 2004). The reactions carried out by P450s in the presence of oxygen, the cofactor NAD(P)H and a corresponding electron transfer system are diverse. These reactions include epoxidation of CeC double bonds and aromatic and sp3 hybridized C-atoms, hydroxylation. P450 enzymes also catalyze oxidative phenol couplings and dealkylation reactions such as alkyl-coumarins dealkylations (Urlacher & Eiben 2006).
* Corresponding author. Tel.: þ54 2652 423789x253. E-mail address: [email protected] 1878-6146/$ e see front matter ª 2010 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.funbio.2010.12.009
246
Levy and Frost suggested that Arthrobacter species metabolized 1 by a sequence of reactions encompassing the enzymatic hydrolysis of the lactone moiety to give o-coumaric acid, and a further double bond reduction to yield melilotic acid (6) mediated by a NADH enzyme (Levy & Frost 1966). On the other hand, Kosuge and Conn suggested that 1 is first hydrogenated to dihydrocoumarin (2) and then hydrolyzed to 6 by white sweet clover (Kosuge & Conn 1962). P450s of the white-rot fungus Phanerochaete chrysosporium hydroxylate 1 at four different positions to form 5-hydroxycoumarin (3), 6-hydroxycoumarin, 7-hydroxycoumarin and 8-hydroxycoumarin (Matsuzaki & Wariishi 2004). On the other hand, 2 is obtained by biotransformation of 1 by several Sac€ ser et al. 2006). These authors charomyces cerevisiae strains (Ha demonstrated that 6 was the biotransformation product which was further cyclized into 2 by distillation in acid media, suggesting that coumarin lactone ring is opened into o-coumaric acid and then a highly specific NADH:o-coumaric acid oxidoreductase, resembling the one of Arthrobacter spp., catalyzes the C3eC4 double bond hydrogenation. It is well known that filamentous fungi have multiple P450 enzymes. Among them, Aspergillus genus showed the largest P450 family, leading the list Aspergillus oryzae and Aspergillus niger which possess 155 and 150 different P450 enzymes, respectively (Lah et al. 2008). This fact supports the idea of an efficient xenobiotic metabolism by these ascomycetes. The biotransformation of 1 by A. niger was conducted more than 40 y ago by means of basic chromatographic techniques. As it was reported the main metabolite was 6 due to the opening of lactone 2. Also, traces of a hydroxylated compound, probably in C3, were identified (Bocks 1967). In the present work we report the metabolism of 1 and derivatives by several Aspergillus spp., especially A. niger. Time course experiments were thoroughly analyzed by GCeMS and NMR. Final products and intermediates were isolated and characterized. Parameters such as oxygen availability, growing and resting cell conditions and their influence in the course of A. niger metabolic routes were studied. In order to determine what enzymes might be involved in the metabolic pathways, specific inhibition assays towards Old Yellow Enzymes (enoate reductases) and P450 monooxygenases were performed in whole cell systems.
C. Aguirre-Pranzoni et al.
Chemicals The commercial chemical reagents, coumarin (1), 3-hydroxy-2Hchromen-2-one, 4-hydroxy-2H-chromen-2-one, 6-hydroxy-2Hchromen-2-one, cyclohex-2-enone, 3,4-dihydrochromen-2-one (2), p-hydroxybenzaldehyde and piperonyl butoxide, (5-[2-(2butoxyethoxy)ethoxymethyl]-6-propyl-1,3-benzodioxole) were purchased from SigmaeAldrich Argentina. Solvents were distilled and dried prior to use.
Preparation of methoxy-2H-chromen-2-one derivatives
Materials and methods
Each of the commercial reagents: 3-hydroxy-2H-chromen-2one, 4-hydroxy-2H-chromen-2-one and 6-hydroxy-2H-chromen-2-one (200 mg, 1.23 mmol) was dissolved in 20 ml of acetone, 596 mg (4.16 mmol) of K2CO3 and 500 ml (5.29 mmol) of Me2SO4 was added, and the solutions were heated to reflux. After reaction completion (3 h) the reaction mixtures were filtered and rinsed with ethyl acetate (EtOAc). Purification was carried out by column chromatography (CC) on Sigel with n-hexane:EtOAc (7:3) as solvent. 3-methoxy-2H-chromen-2-one: yield 164 mg (75 %) 1H NMR (200 MHz, CDCl3): d 4.00 (3-H, s, H3CO), 5.70 (1-H, s, H4), 7.27 (1H, m, H6), 7.31 (1-H, m, H5) 7.54 (1-H, d, J:7.20, H7), 7.80 (1-H, d, J:7.20, H8). 13C NMR (50.2 MHz, CDCl3): d 198.80 (C2); 166.00 (C3); 90.07 (C4); 116.72 (C5); 123.86 (C6); 132.36 (C7); 122.95 (C8); 153.26 (C9); 162.80 (C10); 56.32 (H3CO). MS: m/z values (% relative abundance) Mþ. 176 (50.02), 175 (21.01), 148 (100.00), 133 (48.00), 119 (18.03), 105 (35.10), 89 (61.98), 77 (42.97). 4-methoxy-2H-chromen-2-one: yield 175 mg (81 %). 1H NMR (200 MHz, CDCl3): d 4.02 (3-H, s, H3CO), 5.70 (1-H, s, H3), 7.27 (1-H, m, H6), 7.31 (1-H, m, H5) 7.54 (1-H, d, J:7.20, H7), 7.80 (1-H, d, J:7.20, H8). 13C NMR (50.2 MHz, CDCl3): d162.00 (C2); 90.09 (C3); 166.60 (C4); 116.74 (C5); 123.87 (C6); 132.35 (C7); 122.96 (C8); 153.20 (C9); 115.60 (C10); 56.33 (H3CO). MS: m/z values (% relative abundance) MD. 176 (53.07), 148 (100.00), 133 (47.98), 105 (33.13), 91 (20.60), 89 (57.69), 77 (39.30). 6-methoxy-2H-chromen-2-one (8): yield 92 mg (42 %). 1H NMR (200 MHz, CDCl3): d 3.85 (3-H, s, H3CO), 6.42 (1-H, d, J:10.00 H3), 6.91 (1-H, d, J:2.80, H5), 7.10 (1-H, dd, J:9.10e2.80, H7), 7.27 (1-H, dd, J:9.10e2.80, H8), 7.65 (1-H, d, J:10, H4). 13C NMR (50.2 MHz, CDCl3): d 160.90 (C2); 117.09 (C3), 143.16 (C4); 110.00 (C5); 156.10 (C6); 119.42 (C7); 117.88 (C8); 148.40 (C9); 119.10 (C10); 55.81 (H3CO). MS: m/z values (% relative abundance) MD. 176 (100.00), 161 (34.32), 148 (70.20), 133 (68.49), 105 (46.46), 77 (42.53).
Microorganisms
Analytical methods
Fungal strains were purchased from different collections: American Type Culture Collection (Aspergillus niger ATCC 11394; A. niger ATCC 16404), Instituto Malbran collection (Aspergillus terreus INM 031783), Facultad de Ciencias Exactas-Universidad de Buenos Aires (Aspergillus flavus UBA 294), Departamento de Microbiologıa e Inmunologıa-Universidad Nacional de Rio Cuarto (Aspergillus ochraceus RC 33; Aspergillus fumigatus RC 78). Fungi were maintained on potato dextrose agar (PDA) slants. Spore suspensions were obtained according to reported procedures and adjusted to 107 spores with colony forming ability per millilitre in phosphate buffered saline (PBS) with added Tween-20 (5 ml/ ml) to inoculate the liquid media (Wright et al. 1983).
1
H NMR and 13C NMR spectra were obtained on Bruker AC-200 spectrometer (200 MHz and 50.2 MHz, respectively) in Cl3CD, chemical shifts in ppm are relative to tetramethylsilane (TMS), coupling constants (J ) in Hz. Analytical TLC was performed on Merck precoated silica gel 60 F254 plates. Solvent for TLC were n-hexane:EtOAc mixtures. Column chromatography was carried out on Merck silica gel 60 (230e400 mesh). Gas chromatography-flame ionization detector (GC-FID) analysis was performed in a PerkineElmer Clarus 500 instrument equipped with an Elite 5-column (30 m, 0.25 mm ID, 0.25 mm df) (Dt ¼ 10 C/min), T2 ¼ 320 C for 5 min, T1 ¼ 180 C (Dt ¼ 30 C/min) T3 ¼ 350 C, injector T ¼ 240 C; carrier gas: N2
Coumarin metabolic routes in Aspergillus spp.
35 cm/seg, splitless; FID T ¼ 350 C. Gas chromatographyemass spectrometry (GCeMS) analysis was performed with a GCQ-plus Finnigan Mat equipped with Restek 5-MS column 5 % diphenyl95 % dimethylpolisiloxane (30 m, 0.25 mm ID, 0.25 mm df). T1 ¼ 100 C for 3 min, (15 C/min), T2 ¼ 300 C for 3 min; carrier gas: He 40 cm/seg. The instrument was run in EI mode at energy of 70 eV (tune setting, trap-offset between 10 and 20 v).
Biotransformation procedures Growing cells biotranformation A two-step process was conducted. Spore suspensions from the different Aspergillus strains were incubated in liquid Czapeck medium (sucrose 30 g, yeast extract 5.0 g, KCl 0.5 g, NaNO3 2.0 g, K2HPO4 1.0 g, FeSO4 0.01 g, Mg2SO4.7H2O 0.5 g, distilled water to 1000 ml, pH 5) at 28 C, on rotatory shaker at 180 rpm. Forty-eight-hour-old cultures (5 ml) were subcultured in 125 ml baffled Erlenmeyer flasks containing 30 ml of fresh culture medium in a ten pellets per batch relation. Substrates (10 mg/batch), dissolved in 100 ml of dimethylsulfoxide (DMSO), were added to the cultures 24 h later. Biotransformation progress was monitored by withdrawing 500 ml samples every 24 h and extracted with ethyl acetate (500 ml). Aliquots of the organic layers were analyzed by GC-FID (1 ml) and TLC. After 7 d of incubation, the fermentation broth was filtered and the filtrate was extracted with EtOAc (4 50 ml), dried over anh. Na2SO4 and evaporated under reduced pressure. Blank assays without substrates and without fungi were carried out in parallel. Experiments were performed in triplicate.
