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New perylenequinone derivatives from the endophytic fungus Alternaria tenuissima SS77
Tetrahedron Letters 57 (2016) 3185–3189
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New perylenequinone derivatives from the endophytic fungus Alternaria tenuissima SS77 Fernanda O. Chagas a, Luís G. Dias b, Mônica T. Pupo a,⇑ a Departamento de Ciências Farmacêuticas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Avenida do Café, s/n, 14040-903, Ribeirão Preto, SP, Brazil b Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Avenida Bandeirantes, 3900, 14040-901, Ribeirão Preto, SP, Brazil
a r t i c l e
i n f o
Article history: Received 6 April 2016 Revised 6 June 2016 Accepted 8 June 2016 Available online 9 June 2016 Keywords: Perylenequinones Mycotoxins Phytotoxins Biogenesis Endophyte
a b s t r a c t Three new natural products, 1,4,6b,7,10-pentahydroxy-1,2,6b,7,8,12b-hexahydroperylene-3,9-dione (1), 1,4,9,12a-tetrahydroxy-12-methoxy-1,2,11,12,12a,12b-hexahydroperylene-3,10-dione (2) and 1,4,9-trihydroxy-1,2-dihydroperylene-3,10-dione (3), were obtained from cultures of the endophytic fungus Alternaria tenuissima SS77. Their chemical structures were determined by combined analysis of oneand two-dimensional NMR spectra, UV and ESIHRMS data. Compound 3 is likely an intermediate in the biosynthetic pathway of some perylenequinones produced by this fungus. A new hypothesis for the biosynthesis of partially reduced perylenequinones is suggested. Ó 2016 Elsevier Ltd. All rights reserved.
Introduction Perylenequinones are secondary metabolites characterized by a conjugated aromatic pentacyclic dione; however, natural products with partially reduced perylene core have also been included in this class of compounds.1 Perylenequinone derivatives are found in fungi, plants, insects (aphids), and animals (crinoids).1–10 Different structural features may be attached to the perylenequinone backbone, making perylenequinone derivatives relatively diverse. Most reported perylenequinones have fungal origin, and the simplest naturally occurring perylenequinone known, the 4,9dihydroxyperylene-3,10-quinone (a), was isolated from the fungus Daldinia concentrica in 1956.11 Cercosporin (b) is among the first of isolated mold perylenequinones, which was reported in 1957, even though its structure was only completely determined by 1972.12–14 This compound was deeply studied mainly due its photoactivity.9,10 Cercosporin and other similar compounds, such as hypocrellin A (c) and scutiaquinone A (d) contain carbon substituents attached to perylenequinone backbone1,8–10 (Fig. S1). Another group of mold perylenequinones, in which the parent natural product 4,9-dihydroxyperylene-3,10-quinone is included, does not contain carbon substituents and usually has a partially reduced perylene.1,8 Many mycotoxins produced by Alternaria ⇑ Corresponding author. Tel.: +55 16 3315 4710; fax: +55 16 3315 4178. E-mail addresses: [email protected] (F.O. Chagas), [email protected] (L.G. Dias), [email protected] (M.T. Pupo). http://dx.doi.org/10.1016/j.tetlet.2016.06.035 0040-4039/Ó 2016 Elsevier Ltd. All rights reserved.
species, such as altertoxin II (e), stemphytriol (f), and alterlosin II (g) (Fig. S1), belong to this second group of mold perylenequinones,15 some of which have been reported as phytotoxins16,17 and others have gained importance over the years for causing food and feed contaminations, offering risks to human health due to their mutagenic effects.18–21 In plants, toxic effects of these perylenequinones may occur in the same way that of cercosporin, by generation of free radicals that damage host cells after photoactivation.9 Mutagenicity observed in bacterial and mammal cells have been attributed to various mechanisms of action.20,21 Particular structural features of different perylenequinone derivatives contribute to their photoactive and mutagenic effects, affording compounds with variable levels of bioactivity. As part of our ongoing program for prospecting natural products from endophytic microorganisms, the fungus Alternaria tenuissima SS77 has been cultured in diverse growth conditions, including mixed microbial cultivation. A. tenuissima SS77 has produced polyketides belonging to perylenequinones class, such as stemphyperylenol (h), alterperylenol (i) and altertoxin I (j) (Fig. S1), in single and mixed cultures, and was able to remodel its secondary metabolism in mixed fermentation.22 In this Letter we report the identification of further perylenequinones produced by this fungus. The high structural similarity among those compounds made it possible to undoubtedly characterize them by NMR and UV. Furthermore, we have proposed that the biosynthetic pathways for all those perylenequinones more
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likely share the same precursor in early steps, which later has the perylene core reduced and undergoes peripheral oxidations, affording the final products. Results and discussion Compound 1 was obtained as a pale yellow powder. NMR spectra of 1 were acquired in CD3OD and CDCl3, and the combined analyses allowed the structural determination. Carbons were assigned from gHSQC and gHMBC spectra (CD3OD) (Table S1). The 1H NMR, gHSQC, and gHMBC spectra (Supplementary material) contained resonances for eighteen carbons in a five fused ring structure comprising two aromatic rings with a couple of aromatic ortho coupled hydrogens each [dH 6.99 (1H, d, J = 8.6 Hz, H-5), 8.16 (1H, d, J = 8.6 Hz, H-6), 6.89 (1H, d, J = 8.6 Hz, H-11), 8.20 (1H, d, J = 8.6 Hz, H-12); dC 117.4 (C-5), 136.6 (C-6), 115.7 (C-11), 137.1 (C-12)], two chelated phenolic hydroxyls [dH 12.09 (s, 4-OH), 12.23 (s, 10-OH); dC 161.1 (C-4), 162.0 (C-10)], five non-hydrogenated aromatic carbons [dC 144.0 (C-3b), 133.3 (C-6a), 115.8 (C-9a), 146.0 (C-9b), 133.9 (C-12a)], a carbonyl carbon [dC 204.4 (C-3)], two methylene groups [dH 3.09 (1H, dd, J = 12.4 Hz, 15.6 Hz, H-2a), 3.20 (1H, dd, J = 4.5 Hz, 15.6 Hz, H-2b), 3.29 (1H, dd, J = 12.4 Hz, 15.6 Hz, H-8a), 3.01 (1H, dd, J = 4.5 Hz, 15.6 Hz, H-8b); dC 48.0 (C-2), 43.9 (C-8)], two hydroxylated methine secondary carbons [dH 4.69 (1H, m, H-1), 2.07 (sl, 1-OH), 4.88 (1H, m, H-7), 2.62 (d, J = 6.3 Hz, 7-OH); dC 68.7 (C-1), 69.5 (C-7)], a methine tertiary carbon [dH 4.42 (1H, d, J = 9.8 Hz, H-12b); dC 45.3 (C-12b)], and a hydroxylated tertiary carbon [dH 2.71 (s, 6bOH)]. Data from gHSQC and gHMBC spectra confirmed that each couple of aromatic hydrogen belongs to different phenol groups (rings A and E). The correlations showed the methylene group at position 2 was connected to hydroxylated methine secondary carbon C-1, which was linked to methine tertiary carbon C-12b, constituting part of the cyclohexenone ring B; additionally, the methylene group at position 8 was linked to the hydroxylated methine secondary carbon C-7, which together were part of ring D. The spectral data of 1 were similar to those of partially reduced perylenequinones previously isolated from A. tenuissima SS77.22 In comparison to other analogues, compound 1 possessed five hydroxyls, including two chelated phenolic ones, and no double bond in any cyclohexenone rings. The positions of hydroxyl groups were determined by gHMBC correlations. The ESIHRMS data confirmed the molecular formula of 1 as C20H16O7 by the presence of pseudo-molecular ion at m/z [M H] 367.08510 (calculated m/z 367.08233). The partially reduced perylenequinones from Alternaria spp., and related genera, are divided into two sub-groups according to their structural scaffolds. These compounds exhibit biphenyl or dihydroanthracene frameworks according to relative position of both phenol groups of their structures23,24 (Fig. 1).