Resting cells biotransformation Fungal pellets were harvested from 48 h old cultures, washed with 0.1 M pH 6 phosphate buffer and suspended in 30 ml of fresh buffer. Incubation, substrate addition and work up procedures were carried out as described above.
Time course biotransformation The experiments were performed with growing cell as described above, by collecting 500 ml of media samples from triplicate assays each 24 h. Aqueous media were extracted with EtOAc and evaporated under vacuum. Dried samples were resuspended in 500 ml of Cl2CH2 and analyzed by GCeMS. In order to isolate biotransformation products, several repetitions of the small-scale experiments were carried out. The crude extracts or the isolated metabolites were analyzed by GCeMS, 1H NMR, 13C NMR. 3,4-dihydrochromen-2-one (2): 1H NMR (200 MHz, CDCl3):d 2.71 (2-H, t, J:6.20, H3), 3.01 (2-H, t, J:6.20, H4), 6.86 (1-H, m, H6), 6.89 (1-H, d, J:7.50, H8), 7.10 (1-H, d, J:7.75, H5), 7.16 (1-H, m, H7). 13C NMR (50.2 MHz, CDCl3): d 175.80 (C2), 35.23 (C3), 24.57 (C4), 130.64 (C5), 120.89 (C6), 120.89 (C7), 117.33 (C8), 153.55 (C9), 127.48 (C10). MS: m/z values (% relative abundance) 149 (3.5), Mþ. 148 (27.20), 121 (6.20), 120 (67.80), 92 (11.30), 91 (100.00), 78 (8.90), 77 (13.90), 50 (4.50). 5-hydroxy-2H-chromen-2-one (3): 1H NMR (200 MHz, CDCl3):d 6.19 (1-H, d, J:12.00, H3), 8.04 (1-H, d, J:12.00, H4), 6.66 (1-H, d, J:8.75, H6), 7.22 (1-H, t, J:8.75 H7); 6.69 (1-H, d, J:8.75, H8). 13C NMR (50.2 MHz, CDCl3): d 161.20 (C2), 107.17 (C3), 139.06 (C4), 154.89 (C5), 113.32 (C6), 131.97 (C7), 109.81 (C8),
247
154.57 (C9), 108.61 (C10). MS: m/z values (% relative abundance) Mþ. 162 (39.4), 135 (6.3), 134 (100), 131 (4.6), 121 (4.5), 115 (6.9), 106 (6.0), 95 (5.1), 92 (4.5), 91 (7.3), 79 (8.4), 78 (53.6), 77 (26.1), 63 (8.3), 62 (7.7), 52 (4.6), 51 (18.4), 50 (15.4). 6-hydroxy-3,4-dihydrochromen-2-one (4): 1H NMR (200 MHz, CDCl3): d 2.76 (2-H, dd, J:6.80-5.60, H3), 2.94 (2-H, dd, J:6.80-5.60, H4), 6.67 (1-H, d, J:3.20, H5), 6.71 (1-H, d, J:8.80, H7), 6.93 (1-H, d, J:8.80, H8). 13C NMR (50.2 MHz, CDCl3): d 168.85 (C2), 29.12 (C3), 23.86 (C4), 114.45 (C5), 145.99 (C6), 114.80 (C7), 117.86 (C8), 152.07 (C9), 123.84 (C10). MS: m/z values (% relative abundance) 165 (7.2), MD. 164 (56.9), 136 (100.0), 135 (13.9), 122 (3.8), 121 (5.4), 107 (25.6), 94 (14.6), 91 (4.6), 79 (16.9), 77 (17.7), 66 (10.7), 65 (7.9). 2-(3-hydroxypropyl)phenol (5): 1H NMR (200 MHz, CDCl3): d 1.92 (2-H, m, H20 ), 3.01 (2-H, t, J:7.16, H10 ), 4.11 (2-H, t, J:6.35, H30 ), 6.67 (1-H, d, J:7.99, H6), 6.83 (1-H, t, J:7.99, H4), 7.04 (1-H, d, J:7.75, H3), 7.10 (1-H, t, J:7.50, H5). 13C NMR (50.2 MHz, CDCl3):d 154.31 (C1), 127.11 (C2), 130.31 (C3), 120.83 (C4), 127.39 (C5), 115.24 (C6), 26.10 (C10 ), 28.54 (C20 ), 64.79 (C30 ). MS: m/z values (% relative abundance) Mþ. 152 (3.90), 135 (4.00), 134 (30.50), 133 (30.50 (23.90), 103 (12.40), 91 (100.00), 79 (19.80), 78 (20.20), 77 (62.50), 65 (16.90), 119 (27.80), 115 (29.00), 105 (23.90)). 3-(2-hydroxyphenyl)propanoic acid (melilotic acid; 6): 1H NMR (200 MHz, CDCl3): d 2.77 (2-H, t, J:6.71, H2), 2.93 (2-H, t, J:6.71, H3), 6.83 (1H, d, J:7.41, H6), 7.11 (1-H, t, J:7.41, H8), 7.11 (1-H, d, J:7.41, H9), 7.12 (1-H d:7.41, H7). 13C NMR (50.2 MHz, CDCl3): d 179.32 (C1), 34.40 (C2), 24.60 (C3), 126.75 (C4), 153.92 (C5), 116.64 (C6), 128.06 (C7), 121.52 (C8), 130.52 (C9). MS: m/z values (% relative abundance) Mþ. 166 (0.9), 149 (3.6), 148 (15.3), 147 (2.2), 121 (5.1), 120 (51.1), 119 (18.4), 92 (11.6), 91 (100.0), 77 (17.5), 65 (11.2). 6-methoxy-3,4-dihydrochromen-2-one (9): MS: m/z values (% relative abundance) 179 (12.8), Mþ. 178 (49.1), 151 (6.4), 150 (70.6), 137 (1.8), 136 (19.6), 135 (100.0), 108 (19.3), 107 (20.9), 91 (2.9), 79 (30.3), 78 (17.3), 77 (53.3). Hydroxy derivative of 6-methoxy-3,4-dihydrochromen2-one (10): MS: m/z values (% relative abundance) 195 (9.9), Mþ. 194 (66.9), 166 (82.5), 152 (100.0), 151 (35.5), 136 (20.4), 124 (48.3), 123 (36.4), 109 (25.9), 95 (34.4), 77 (21.7).
Enzyme inhibition assays The experiments were performed with resting cell systems consisting of 30 ml phosphate buffer (pH 6) inoculated with a centrifugated 24 h culture of Saccharomyces cerevisiae or fungal pellets harvested from 48 h old Aspergillus niger cultures, with the addition of different concentrations of the inhibitors, p-hydroxybenzaldehyde or piperonyl butoxide, ranged between 1 and 10 mM. Three hours later the corresponding substrate, cyclohex-2-enone or 1, was added at final concentrations of 0.7 mM. Incubations were carried out under the above described conditions. Samples were withdrawn every 24 h until 164 h.
Results Coumarin metabolism by several Aspergillus strains A 7-d-biotransformation screening with growing cells was carried out. Samples were analyzed by GC-FID and are expressed
248
as percentage of biotransformed substrate (Fig 1). As depicted in the figure, Aspergillus niger, Aspergillus flavus and Aspergillus ochraceus, resulted the most active species whereas the substrate was recovered untouched from the experiments performed with Aspergillus fumigatus and Aspergillus terreus. Since the metabolite profiles for A. niger, A. flavus and A. ochraceus seemed to be quite different, 7-d-biotransformation experiments were performed to isolate and identify the main products (Fig 2), either with growing or resting cells. Surprisingly, when A. ochraceus resting cells were used, 1 was gradually consumed until completion in the fifth day, without the appearance of biotransformation products. This fact, together with the observed variation in the pellets morphology and a slight increasing in the biomass amount, led us to think that 1 was probably being used as carbon source by the fungus under these starving conditions. On the contrary, with growing cells, 1 was transformed into a sole metabolite, 2, in 80 % conversion rate at day 7. In the 1H NMR spectrum, the main characteristic was the disappearance of the typical doublets at 6.41 and 7.71 ppm of the hydrogens on the double bond C3eC4 of 1 and the appearance of two new triplets integrating for two hydrogens each at higher fields, 2.71 and 3.01 ppm, corresponding to H3 and H4, respectively. The 13C NMR spectrum shows two signals corresponding to the two new saturated carbons at 24.57 and 35.23 for C4 and C3, respectively. These data were confirmed by homo- and heteronuclear bidimensional NMR experiments. The MS spectrum showed an increment of two mass units for the molecular ion of the new compound (m/z 148 Mþ) respect to molecular ion of 1 (m/z 146 Mþ). Aspergillus flavus also showed a different metabolism, being resting cells more efficient than growing cells to convert 1 into a more polar compound in 92 % conversion rate. The main biotransformation product of the resting cell experiment was purified by CC and identified by GCeMS and NMR as 5-hydroxycoumarin (3). In the 1H NMR spectrum it can be seen as a triplet at 7.57 ppm (J ¼ 8.75 Hz) corresponding to H7, and a pair of doublets with the same coupling constants at 6.99 and 6.93 ppm of H6 and H8, respectively. These data were confirmed by bidimensional homo- and heteronuclear experiments.