Determining whether compound 1 has a biphenyl or a dihydroanthracene framework was not possible by gHMBC, since no key correlations were observed. However, the UV spectra of compounds presenting one or another scaffold are quite characteristic (Fig. 1). The main difference between UV spectra is the presence of a shoulder (sh) band at 285 nm in perylenequinones displaying dihydroanthracene framework, which is absent in perylenequinones with biphenyl framework. Besides, when compared to biphenyl perylenequinones, dihydroanthracene perylenequinones present a characteristic bathochromic shift in the 300–400 nm band. Also, dihydroanthracene perylenequinones display the maximum molar absorptivity (e) at 258 nm, followed by 214 nm, 285 (sh) and 357 nm, while biphenyl perylenequinones have the maximum e bellow 200 nm, followed by 215 nm, 260 and 342 nm, as shown in the UV spectra of stemphyperylenol (h) and alterperylenol (i) (Fig. 1). Therefore, the relative intensities between bands around 250 and 350 nm can be used to indicate the correct framework. This is the first Letter correlating perylenequinone UV absorptions with their respective frameworks. These characteristic UV spectra were observed in all isolated reduced perylenequinones from A. tenuissima SS77 (Fig. S28) and also in previously reported compounds.16,17,25–27 Thus, based on UV spectra, it was determined that compound 1 possesses a dihydroanthracene framework. Therefore, compound 1 is different from the previously reported stemphytriol (f) (Fig. S1), which possesses a biphenyl framework.26 The relative stereochemistry of 1 was established based on hydrogen coupling constants. The coupling constants of H-2a and H-12b hydrogens enabled presuming the spatial orientation of hydroxyl at C-1 and hydrogen H-12b. The coupling constants of 12.4 Hz and 9.8 Hz indicated that H-2a presents a pseudo-diaxial correlation to H-1, and H-1 presents a pseudo-diaxial correlation to H-12b, respectively. Thus, the spatial orientation of hydroxyl at C-1 and hydrogen H-12b were pseudo-equatorial and pseudo-axial, respectively. In the same way, by the coupling constants between H-7 and H-8a hydrogens (J = 12.4 Hz) that indicated a pseudo-diaxial correlation, the orientation of hydroxyl at C-7 was determined as pseudo-equatorial. Such as found in all structures of partially reduced perylenequinones, the hydroxyl at C-6b is suggested to be in pseudo-axial, likewise hydrogen H-12b, maintaining the relative planarity of the five fused ring structure. The relative configuration between C-12b and C-6b could not be determined. However, considering all isolated reduced perylenequinones, it was assumed that H-12b and OH-6b were in the same side of molecule. Then, the relative configurations of the stereogenic centers at position 1, 12b, 6b and 7 were S⁄, S⁄, R⁄ and S⁄, respectively. Compound 1 is very similar to stemphyperylenol (h) (Fig. S1), of which we have previously determined the absolute configuration of stereogenic centers as 1R, 12bR, 6bR, and 7R, by comparing experimental ECD with theoretical calculations.22 However, due
Figure 1. Basic structures of perylenequinones presenting dihydroanthracene (I) and biphenyl (II) scaffolds, and characteristic experimental UV spectra of both types of perylenequinones.
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to the tiny amount of compound 1 and required further purifications, ECD was not performed and absolute configuration was not determined.
Compound 2 was obtained as a yellow powder. The carbons were assigned from gHSQC and gHMBC spectra (Table S1). The interpretation of 1H NMR, gHSQC, and gCOSY spectra (Supplementary material) showed compound 2 possesses structural similarities to partially reduced perylenequinones: presence of two aromatic rings with a couple of aromatic ortho coupled hydrogens each [dH 7.04 (1H, d, J = 8.9 Hz, H-5), 7.84 (1H, d, J = 8.9 Hz, H-6), 7.84 (1H, d, J = 8.9 Hz, H-7), 7.11 (1H, d, J = 8.9 Hz, H-8); dC 117.6 (C-5), 132.7 (C-6), 132.7 (C-7), 119.6 (C-8)], two chelated phenolic hydroxyls [dH 12.46 (s, 4-OH), 12.42 (s, 9-OH); dC 162.4 (C-4), 161.6 (C-9)], six non-hydrogenated aromatic carbons [dC 117.3 (C-3a), 135.6 (C-3b), 123.6 (C-6a), 123.2 (C-6b), 113.9 (C-9a), 139.1 (C9b)], two carbonyl carbons [dC 202.4 (C-3), 202.4 (C-10)], two methylene groups [dH 2.91 (1H, dd, J = 12.2 Hz, 16.2 Hz, H-2a), 3.17 (1H, dd, J = 4.6 Hz, 16.2 Hz, H-2b), 3.18 (1H, dd, J = 3.5 Hz, 17.3 Hz, H-11a), 3.39 (1H, dd, J = 2.2 Hz, 17.3 Hz, H-11b); dC 47.7 (C-2), 36.7 (C-11)], a hydroxylated methine secondary carbon [dH 4.68 (1H, m, H-1), 2.83 (d, J = 6.0 Hz, 1-OH); dC 65.9 (C-1)], a methoxylated methine secondary carbon [dH 4.67 (1H, dd, J = 2.2 Hz, 3.5 Hz, H-12), 3.45 (3H, s, 12-OMe); dC 65.9 (C-12), 55.7 (12-OMe)], a methine tertiary carbon [dH 3.56 (1H, d, J = 9.6 Hz, H-12b); dC 48.0 (C-12b)], and a hydroxylated tertiary carbon [dH 1.98 (s, 12a-OH); dC 71.1 (C-12a)]. Differing from 1, compound 2 possesses only four hydroxyls. Instead, a methoxyl group (12-OMe) was attached to carbon C-12. The molecular formula of 2 was confirmed by ESIHRMS as C21H18O7 by the presence of the pseudo-molecular ion at m/z [M H] 381.10081 (calculated m/z 381.09798). The biphenyl framework of compound 2 was determined based on its UV spectrum (Fig. S28). The relative stereochemistry of 2 was established based on hydrogen coupling constants. The coupling constant between hydrogens H-1 and H-2a (J = 12.2 Hz) indicated a pseudo-diaxial correlation between them, and the orientation of hydroxyl attached to C-1 was determined as pseudo-equatorial. A pseudodiaxial correlation was also observed between hydrogens H-1and H-12b (J = 9.6 Hz), indicating H-12b presented pseudo-axial orientation. Similarly, the coupling constants of hydrogen H-11a with H-12 (J = 3.5 Hz) and hydrogen H-11b with H-12 (J = 2.2 Hz) indicated that H-12 had a pseudo-equatorial orientation. Thus, methoxyl group attached to C-12 was in a pseudo-axial orientation. Accordingly to other partially reduced perylenequinones, the hydroxyl attached to C-12a was suggested to have pseudo-axial orientation, and H-12b and 12a-OH were in opposite sides of the molecular plane. Then, the relative configurations of the stereogenic centers at position 1, 12b, 12a, and 12 were determined as S⁄, S⁄, R⁄ and R⁄, respectively. Compound 2 was believed to be a methylated derivative of stemphytriol (f) (Fig. S1) at first, but the opposite relative configuration at C-12a brought doubts about compound 2 being a natural product or, instead, a solvent derivative artifact formed during extraction and fractionation with methanol. Since O-methylation