C. Aguirre-Pranzoni et al.
In the experiments with growing cells of A. niger ATCC 11394, it was possible to detect that 1 was entirely converted into 2 before 48 h of biotransformation. However, at 7 d, two different metabolites were isolated: 2-(3-hydroxypropyl)phenol (5) and 6-hydroxy-3,4-dihydrochromen-2-one (4). It was particularly noticeable in the 1H NMR spectrum of 5 the occurrence of a multiplet that integrates for two hydrogens at 1.92 ppm (H20 ), together with the presence of two triplets at 3.01 ppm (J ¼ 7.16 Hz) and 4.11 ppm (J ¼ 6.35 Hz) corresponding to H10 and H30 , respectively. No modifications were evident on the aromatic ring and there was no evidence of the existence of the lactone moiety. For the metabolite, 4, it was easy to assume that it was a derivative of 2 due to the presence of two triplets between 2.70 and 3.00 ppm corresponding to H10 and H20 , and the absence of signals of the hydrogens on the double bond. The assignment of the hydroxylation position on the aromatic ring was achieved by bidimensional homo- and heteronuclear NMR spectroscopy. The signals at 6.67 ppm (d, J:2.80 Hz), 6.71 ppm (dd, J:8.80 and 2.80 Hz) and 6.92 ppm (d, J:8.80 Hz) were attributed to hydrogens 5, 7 and 8, respectively, due to a long distance correlation observed between H5 and H3 in the COSY-45 spectrum and H3 and C5 (114.45 ppm) in the HMBC.
Time course biotransformation experiments by Aspergillus niger ATCC 11394 Time course experiments allowed us detecting compounds derived from the opening of the dihydrocoumarin lactone ring and the further reduction of the carboxylic acid into the corresponding aldehyde, confirm by the presence of a singlet
OH
2
O
O
5
A. ochraceus
6
10
3
4
O
O
O
O
A. flavus
3 2
7 8
9
1
O
O 1
A. niger HO
2
O
O 3 4
1'
2' 3'
5 6
Fig 1 e Screening of coumarin (1) biotransformation by different Aspergillus species in an optimized two-step process with growing cells carried out for 168 h. Remanent substrate was determined by GC-FID and expressed as percentage of converted substrate.
2
4
1
OH
OH
5 Fig 2 e Biotransformation of coumarin (1) by selected Aspergillus species. Optimized two-step process with growing cells were carried out for 168 h. Metabolites were isolated and determined by MS and NMR.
Coumarin metabolic routes in Aspergillus spp.
249
at 9.87 ppm in the 1H NMR spectrum of the 72-h-extract. This was in agreement with the occurrence of a metabolite of Mþ. 150 in the GCeMS analysis, which was assigned as 3-(2hydroxyphenyl)propanal (7). Compound 7 disappeared at longer incubation times (96 and 144 h), whereas 5 (Mþ. 152) resulted the main metabolite detected. In addition, the presence of the hydroxylated metabolite was noticeable only after 96 and 144 h when a peak of Mþ. 164 appeared in the GCeMS analysis (Fig 3). To corroborate this route, 2 was added as substrate. After 7 d, two metabolites were isolated, 4 and 5. In order to study the metabolic pathway for related compounds, some monohydroxylated commercial coumarins, such as 3-hydroxycoumarin, 4-hydroxycoumarin and 6-hydroxycoumarin were added as substrates to growing cells. To our surprise, the three compounds were recovered untransformed at the end of the experiments (7 d). The methoxy derivatives were further used as substrates. Only 6-methoxycoumarin (8) was biotransformed into three main metabolites with molecular ions of m/z 178 (9), m/z 194 (10) and m/z 164 (4) as depicted in Fig 4. On the contrary, the other two substrates were recovered untouched. Similar to the study carried out with 1, a time course experiment was carried out with 8. The crude extract of the biotransformation was monitored by GCeMS. We observed that at 24 h, the substrate was completely converted into 9, a metabolite of molecular ion of m/z 178, corresponding to hydrogenation on the C3eC4 double bond, in agreement with the results observed for 1. After 72 h, two new metabolites were detected. Their
1
O
O
molecular ions were m/z 164 and 194. The first is in agreement with demethylation of the intermediate 9 (m/z 178) and the latter is attributable to hydroxylation on the aromatic ring. Since structural elucidation was performed by GCeMS, it was not possible to assign the position of the new hydroxyl group. However, we can assure that the hydroxylation occurred on the aromatic ring, since no peaks corresponding to [MeH2O]þ. and/or [MeHO]þ. appeared, as expected if hydroxylation would have generated a secondary alcohol. Moreover, two peaks of m/z 166 [MeCO]þ. and m/z 152 corresponding to the loss of the ketene moiety presented important intensities in the MS spectrum of 10. Since no lactone ring opening products were detected, we can assume that electron-donating groups on the aromatic ring, such as methoxy, can either electronically modulate the lability of the intramolecular ester bond or exert detrimental interaction with the hydrolytic enzyme responsible for lactone opening. Nevertheless, further experiments with other substituents on the aromatic ring will be necessary to demonstrate this hypothesis. To compare the influence of oxygen availability in the biotransformation process, a comparative assay using different medium volumes was performed (30 ml and 60 ml in 125 ml closed flasks). All other experimental variables, such as inocula, substrate concentrations, agitation and temperature were preserved. The GCeMS analysis of the assays with higher oxygenation (minor medium volume), gave 2, 4 and 5 as the main metabolites at the end of the process. On the contrary, with lower oxygen conditions (major medium volume) 2, 4 and 6 were identified, but no further hydroxylated metabolites were detected.
Enzyme inhibitors influence A well-known inhibitor of the enoate reductases belonging to the OYE family, p-hydroxybenzaldehyde, was assayed in yeast
O
O
2 O
H3CO HO
OH O
OH
6
8
O
4
O
O
O
O
H3CO
7
OH
9
O
H3CO
HO
HO
5
OH
OH
Fig 3 e Pathway proposed for coumarin (1) metabolism by A. niger ATCC 11394, based on time course experiments with growing and resting cells, analyzed by GCeMS and NMR.
O
10
O
O
O
4
Fig 4 e Pathway proposed for 6-methoxycoumarin (8) metabolism by A. niger ATCC 11394 based on growing cell time course experiments, analyzed by GCeMS and NMR.
250
C. Aguirre-Pranzoni et al.
Table 1 e Effects of enoate reductase inhibitor ( p-hydroxybenzaldehyde) on coumarin (1) and cyclohexen-2-one biotransformation by S. cerevisiae and A. niger ATCC 11394 Biocatalytic system
Inhibitor concentration ( p-hydroxybenzaldehyde)
Substrate: coumarin (1)
Substrate: cyclohexen-2-one
S. cerevisiae
0.7 mM 1.4 mM 2.0 mM 2.5 mM 3.0 mM
2 2 e e e
e e e e e
A. niger
2.0 mM 4.0 mM 6.0 mM 8.0 mM 10.0 mM
2 2 2 2 2
Cyclohexanoneecyclohexanol Cyclohexanoneecyclohexanol Cyclohexanoneecyclohexanol Cyclohexanoneecyclohexanol Cyclohexanoneecyclohexanol
Biotransformation productsa
a Determined by GCeMS.
(Williams & Bruce 2002). This inhibitor was tested in our system in order to find out if the enzyme responsible for the first reaction in the coumarin metabolic pathway belongs to the OYE family. p-Hydroxybenzaldehyde was added to resting cells 3 h prior to the addition of 1 in five different concentrations. As in all the other processes, the bio-reactions were checked every 24 h until day 7. As a positive control, the biotransformation of cyclohex-2-enone with and without the addition of the inhibitor was performed. Additional control assays, involving the use of Saccharomyces cerevisiae towards both substrates were performed demonstrating that concentrations of p-hydroxybenzaldehyde up to 2 mM inhibited the reduction of both the coumarin C3eC4 double bond and the cyclohexen-2-one (Table 1). Conversely, with Aspergillus niger cultures, no inhibitions were observed, neither when the substrate was 1 nor cyclohex-2-enone. It is worth to point out that p-hydroxybenzaldehyde was metabolized by other A. niger enzyme/s into several non-identified more polar products. Consequently, a higher inhibitor concentration was used (10 mM). In this condition, 1 was totally transformed into 2, although a reasonable quantity of the OYE inhibitor still remained in the bio-reaction medium after 48 h (Fig 5, Table 1). This suggests that the reduction of the C3eC4 double bond of coumarin derivatives in this A. niger strain might be mediated by isoenzymes which are p-hydroxybenzaldehyde non-sensitive or by other kind of reductases not belonging to the OYE family. We also developed a P450 inhibition assay working with A. niger resting cells, adding 0.7 mM of 1 and different concentrations of piperonyl butoxide. As expected, 1 was totally transformed into 2 at 24 h and the only metabolites detected at 164 h were 2 and mainly 5. When 2 was added as substrate, no changes were observed at 24 h; meanwhile at 164 h, no hydroxylated metabolites were detected. Consequently, we can assert that the occurrence of metabolite 4, in the coumarin metabolic pathway in A. niger was mediated by P450s (Table 2). In order to discard the possibility that exoenzymes like lignin and manganese peroxidases could metabolize 1, we carried out a simple assay consisting in the substrate addition (0.7 mM) to aseptically filtered culture broth after 4 d of incubation with the fungal development. Samples were taken after
24, 48 and 72 h and no transformation products were detected and 1 was recovered untouched.
Discussion Taking into account the high expression of P450 enzymes reported for the genus Aspergillus and their vast acceptance of xenobiotic substrates (Lah et al. 2008), the first step of this work was a biotransformation screening by several Aspergillus strains towards 1. Aspergillus niger, Aspergillus flavus and Aspergillus ochraceus resulted the most active species (Figs 1 and 2). In the conditions of these experiments no differences were observed in biomass rates (data not shown), although it is worth to consider that the xenobiotic substrate was always added in the stationary phase of the growing cell cultures (48 h old). Aspergillus niger performed the quantitative reduction of the C3eC4 double bond of 1 to yield 2 in 24 h. After that, two divergent metabolic pathways took place: (a) the lactone moiety opening and further reduction of the carboxylic acid, furnishing the primary alcohol 5 as the main terminal metabolite and, (b) the hydroxylation on the aromatic ring of 2 in a specific position to give 4 (Fig 3). This pathway was corroborated by the biotransformation of the main postulated
Fig 5 e Relative quantities of coumarin (1), dihydrocoumarin (2) and p-hydroxybenzaldehyde (OYE-inhibitor) in the time course inhibition assay developed with resting cells of A. niger ATCC 11394. Initial concentrations: coumarin 0.7 mM and p-hydroxybenzaldehyde 10.0 mM.
Coumarin metabolic routes in Aspergillus spp.
251
Table 2 e Effects of P450 monooxygenase inhibitor (piperonyl butoxide) on coumarin (1) and dihydrocoumarin (2) biotransformations by A. niger ATCC 11394 Substrate (0.7 mM)
Inhibitor concentration (piperonyl butoxide)
Biotransformation productsa 24 h
Coumarin (1)
1.0 mM 1.5 mM 2.0 mM 2.5 mM
2 2 2 2
Dihydrocoumarin (2)
1.0 mM 1.5 mM 2.0 mM 2.5 mM
e e e e
164 h 2 2 2 2
and and and and
5 5 5 5
5 5 5 5
a Determined by GCeMS.
intermediate 2, which yielded the same terminal metabolites, 4 and 5. These results are partially in agreement with the ones published for the biotransformation of 1 and 6-methylcoumarin by Colletotrichum capsici, which catalyzed the reductive pathway, although no hydroxylation processes occurred (Kumari et al. 2004). On the contrary, the hydroxy derivatives of coumarin on positions 3, 4 and 6 were not metabolized by the fungal system. This behaviour may be rationalized by considering that free phenolic group may exert certain toxicity over the fungus, in comparison with the O-methylated derivatives as previously reported (Bisogno et al. 2007). On the other hand, phenolic substrates might inhibit enoate reductase type enzymes as it is reported for the OYE (Matthews & Massey 1975). In addition, the C3eC4 double bond of 8 was reduced into 9 and, in further independent steps, 9 was hydroxylated at the aromatic ring yielding 10 and demethylated into 4 (Fig 4). This couple of processes is clearly mediated by P450 enzymes (Roberts et al. 2002). O-dealkylations mediated by A. niger have already been published on five derivatives of 4-methylumbelliferone, 7-methoxy, 7-ethoxy, 7-butoxy, 7-O-benzyloxy and 7-acetoxy (Kumar et al. 1998). According to Sariaslani and Rosazza, biotransformation of 7-ethoxycoumarin by Streptomyces griseus resulted in the accumulation of 7-hydroxycoumarin and 7-hydroxy-6-methoxycoumarin, involving a series of reactions that includes O-deethylation and 6-hydroxylation to form a 6,7-dihydroxycoumarin, and subsequent O-methylation (Sariaslani & Rosazza 1983). The fact that a well-known OYE enzyme inhibitor, p-hydroxybenzaldehyde, did not inhibit the C3eC4 double bond hydrogenation, revealed that this step might be catalyzed by an enzyme not belonging to the OYE family described for bacteria and for Saccharomyces cerevisiae towards the same substrate or, by isoenzymes which are p-hydroxybenzaldehyde non-sensitive. The pesticide synergist piperonyl butoxide, which was reported as a potent P450 inhibitor in filamentous fungi (Hiratsuka et al. 2001; Sato et al. 2002), allowed us to corroborate that the hydroxylation was mediated by this kind of enzymes since different concentrations of it prevented the appearance of the hydroxylated metabolite 4. Other evidence in favour to this postulate is that hydroxylation is inhibited in the lowest
oxygen concentration assay because this kind of reactions needs oxygen as oxidant. The other two analyzed Aspergillus strains demonstrated to have similar but no identical metabolism pathways for 1. Like A. niger, A. ochraceus carried out the reduction of the C3eC4 double bond to yield 2 but there were no further detectable transformation products. Meanwhile, A. flavus did not perform double bond reductions, and only produced oxygenated metabolites, mainly 3. In summary, this paper is another contribution to the understanding of the ability of some members of the Aspergillus genus to metabolize xenobiotic substrates, particularly coumarins. It proved that A. ochraceus is able to reduce the C3eC4 double bond of coumarin and that A. flavus showed monooxygenase activity that hydroxylates selectively the position 5 of the same substrate. In A. niger two divergent paths take place from the generation of dihydrocoumarin: (a) the lactone moiety opening and further reduction of the carboxylic acid furnishing the primary alcohol 2-(3-hydroxypropyl)phenol and, the hydroxylation on the aromatic ring, herein selectively on the position 6. It was shown that the latter species lacks the ability to metabolize the tested hydroxycoumarin analogues and, that among the methoxylated derivatives, only 6-methoxycoumarin was reduced into its 3,4-dihydroderivative. This was further hydroxylated on the aromatic ring and demethylated, most likely by the action of P450-type enzymes. This knowledge would be also applicable for the biocatalytic preparation of interesting coumarin derivatives.
Acknowledgements This work was supported by grants from Universidad Nacional de San Luis 7301; PIP6228 Consejo Nacional de Investigacnicas (CONICET) and PICT352 ciones Cientıficas y Te n Cientıfica y Tecnolo gica. Agencia Nacional de Promocio C.A.P. is a doctoral CONICET fellow. A.A.O is a postdoctoral CONICET fellow. C.E.T and M.K.S are members of the Research nica Ferrari and Dr Hugo Career of CONICET. We thank Lic Mo Lancelle for the technical assistance.
references
Adfa M, Yoshimura T, Komura K, Koketsu M, 2010. Antitermite activities of coumarin derivatives and scopoletin from Protium javanicum Burm. f. Journal of Chemical Ecology 36: 720e726. Bisogno F, Mascotti L, Sanchez C, Garibotto F, Giannini F, Kurina-Sanz M, Enriz RD, 2007. Structureeantifungal activity relationship of related cinnamic acid derivatives. Journal of Agricultural and Food Chemistry 55: 10635e10640. Bocks S, 1967. Fungal metabolism: the transformations of coumarin, o-coumaric acid and trans-cinnamic acid by Aspergillus niger. Phytochemistry 6: 127e130. € ser K, Henning-Wenk H, Schwab W, 2006. Biocatalytic proHa duction of dihydrocoumarin from coumarin by Saccharomyces cerevisiae. Journal of Agricultural and Food Chemistry 54: 6236e6240. Hiratsuka N, Wariishi H, Tanaka H, 2001. Degradation of diphenyl ether herbicides by the lignin-degrading basidomycetes Coriolus versicolor. Applied Microbiology and Biotechnology 57: 563e571.
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Kosuge T, Conn EE, 1962. The metabolism of aromatic compounds in higher plants. Journal of Biological Chemistry 237: 1653e1656. Kumar KA, Vijay Kumarb BS, Laxminarayanab B, Ananthanarayanana S, 1998. Biotransformations of 4-methyl umbelliferone derivatives. Fungal mediated O-dealkylations. Studies in Surface Science and Catalysis 113: 541e546. Kumari GNK, Ganesh MR, Anitha R, Aravind S, 2004. Complete reduction of H-pyran-2-one moiety of coumarin and 6-methylcoumarin by Colletotrichum capsici. Zeitschrift € r Naturforschung C 59: 405e407. fu Lah L, Krasevec N, Trontelj P, Komel R, 2008. High diversity and complex evolution of fungal cytochrome P450 reductase: cytochrome P450 systems. Fungal Genetics and Biology 45: 446e458. Levy CC, Frost P, 1966. The metabolism of coumarin by microorganism. The Journal of Biological Chemistry 241: 997e1003. Li W, Schuler MA, Berenbaum MR, 2003. Diversifications of furanocoumarin-metabolizing cytochrome P450 monooxygenases in two papilionids: specificity and substrate encounter rate. Proceedings of the National Academy of Sciences of the United States of America 100: 14593e14598. Matsuzaki F, Wariishi H, 2004. Functional diversity of cytochrome P450s of the white-rot fungus Phanerochaete chrysosporium. Biochemical and Biophysical Research Communications 324: 387e393. Matthews RG, Massey V, 1975. Identification of p-hydroxybenzaldehyde as the ligand in the green of Old Yellow Enzyme. The Journal of Biological Chemistry 250: 9294e9298.