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process is not expected to change the configuration of the center in which the hydroxyl group is attached, we considered compound 2 might be a solvent artifact. However, an epimer of stemphytriol possessing R⁄-configuration at C-12a has been isolated from an endophytic Alternaria alternate.4 Thus, compound 2 possibly would be a methylated analogue of 12a-epi-stemphytriol. Indeed, enzymes O-methyltransferases are involved in the biosynthesis of some mycotoxins, such as cercosporin and alternariol.28,29 The presence of genes encoding such enzymes in the biosynthetic gene cluster responsible for the biosynthesis of partially reduced perylenequinones could confirm compound 2 is a natural product instead a solvent artifact. Nevertheless, genetic studies on those compounds have never been performed, and the doubt remains. Compound 3 was obtained as a yellow brownish powder. Carbons were assigned from gHSQC and gHMBC spectra (Table S1). The interpretation of 1H NMR, gHSQC, and gCOSY spectra (Supplementary material) showed compound 2 possesses structural similarities to partially reduced perylenequinones and some additional features: presence of two aromatic rings with a couple of aromatic ortho coupled hydrogens each [dH 7.53 (1H, d, J = 9.4 Hz, H-5), 8.86 (1H, d, J = 9.4 Hz, H-6), 8.87 (1H, d, J = 9.4 Hz, H-7), 7.50 (1H, d, J = 9.4 Hz, H-8); dC 122.7 (C-5), 130.9 (C-6), 132.5 (C-7), 120.8 (C8)], two chelated phenolic hydroxyls [dH 13.23 (s, 4-OH), 15.20 (s, 9-OH); dC 162.6 (C-4), 168.0 (C-9)], eight non-hydrogenated aromatic carbons [dC 110.8 (C-3a), 127.7 (C-3b), 121.8 (C-6a), 129.2 (C-6b), 112.6 (C-9a), 125.5 (C-9b), 124.3 (C-12a), 134.9 (C-12b)], a carbonyl carbon [dC 201.1 (C-3)], a conjugated carbonyl carbon [dC 187.9 (C-10)], two olefinic hydrogens [dH 7.14 (1H, d, J = 10.1 Hz, H-11), 8.56 (1H, d, J = 10.1 Hz, H-12); dC 129.3 (C-11), 136.9 (C-12)], a methylene group [dH 3.27 (1H, dd, J = 3.9 Hz, 16.9 Hz, H-2a), 3.42 (1H, dd, J = 2.6 Hz, 16.9 Hz, H-2b); dC 44.8 (C2)], and a hydroxylated methine secondary carbon [dH 6.17 (1H, dd, J = 2.6 Hz, 3.9 Hz, H-1); dC 63.9 (C-1)]. Data from gHSQC and gHMBC spectra confirmed that each couple of aromatic hydrogens belongs to different phenol groups (rings A and D) and the olefinic hydrogens are part of a conjugated carbonyl system (ring E). These NMR data strongly suggested compound 3 was a perylenequinone. However, all mentioned hydrogens were deshielded compared to those of similar compounds, such as 1 and 2, and other partially reduced perylenequinones isolated from A. tenussima,22 indicating compound 3 exhibited a particular structural feature. Besides, compound 3 possessed two additional olefinic/aromatic carbons (C-12a, C12b). The methylene group at position 2 was connected to the hydroxylated methine secondary carbon C-1, constituting part of the cyclohexenone ring B. A hydroxyl attached to C-1, which is common in perylenequinones, was partially responsible for the carbon chemical shift of C-1; however, the hydrogen chemical shift of H-1 was more deshielded than expected, since the chemical shift of the referred hydrogen in other perylenequinones is about dH 4.50–4.80. The gHMBC correlation from methylene hydrogen H2b to the carbonyl C-3 confirmed their vicinity, and also the ring B features, whereas the correlation from the hydrogen H-2b to the carbon C-12b indicated the presence of a double bond connecting rings B and E. Thus, differing from other perylenequinones isolated from A. tenuissima SS77, compound 3 possesses a planar aromatic ring C, such as in cercosporin (b) (Fig. S1). The anisotropic effects of ring C on hydrogen H-1 were likely responsible for the additional shift to downfield observed in this hydrogen. The ESIHRMS data confirmed the molecular formula of 3 as C20H12O5 by the presence of pseudo-molecular ion at m/z [M H] 331.0612 (calculated m/z 331.06120). The gHMBC correlations from hydrogens H-7 and H-12 to carbon C-9b confirmed the rings D and E were fused. However, based on NMR data, it was not possible to determine whether compound