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Roberts GA, Grogan G, Greter A, Flitsch SL, Turner NJ, 2002. Identification of a new class of cytochrome P450 from a Rhodococcus sp. Journal of Bacteriology 184: 3898e3908. Sariaslani FS, Rosazza JP, 1983. Novel biotransformations of 7-ethoxycoumarin by Streptomyces griseus. Applied and Environmental Microbiology 46: 468e474. Sato A, Watanabe T, Watanabe Y, Harazono K, Fukatsu T, 2002. Screening for basidiomycetous fungi capable of degrading 2,7-dichlorodibenzo-p-dioxin. FEMS Microbiology Letters 213: 213e217. Subramanian V, Yadav JS, 2008. Regulation and heterologous expression of P450 enzyme system components of the white rot fungus Phanerochaete chrysosporium. Enzyme and Microbial Technology 43: 141e229. Urlacher VB, Eiben S, 2006. Cytochrome P450 monooxygenases: perspectives for synthetic application. Trends in Biotechnology 24: 324e330. Vassallo JD, Hicks SM, Daston GP, Lehman-McKeeman LD, 2004. Metabolic detoxification determines species differences in coumarin-induced hepatotoxicity. Toxicological Sciences 80: 249e257. Williams RE, Bruce NC, 2002. New uses for an old enzyme e the Old Yellow Enzyme family of flavoenzymes. Microbiology 148: 1607e1614. Wright L, Scott E, Gorman S, 1983. The sensitivity of mycelium, arthrospores and microconidia of Trichophyton mentagrophytes to imidazoles determined by in vitro tests. Journal of Antimicrobial Chemotherapy 12: 317e323.
journal homepage: www.elsevier.com/locate/funbio
Coumarin metabolic routes in Aspergillus spp. Celeste AGUIRRE-PRANZONI, Alejandro A. ORDEN, Fabricio R. BISOGNO, Carlos E. ARDANAZ, Carlos E. TONN, Marcela KURINA-SANZ* INTEQUI-CONICET, Facultad de Quımica, Bioquımica y Farmacia, Universidad Nacional de San Luis, Chacabuco y Pedernera, CP 5700 San Luis, Argentina
article info
abstract
Article history:
Coumarin metabolism by several Aspergillus strains was studied. Aspergillus ochraceus and
Received 29 June 2010
Aspergillus niger carried out the reduction of the C3eC4 double bond to yield dihydrocou-
Received in revised form
marin in 24 h. Meanwhile, the first strain did not transform dihydrocoumarin after 7 d,
16 November 2010
A. niger demonstrated to have two divergent catabolic pathways: (a) the lactone
Accepted 19 December 2010
moiety opening and further reduction of the carboxylic acid furnishing the primary alcohol
Available online 29 December 2010
2-(3-hydroxypropyl)phenol and, (b) the hydroxylation of the aromatic ring of dihydrocou-
Corresponding Editor: Steven Harris
marin at a specific position to give 6-hydroxy-3,4-dihydrochromen-2-one. Aspergillus flavus did not perform double bond reductions, and only produced oxygenated metabolites,
Keywords:
mainly 5-hydroxycoumarin. Enzyme-specific inhibitors and a coumarin analogous were
Aspergillus
useful to confirm the A. niger catabolic route.
Coumarin
ª 2010 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Dihydrocoumarin Hydroxycoumarin Metabolism
Introduction In nature, coumarin (1) and its derivatives occur in a huge number of living organisms. A possible explanation for such a vast distribution among plants is the ability of 1 to dissuade herbivores from eating them (Adfa et al. 2010). It has been proposed that toxicity and bitter taste are the main reasons for deterrence. In terms of coevolution, herbivores had to evolve in order to circumvent this situation by expressing several metabolic pathways to detoxify xenobiotic substances (Li et al. 2003). On the other hand, a similar scenario becomes evident between plants and fungi. It has been reported that when plants are attacked by different fungal strains, their metabolite profile is drastically perturbed, displaying the induction of defense metabolic routes. Likewise, fungi express several detoxification strategies, being the cytochrome P450 usually involved (Subramanian & Yadav 2008).
Concerning 1 metabolism, several studies have been carried out in mammals such as mice, rats and humans. Cytochrome P450 monooxygenases (P450s) are the enzymes responsible for the two main oxygenations reported: the epoxidation of the double bond C3eC4 to yield coumarin 3,4-epoxide and the hydroxylations at the aromatic ring, with preference in C7. The epoxide may be further conjugated with glutathione or be rearranged to o-hydroxyphenylacetaldehyde which is subsequently oxidized to o-hydroxyphenylacetic acid or reduced to o-hydroxyphenylethanol by a NADþ aldehyde dehydrogenase (Vassallo et al. 2004). The reactions carried out by P450s in the presence of oxygen, the cofactor NAD(P)H and a corresponding electron transfer system are diverse. These reactions include epoxidation of CeC double bonds and aromatic and sp3 hybridized C-atoms, hydroxylation. P450 enzymes also catalyze oxidative phenol couplings and dealkylation reactions such as alkyl-coumarins dealkylations (Urlacher & Eiben 2006).
* Corresponding author. Tel.: þ54 2652 423789x253. E-mail address: [email protected] 1878-6146/$ e see front matter ª 2010 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.funbio.2010.12.009
246
Levy and Frost suggested that Arthrobacter species metabolized 1 by a sequence of reactions encompassing the enzymatic hydrolysis of the lactone moiety to give o-coumaric acid, and a further double bond reduction to yield melilotic acid (6) mediated by a NADH enzyme (Levy & Frost 1966). On the other hand, Kosuge and Conn suggested that 1 is first hydrogenated to dihydrocoumarin (2) and then hydrolyzed to 6 by white sweet clover (Kosuge & Conn 1962). P450s of the white-rot fungus Phanerochaete chrysosporium hydroxylate 1 at four different positions to form 5-hydroxycoumarin (3), 6-hydroxycoumarin, 7-hydroxycoumarin and 8-hydroxycoumarin (Matsuzaki & Wariishi 2004). On the other hand, 2 is obtained by biotransformation of 1 by several Sac€ ser et al. 2006). These authors charomyces cerevisiae strains (Ha demonstrated that 6 was the biotransformation product which was further cyclized into 2 by distillation in acid media, suggesting that coumarin lactone ring is opened into o-coumaric acid and then a highly specific NADH:o-coumaric acid oxidoreductase, resembling the one of Arthrobacter spp., catalyzes the C3eC4 double bond hydrogenation. It is well known that filamentous fungi have multiple P450 enzymes. Among them, Aspergillus genus showed the largest P450 family, leading the list Aspergillus oryzae and Aspergillus niger which possess 155 and 150 different P450 enzymes, respectively (Lah et al. 2008). This fact supports the idea of an efficient xenobiotic metabolism by these ascomycetes. The biotransformation of 1 by A. niger was conducted more than 40 y ago by means of basic chromatographic techniques. As it was reported the main metabolite was 6 due to the opening of lactone 2. Also, traces of a hydroxylated compound, probably in C3, were identified (Bocks 1967). In the present work we report the metabolism of 1 and derivatives by several Aspergillus spp., especially A. niger. Time course experiments were thoroughly analyzed by GCeMS and NMR. Final products and intermediates were isolated and characterized. Parameters such as oxygen availability, growing and resting cell conditions and their influence in the course of A. niger metabolic routes were studied. In order to determine what enzymes might be involved in the metabolic pathways, specific inhibition assays towards Old Yellow Enzymes (enoate reductases) and P450 monooxygenases were performed in whole cell systems.
C. Aguirre-Pranzoni et al.
Chemicals The commercial chemical reagents, coumarin (1), 3-hydroxy-2Hchromen-2-one, 4-hydroxy-2H-chromen-2-one, 6-hydroxy-2Hchromen-2-one, cyclohex-2-enone, 3,4-dihydrochromen-2-one (2), p-hydroxybenzaldehyde and piperonyl butoxide, (5-[2-(2butoxyethoxy)ethoxymethyl]-6-propyl-1,3-benzodioxole) were purchased from SigmaeAldrich Argentina. Solvents were distilled and dried prior to use.
Preparation of methoxy-2H-chromen-2-one derivatives
Materials and methods
Each of the commercial reagents: 3-hydroxy-2H-chromen-2one, 4-hydroxy-2H-chromen-2-one and 6-hydroxy-2H-chromen-2-one (200 mg, 1.23 mmol) was dissolved in 20 ml of acetone, 596 mg (4.16 mmol) of K2CO3 and 500 ml (5.29 mmol) of Me2SO4 was added, and the solutions were heated to reflux. After reaction completion (3 h) the reaction mixtures were filtered and rinsed with ethyl acetate (EtOAc). Purification was carried out by column chromatography (CC) on Sigel with n-hexane:EtOAc (7:3) as solvent. 3-methoxy-2H-chromen-2-one: yield 164 mg (75 %) 1H NMR (200 MHz, CDCl3): d 4.00 (3-H, s, H3CO), 5.70 (1-H, s, H4), 7.27 (1H, m, H6), 7.31 (1-H, m, H5) 7.54 (1-H, d, J:7.20, H7), 7.80 (1-H, d, J:7.20, H8). 13C NMR (50.2 MHz, CDCl3): d 198.80 (C2); 166.00 (C3); 90.07 (C4); 116.72 (C5); 123.86 (C6); 132.36 (C7); 122.95 (C8); 153.26 (C9); 162.80 (C10); 56.32 (H3CO). MS: m/z values (% relative abundance) Mþ. 176 (50.02), 175 (21.01), 148 (100.00), 133 (48.00), 119 (18.03), 105 (35.10), 89 (61.98), 77 (42.97). 4-methoxy-2H-chromen-2-one: yield 175 mg (81 %). 1H NMR (200 MHz, CDCl3): d 4.02 (3-H, s, H3CO), 5.70 (1-H, s, H3), 7.27 (1-H, m, H6), 7.31 (1-H, m, H5) 7.54 (1-H, d, J:7.20, H7), 7.80 (1-H, d, J:7.20, H8). 13C NMR (50.2 MHz, CDCl3): d162.00 (C2); 90.09 (C3); 166.60 (C4); 116.74 (C5); 123.87 (C6); 132.35 (C7); 122.96 (C8); 153.20 (C9); 115.60 (C10); 56.33 (H3CO). MS: m/z values (% relative abundance) MD. 176 (53.07), 148 (100.00), 133 (47.98), 105 (33.13), 91 (20.60), 89 (57.69), 77 (39.30). 6-methoxy-2H-chromen-2-one (8): yield 92 mg (42 %). 1H NMR (200 MHz, CDCl3): d 3.85 (3-H, s, H3CO), 6.42 (1-H, d, J:10.00 H3), 6.91 (1-H, d, J:2.80, H5), 7.10 (1-H, dd, J:9.10e2.80, H7), 7.27 (1-H, dd, J:9.10e2.80, H8), 7.65 (1-H, d, J:10, H4). 13C NMR (50.2 MHz, CDCl3): d 160.90 (C2); 117.09 (C3), 143.16 (C4); 110.00 (C5); 156.10 (C6); 119.42 (C7); 117.88 (C8); 148.40 (C9); 119.10 (C10); 55.81 (H3CO). MS: m/z values (% relative abundance) MD. 176 (100.00), 161 (34.32), 148 (70.20), 133 (68.49), 105 (46.46), 77 (42.53).
Microorganisms
Analytical methods
Fungal strains were purchased from different collections: American Type Culture Collection (Aspergillus niger ATCC 11394; A. niger ATCC 16404), Instituto Malbran collection (Aspergillus terreus INM 031783), Facultad de Ciencias Exactas-Universidad de Buenos Aires (Aspergillus flavus UBA 294), Departamento de Microbiologıa e Inmunologıa-Universidad Nacional de Rio Cuarto (Aspergillus ochraceus RC 33; Aspergillus fumigatus RC 78). Fungi were maintained on potato dextrose agar (PDA) slants. Spore suspensions were obtained according to reported procedures and adjusted to 107 spores with colony forming ability per millilitre in phosphate buffered saline (PBS) with added Tween-20 (5 ml/ ml) to inoculate the liquid media (Wright et al. 1983).
1
H NMR and 13C NMR spectra were obtained on Bruker AC-200 spectrometer (200 MHz and 50.2 MHz, respectively) in Cl3CD, chemical shifts in ppm are relative to tetramethylsilane (TMS), coupling constants (J ) in Hz. Analytical TLC was performed on Merck precoated silica gel 60 F254 plates. Solvent for TLC were n-hexane:EtOAc mixtures. Column chromatography was carried out on Merck silica gel 60 (230e400 mesh). Gas chromatography-flame ionization detector (GC-FID) analysis was performed in a PerkineElmer Clarus 500 instrument equipped with an Elite 5-column (30 m, 0.25 mm ID, 0.25 mm df) (Dt ¼ 10 C/min), T2 ¼ 320 C for 5 min, T1 ¼ 180 C (Dt ¼ 30 C/min) T3 ¼ 350 C, injector T ¼ 240 C; carrier gas: N2
Coumarin metabolic routes in Aspergillus spp.
35 cm/seg, splitless; FID T ¼ 350 C. Gas chromatographyemass spectrometry (GCeMS) analysis was performed with a GCQ-plus Finnigan Mat equipped with Restek 5-MS column 5 % diphenyl95 % dimethylpolisiloxane (30 m, 0.25 mm ID, 0.25 mm df). T1 ¼ 100 C for 3 min, (15 C/min), T2 ¼ 300 C for 3 min; carrier gas: He 40 cm/seg. The instrument was run in EI mode at energy of 70 eV (tune setting, trap-offset between 10 and 20 v).
Biotransformation procedures Growing cells biotranformation A two-step process was conducted. Spore suspensions from the different Aspergillus strains were incubated in liquid Czapeck medium (sucrose 30 g, yeast extract 5.0 g, KCl 0.5 g, NaNO3 2.0 g, K2HPO4 1.0 g, FeSO4 0.01 g, Mg2SO4.7H2O 0.5 g, distilled water to 1000 ml, pH 5) at 28 C, on rotatory shaker at 180 rpm. Forty-eight-hour-old cultures (5 ml) were subcultured in 125 ml baffled Erlenmeyer flasks containing 30 ml of fresh culture medium in a ten pellets per batch relation. Substrates (10 mg/batch), dissolved in 100 ml of dimethylsulfoxide (DMSO), were added to the cultures 24 h later. Biotransformation progress was monitored by withdrawing 500 ml samples every 24 h and extracted with ethyl acetate (500 ml). Aliquots of the organic layers were analyzed by GC-FID (1 ml) and TLC. After 7 d of incubation, the fermentation broth was filtered and the filtrate was extracted with EtOAc (4 50 ml), dried over anh. Na2SO4 and evaporated under reduced pressure. Blank assays without substrates and without fungi were carried out in parallel. Experiments were performed in triplicate.
Resting cells biotransformation Fungal pellets were harvested from 48 h old cultures, washed with 0.1 M pH 6 phosphate buffer and suspended in 30 ml of fresh buffer. Incubation, substrate addition and work up procedures were carried out as described above.
Time course biotransformation The experiments were performed with growing cell as described above, by collecting 500 ml of media samples from triplicate assays each 24 h. Aqueous media were extracted with EtOAc and evaporated under vacuum. Dried samples were resuspended in 500 ml of Cl2CH2 and analyzed by GCeMS. In order to isolate biotransformation products, several repetitions of the small-scale experiments were carried out. The crude extracts or the isolated metabolites were analyzed by GCeMS, 1H NMR, 13C NMR. 3,4-dihydrochromen-2-one (2): 1H NMR (200 MHz, CDCl3):d 2.71 (2-H, t, J:6.20, H3), 3.01 (2-H, t, J:6.20, H4), 6.86 (1-H, m, H6), 6.89 (1-H, d, J:7.50, H8), 7.10 (1-H, d, J:7.75, H5), 7.16 (1-H, m, H7). 13C NMR (50.2 MHz, CDCl3): d 175.80 (C2), 35.23 (C3), 24.57 (C4), 130.64 (C5), 120.89 (C6), 120.89 (C7), 117.33 (C8), 153.55 (C9), 127.48 (C10). MS: m/z values (% relative abundance) 149 (3.5), Mþ. 148 (27.20), 121 (6.20), 120 (67.80), 92 (11.30), 91 (100.00), 78 (8.90), 77 (13.90), 50 (4.50). 5-hydroxy-2H-chromen-2-one (3): 1H NMR (200 MHz, CDCl3):d 6.19 (1-H, d, J:12.00, H3), 8.04 (1-H, d, J:12.00, H4), 6.66 (1-H, d, J:8.75, H6), 7.22 (1-H, t, J:8.75 H7); 6.69 (1-H, d, J:8.75, H8). 13C NMR (50.2 MHz, CDCl3): d 161.20 (C2), 107.17 (C3), 139.06 (C4), 154.89 (C5), 113.32 (C6), 131.97 (C7), 109.81 (C8),
247
154.57 (C9), 108.61 (C10). MS: m/z values (% relative abundance) Mþ. 162 (39.4), 135 (6.3), 134 (100), 131 (4.6), 121 (4.5), 115 (6.9), 106 (6.0), 95 (5.1), 92 (4.5), 91 (7.3), 79 (8.4), 78 (53.6), 77 (26.1), 63 (8.3), 62 (7.7), 52 (4.6), 51 (18.4), 50 (15.4). 6-hydroxy-3,4-dihydrochromen-2-one (4): 1H NMR (200 MHz, CDCl3): d 2.76 (2-H, dd, J:6.80-5.60, H3), 2.94 (2-H, dd, J:6.80-5.60, H4), 6.67 (1-H, d, J:3.20, H5), 6.71 (1-H, d, J:8.80, H7), 6.93 (1-H, d, J:8.80, H8). 13C NMR (50.2 MHz, CDCl3): d 168.85 (C2), 29.12 (C3), 23.86 (C4), 114.45 (C5), 145.99 (C6), 114.80 (C7), 117.86 (C8), 152.07 (C9), 123.84 (C10). MS: m/z values (% relative abundance) 165 (7.2), MD. 164 (56.9), 136 (100.0), 135 (13.9), 122 (3.8), 121 (5.4), 107 (25.6), 94 (14.6), 91 (4.6), 79 (16.9), 77 (17.7), 66 (10.7), 65 (7.9). 2-(3-hydroxypropyl)phenol (5): 1H NMR (200 MHz, CDCl3): d 1.92 (2-H, m, H20 ), 3.01 (2-H, t, J:7.16, H10 ), 4.11 (2-H, t, J:6.35, H30 ), 6.67 (1-H, d, J:7.99, H6), 6.83 (1-H, t, J:7.99, H4), 7.04 (1-H, d, J:7.75, H3), 7.10 (1-H, t, J:7.50, H5). 13C NMR (50.2 MHz, CDCl3):d 154.31 (C1), 127.11 (C2), 130.31 (C3), 120.83 (C4), 127.39 (C5), 115.24 (C6), 26.10 (C10 ), 28.54 (C20 ), 64.79 (C30 ). MS: m/z values (% relative abundance) Mþ. 152 (3.90), 135 (4.00), 134 (30.50), 133 (30.50 (23.90), 103 (12.40), 91 (100.00), 79 (19.80), 78 (20.20), 77 (62.50), 65 (16.90), 119 (27.80), 115 (29.00), 105 (23.90)). 3-(2-hydroxyphenyl)propanoic acid (melilotic acid; 6): 1H NMR (200 MHz, CDCl3): d 2.77 (2-H, t, J:6.71, H2), 2.93 (2-H, t, J:6.71, H3), 6.83 (1H, d, J:7.41, H6), 7.11 (1-H, t, J:7.41, H8), 7.11 (1-H, d, J:7.41, H9), 7.12 (1-H d:7.41, H7). 13C NMR (50.2 MHz, CDCl3): d 179.32 (C1), 34.40 (C2), 24.60 (C3), 126.75 (C4), 153.92 (C5), 116.64 (C6), 128.06 (C7), 121.52 (C8), 130.52 (C9). MS: m/z values (% relative abundance) Mþ. 166 (0.9), 149 (3.6), 148 (15.3), 147 (2.2), 121 (5.1), 120 (51.1), 119 (18.4), 92 (11.6), 91 (100.0), 77 (17.5), 65 (11.2). 6-methoxy-3,4-dihydrochromen-2-one (9): MS: m/z values (% relative abundance) 179 (12.8), Mþ. 178 (49.1), 151 (6.4), 150 (70.6), 137 (1.8), 136 (19.6), 135 (100.0), 108 (19.3), 107 (20.9), 91 (2.9), 79 (30.3), 78 (17.3), 77 (53.3). Hydroxy derivative of 6-methoxy-3,4-dihydrochromen2-one (10): MS: m/z values (% relative abundance) 195 (9.9), Mþ. 194 (66.9), 166 (82.5), 152 (100.0), 151 (35.5), 136 (20.4), 124 (48.3), 123 (36.4), 109 (25.9), 95 (34.4), 77 (21.7).
Enzyme inhibition assays The experiments were performed with resting cell systems consisting of 30 ml phosphate buffer (pH 6) inoculated with a centrifugated 24 h culture of Saccharomyces cerevisiae or fungal pellets harvested from 48 h old Aspergillus niger cultures, with the addition of different concentrations of the inhibitors, p-hydroxybenzaldehyde or piperonyl butoxide, ranged between 1 and 10 mM. Three hours later the corresponding substrate, cyclohex-2-enone or 1, was added at final concentrations of 0.7 mM. Incubations were carried out under the above described conditions. Samples were withdrawn every 24 h until 164 h.
Results Coumarin metabolism by several Aspergillus strains A 7-d-biotransformation screening with growing cells was carried out. Samples were analyzed by GC-FID and are expressed
248
as percentage of biotransformed substrate (Fig 1). As depicted in the figure, Aspergillus niger, Aspergillus flavus and Aspergillus ochraceus, resulted the most active species whereas the substrate was recovered untouched from the experiments performed with Aspergillus fumigatus and Aspergillus terreus. Since the metabolite profiles for A. niger, A. flavus and A. ochraceus seemed to be quite different, 7-d-biotransformation experiments were performed to isolate and identify the main products (Fig 2), either with growing or resting cells. Surprisingly, when A. ochraceus resting cells were used, 1 was gradually consumed until completion in the fifth day, without the appearance of biotransformation products. This fact, together with the observed variation in the pellets morphology and a slight increasing in the biomass amount, led us to think that 1 was probably being used as carbon source by the fungus under these starving conditions. On the contrary, with growing cells, 1 was transformed into a sole metabolite, 2, in 80 % conversion rate at day 7. In the 1H NMR spectrum, the main characteristic was the disappearance of the typical doublets at 6.41 and 7.71 ppm of the hydrogens on the double bond C3eC4 of 1 and the appearance of two new triplets integrating for two hydrogens each at higher fields, 2.71 and 3.01 ppm, corresponding to H3 and H4, respectively. The 13C NMR spectrum shows two signals corresponding to the two new saturated carbons at 24.57 and 35.23 for C4 and C3, respectively. These data were confirmed by homo- and heteronuclear bidimensional NMR experiments. The MS spectrum showed an increment of two mass units for the molecular ion of the new compound (m/z 148 Mþ) respect to molecular ion of 1 (m/z 146 Mþ). Aspergillus flavus also showed a different metabolism, being resting cells more efficient than growing cells to convert 1 into a more polar compound in 92 % conversion rate. The main biotransformation product of the resting cell experiment was purified by CC and identified by GCeMS and NMR as 5-hydroxycoumarin (3). In the 1H NMR spectrum it can be seen as a triplet at 7.57 ppm (J ¼ 8.75 Hz) corresponding to H7, and a pair of doublets with the same coupling constants at 6.99 and 6.93 ppm of H6 and H8, respectively. These data were confirmed by bidimensional homo- and heteronuclear experiments.
C. Aguirre-Pranzoni et al.
In the experiments with growing cells of A. niger ATCC 11394, it was possible to detect that 1 was entirely converted into 2 before 48 h of biotransformation. However, at 7 d, two different metabolites were isolated: 2-(3-hydroxypropyl)phenol (5) and 6-hydroxy-3,4-dihydrochromen-2-one (4). It was particularly noticeable in the 1H NMR spectrum of 5 the occurrence of a multiplet that integrates for two hydrogens at 1.92 ppm (H20 ), together with the presence of two triplets at 3.01 ppm (J ¼ 7.16 Hz) and 4.11 ppm (J ¼ 6.35 Hz) corresponding to H10 and H30 , respectively. No modifications were evident on the aromatic ring and there was no evidence of the existence of the lactone moiety. For the metabolite, 4, it was easy to assume that it was a derivative of 2 due to the presence of two triplets between 2.70 and 3.00 ppm corresponding to H10 and H20 , and the absence of signals of the hydrogens on the double bond. The assignment of the hydroxylation position on the aromatic ring was achieved by bidimensional homo- and heteronuclear NMR spectroscopy. The signals at 6.67 ppm (d, J:2.80 Hz), 6.71 ppm (dd, J:8.80 and 2.80 Hz) and 6.92 ppm (d, J:8.80 Hz) were attributed to hydrogens 5, 7 and 8, respectively, due to a long distance correlation observed between H5 and H3 in the COSY-45 spectrum and H3 and C5 (114.45 ppm) in the HMBC.
Time course biotransformation experiments by Aspergillus niger ATCC 11394 Time course experiments allowed us detecting compounds derived from the opening of the dihydrocoumarin lactone ring and the further reduction of the carboxylic acid into the corresponding aldehyde, confirm by the presence of a singlet
OH
2
O
O
5
A. ochraceus
6
10
3
4
O
O
O
O
A. flavus
3 2
7 8
9
1
O
O 1
A. niger HO
2
O
O 3 4
1'
2' 3'
5 6
Fig 1 e Screening of coumarin (1) biotransformation by different Aspergillus species in an optimized two-step process with growing cells carried out for 168 h. Remanent substrate was determined by GC-FID and expressed as percentage of converted substrate.
2
4
1
OH
OH
5 Fig 2 e Biotransformation of coumarin (1) by selected Aspergillus species. Optimized two-step process with growing cells were carried out for 168 h. Metabolites were isolated and determined by MS and NMR.
Coumarin metabolic routes in Aspergillus spp.
249
at 9.87 ppm in the 1H NMR spectrum of the 72-h-extract. This was in agreement with the occurrence of a metabolite of Mþ. 150 in the GCeMS analysis, which was assigned as 3-(2hydroxyphenyl)propanal (7). Compound 7 disappeared at longer incubation times (96 and 144 h), whereas 5 (Mþ. 152) resulted the main metabolite detected. In addition, the presence of the hydroxylated metabolite was noticeable only after 96 and 144 h when a peak of Mþ. 164 appeared in the GCeMS analysis (Fig 3). To corroborate this route, 2 was added as substrate. After 7 d, two metabolites were isolated, 4 and 5. In order to study the metabolic pathway for related compounds, some monohydroxylated commercial coumarins, such as 3-hydroxycoumarin, 4-hydroxycoumarin and 6-hydroxycoumarin were added as substrates to growing cells. To our surprise, the three compounds were recovered untransformed at the end of the experiments (7 d). The methoxy derivatives were further used as substrates. Only 6-methoxycoumarin (8) was biotransformed into three main metabolites with molecular ions of m/z 178 (9), m/z 194 (10) and m/z 164 (4) as depicted in Fig 4. On the contrary, the other two substrates were recovered untouched. Similar to the study carried out with 1, a time course experiment was carried out with 8. The crude extract of the biotransformation was monitored by GCeMS. We observed that at 24 h, the substrate was completely converted into 9, a metabolite of molecular ion of m/z 178, corresponding to hydrogenation on the C3eC4 double bond, in agreement with the results observed for 1. After 72 h, two new metabolites were detected. Their
1
O
O
molecular ions were m/z 164 and 194. The first is in agreement with demethylation of the intermediate 9 (m/z 178) and the latter is attributable to hydroxylation on the aromatic ring. Since structural elucidation was performed by GCeMS, it was not possible to assign the position of the new hydroxyl group. However, we can assure that the hydroxylation occurred on the aromatic ring, since no peaks corresponding to [MeH2O]þ. and/or [MeHO]þ. appeared, as expected if hydroxylation would have generated a secondary alcohol. Moreover, two peaks of m/z 166 [MeCO]þ. and m/z 152 corresponding to the loss of the ketene moiety presented important intensities in the MS spectrum of 10. Since no lactone ring opening products were detected, we can assume that electron-donating groups on the aromatic ring, such as methoxy, can either electronically modulate the lability of the intramolecular ester bond or exert detrimental interaction with the hydrolytic enzyme responsible for lactone opening. Nevertheless, further experiments with other substituents on the aromatic ring will be necessary to demonstrate this hypothesis. To compare the influence of oxygen availability in the biotransformation process, a comparative assay using different medium volumes was performed (30 ml and 60 ml in 125 ml closed flasks). All other experimental variables, such as inocula, substrate concentrations, agitation and temperature were preserved. The GCeMS analysis of the assays with higher oxygenation (minor medium volume), gave 2, 4 and 5 as the main metabolites at the end of the process. On the contrary, with lower oxygen conditions (major medium volume) 2, 4 and 6 were identified, but no further hydroxylated metabolites were detected.
Enzyme inhibitors influence A well-known inhibitor of the enoate reductases belonging to the OYE family, p-hydroxybenzaldehyde, was assayed in yeast
O
O
2 O
H3CO HO
OH O
OH
6
8
O
4
O
O
O
O
H3CO
7
OH
9
O
H3CO
HO
HO
5
OH
OH
Fig 3 e Pathway proposed for coumarin (1) metabolism by A. niger ATCC 11394, based on time course experiments with growing and resting cells, analyzed by GCeMS and NMR.
O
10
O
O
O
4
Fig 4 e Pathway proposed for 6-methoxycoumarin (8) metabolism by A. niger ATCC 11394 based on growing cell time course experiments, analyzed by GCeMS and NMR.
250
C. Aguirre-Pranzoni et al.
Table 1 e Effects of enoate reductase inhibitor ( p-hydroxybenzaldehyde) on coumarin (1) and cyclohexen-2-one biotransformation by S. cerevisiae and A. niger ATCC 11394 Biocatalytic system
Inhibitor concentration ( p-hydroxybenzaldehyde)
Substrate: coumarin (1)
Substrate: cyclohexen-2-one
S. cerevisiae
0.7 mM 1.4 mM 2.0 mM 2.5 mM 3.0 mM
2 2 e e e
e e e e e
A. niger
2.0 mM 4.0 mM 6.0 mM 8.0 mM 10.0 mM
2 2 2 2 2
Cyclohexanoneecyclohexanol Cyclohexanoneecyclohexanol Cyclohexanoneecyclohexanol Cyclohexanoneecyclohexanol Cyclohexanoneecyclohexanol
Biotransformation productsa
a Determined by GCeMS.
(Williams & Bruce 2002). This inhibitor was tested in our system in order to find out if the enzyme responsible for the first reaction in the coumarin metabolic pathway belongs to the OYE family. p-Hydroxybenzaldehyde was added to resting cells 3 h prior to the addition of 1 in five different concentrations. As in all the other processes, the bio-reactions were checked every 24 h until day 7. As a positive control, the biotransformation of cyclohex-2-enone with and without the addition of the inhibitor was performed. Additional control assays, involving the use of Saccharomyces cerevisiae towards both substrates were performed demonstrating that concentrations of p-hydroxybenzaldehyde up to 2 mM inhibited the reduction of both the coumarin C3eC4 double bond and the cyclohexen-2-one (Table 1). Conversely, with Aspergillus niger cultures, no inhibitions were observed, neither when the substrate was 1 nor cyclohex-2-enone. It is worth to point out that p-hydroxybenzaldehyde was metabolized by other A. niger enzyme/s into several non-identified more polar products. Consequently, a higher inhibitor concentration was used (10 mM). In this condition, 1 was totally transformed into 2, although a reasonable quantity of the OYE inhibitor still remained in the bio-reaction medium after 48 h (Fig 5, Table 1). This suggests that the reduction of the C3eC4 double bond of coumarin derivatives in this A. niger strain might be mediated by isoenzymes which are p-hydroxybenzaldehyde non-sensitive or by other kind of reductases not belonging to the OYE family. We also developed a P450 inhibition assay working with A. niger resting cells, adding 0.7 mM of 1 and different concentrations of piperonyl butoxide. As expected, 1 was totally transformed into 2 at 24 h and the only metabolites detected at 164 h were 2 and mainly 5. When 2 was added as substrate, no changes were observed at 24 h; meanwhile at 164 h, no hydroxylated metabolites were detected. Consequently, we can assert that the occurrence of metabolite 4, in the coumarin metabolic pathway in A. niger was mediated by P450s (Table 2). In order to discard the possibility that exoenzymes like lignin and manganese peroxidases could metabolize 1, we carried out a simple assay consisting in the substrate addition (0.7 mM) to aseptically filtered culture broth after 4 d of incubation with the fungal development. Samples were taken after
24, 48 and 72 h and no transformation products were detected and 1 was recovered untouched.
Discussion Taking into account the high expression of P450 enzymes reported for the genus Aspergillus and their vast acceptance of xenobiotic substrates (Lah et al. 2008), the first step of this work was a biotransformation screening by several Aspergillus strains towards 1. Aspergillus niger, Aspergillus flavus and Aspergillus ochraceus resulted the most active species (Figs 1 and 2). In the conditions of these experiments no differences were observed in biomass rates (data not shown), although it is worth to consider that the xenobiotic substrate was always added in the stationary phase of the growing cell cultures (48 h old). Aspergillus niger performed the quantitative reduction of the C3eC4 double bond of 1 to yield 2 in 24 h. After that, two divergent metabolic pathways took place: (a) the lactone moiety opening and further reduction of the carboxylic acid, furnishing the primary alcohol 5 as the main terminal metabolite and, (b) the hydroxylation on the aromatic ring of 2 in a specific position to give 4 (Fig 3). This pathway was corroborated by the biotransformation of the main postulated
Fig 5 e Relative quantities of coumarin (1), dihydrocoumarin (2) and p-hydroxybenzaldehyde (OYE-inhibitor) in the time course inhibition assay developed with resting cells of A. niger ATCC 11394. Initial concentrations: coumarin 0.7 mM and p-hydroxybenzaldehyde 10.0 mM.
Coumarin metabolic routes in Aspergillus spp.
251
Table 2 e Effects of P450 monooxygenase inhibitor (piperonyl butoxide) on coumarin (1) and dihydrocoumarin (2) biotransformations by A. niger ATCC 11394 Substrate (0.7 mM)
Inhibitor concentration (piperonyl butoxide)
Biotransformation productsa 24 h
Coumarin (1)
1.0 mM 1.5 mM 2.0 mM 2.5 mM
2 2 2 2
Dihydrocoumarin (2)
1.0 mM 1.5 mM 2.0 mM 2.5 mM
e e e e
164 h 2 2 2 2
and and and and
5 5 5 5
5 5 5 5
a Determined by GCeMS.
intermediate 2, which yielded the same terminal metabolites, 4 and 5. These results are partially in agreement with the ones published for the biotransformation of 1 and 6-methylcoumarin by Colletotrichum capsici, which catalyzed the reductive pathway, although no hydroxylation processes occurred (Kumari et al. 2004). On the contrary, the hydroxy derivatives of coumarin on positions 3, 4 and 6 were not metabolized by the fungal system. This behaviour may be rationalized by considering that free phenolic group may exert certain toxicity over the fungus, in comparison with the O-methylated derivatives as previously reported (Bisogno et al. 2007). On the other hand, phenolic substrates might inhibit enoate reductase type enzymes as it is reported for the OYE (Matthews & Massey 1975). In addition, the C3eC4 double bond of 8 was reduced into 9 and, in further independent steps, 9 was hydroxylated at the aromatic ring yielding 10 and demethylated into 4 (Fig 4). This couple of processes is clearly mediated by P450 enzymes (Roberts et al. 2002). O-dealkylations mediated by A. niger have already been published on five derivatives of 4-methylumbelliferone, 7-methoxy, 7-ethoxy, 7-butoxy, 7-O-benzyloxy and 7-acetoxy (Kumar et al. 1998). According to Sariaslani and Rosazza, biotransformation of 7-ethoxycoumarin by Streptomyces griseus resulted in the accumulation of 7-hydroxycoumarin and 7-hydroxy-6-methoxycoumarin, involving a series of reactions that includes O-deethylation and 6-hydroxylation to form a 6,7-dihydroxycoumarin, and subsequent O-methylation (Sariaslani & Rosazza 1983). The fact that a well-known OYE enzyme inhibitor, p-hydroxybenzaldehyde, did not inhibit the C3eC4 double bond hydrogenation, revealed that this step might be catalyzed by an enzyme not belonging to the OYE family described for bacteria and for Saccharomyces cerevisiae towards the same substrate or, by isoenzymes which are p-hydroxybenzaldehyde non-sensitive. The pesticide synergist piperonyl butoxide, which was reported as a potent P450 inhibitor in filamentous fungi (Hiratsuka et al. 2001; Sato et al. 2002), allowed us to corroborate that the hydroxylation was mediated by this kind of enzymes since different concentrations of it prevented the appearance of the hydroxylated metabolite 4. Other evidence in favour to this postulate is that hydroxylation is inhibited in the lowest
oxygen concentration assay because this kind of reactions needs oxygen as oxidant. The other two analyzed Aspergillus strains demonstrated to have similar but no identical metabolism pathways for 1. Like A. niger, A. ochraceus carried out the reduction of the C3eC4 double bond to yield 2 but there were no further detectable transformation products. Meanwhile, A. flavus did not perform double bond reductions, and only produced oxygenated metabolites, mainly 3. In summary, this paper is another contribution to the understanding of the ability of some members of the Aspergillus genus to metabolize xenobiotic substrates, particularly coumarins. It proved that A. ochraceus is able to reduce the C3eC4 double bond of coumarin and that A. flavus showed monooxygenase activity that hydroxylates selectively the position 5 of the same substrate. In A. niger two divergent paths take place from the generation of dihydrocoumarin: (a) the lactone moiety opening and further reduction of the carboxylic acid furnishing the primary alcohol 2-(3-hydroxypropyl)phenol and, the hydroxylation on the aromatic ring, herein selectively on the position 6. It was shown that the latter species lacks the ability to metabolize the tested hydroxycoumarin analogues and, that among the methoxylated derivatives, only 6-methoxycoumarin was reduced into its 3,4-dihydroderivative. This was further hydroxylated on the aromatic ring and demethylated, most likely by the action of P450-type enzymes. This knowledge would be also applicable for the biocatalytic preparation of interesting coumarin derivatives.
Acknowledgements This work was supported by grants from Universidad Nacional de San Luis 7301; PIP6228 Consejo Nacional de Investigacnicas (CONICET) and PICT352 ciones Cientıficas y Te n Cientıfica y Tecnolo gica. Agencia Nacional de Promocio C.A.P. is a doctoral CONICET fellow. A.A.O is a postdoctoral CONICET fellow. C.E.T and M.K.S are members of the Research nica Ferrari and Dr Hugo Career of CONICET. We thank Lic Mo Lancelle for the technical assistance.
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