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3 presents similarity to biphenyl or dihydroanthracene scaffolds, since additional gHMBC correlations connecting rings A/B to D/E were not observed. Actually, in the case of perylenequinones presenting a non-reduced ring C, such as compound 3, the scaffolds are called phenanthrene or anthracene, more specifically (Fig. 2). We performed electronic structure calculations to estimate carbon and hydrogen chemical shifts of the two possible isomers and to determine the correct framework of compound 3 by comparison to experimental NMR data. However, the isomers were not distinguished (data not shown). Differing from partially reduced perylenequinones, both isomeric forms of non-reduced perylenequinones are in a tautomeric equilibrium in solution. Tautomerism was studied in natural perylenequinones such as hypocrellin A (c), cercosporin (b) and analogues30–33 (Fig. S1). Arnone and co-workers found that tautomeric equilibrium seems to be affected by substituent effects, strength of the intramolecular phenol–quinone hydrogen bond, distortion from the planarity of the perylenequinone system, solvation and aggregation effects.33 We also performed computational statistical thermodynamics studies to determine the stability of both tautomers T1 and T2 in chloroform (Fig. 2). Differing from other studied non-reduced perylenequinones, compound 3 possesses only one substituent in ring B, a hydroxyl group. Additionally, this ring is partially reduced resulting in the absence of tautomerism between rings A and B. The absence of electronic and steric effects of substituents in rings D and E makes tautomeric equilibrium being very effective in this part of molecule. The planarity of naphthalene moiety favors the formation of a strong intramolecular hydrogen bond, which is confirmed by the hydrogen chemical shift of phenolic hydroxyl at position 9 (dH 15.20). After energy calculations, considering solvation effects and no aggregation effects (see Supplementary material for detail), we found tautomer T1 a little more stable then T2, with a free energy difference of 2 kcal mol 1 between them. This result is in agreement with calculations performed to 4,9-dihydroxyperylene3,10-quinone (a) (Fig. S1), showing this tautomer, displaying phenanthrene framework, is more stable than 3,9-dihydroxyperylene-4,10-quinone, which possesses anthracene framework, for about 3 kcal mol 133. Based on the difference of energy, we could predict that both tautomers T1 and T2 co-exist in solution, but the relative abundance of each one has not been calculated. Furthermore, polar solvents, such as chloroform, favor tautomeric equilibrium and may contribute to the mixture of compounds observed in the 1H NMR spectrum. Therefore, based on thermodynamic analyses, compound 3 preferably assumes a phenanthrene framework (T1) in solution. Although compound 3 possesses only one stereogenic center, its absolute configuration was not determined since the inability to purify this minority compound precluded ECD analyses. Nevertheless, by coupling constant of H-1 with hydrogens H-2a and H-2b (J = 3.9 Hz, 2.6 Hz, respectively), we determined the hydrogen H1 was in a pseudo-equatorial orientation. Thus, hydroxyl attached
Figure 2. Non-reduced perylenequinone scaffolds and tautomeric forms of compound 3.
to C-1 had a pseudo-axial orientation, differing from all other partially reduced perylenequinones isolated from A. tenuissima SS77, including 1 and 2, which had pseudo-equatorial hydroxyl groups. The isolation of 3 from culture broth of A. tenuissima SS77 raised questions about its biosynthesis and biosynthesis of partially reduced perylenequinones. Based on the literature, the biosynthesis of partially reduced perylenequinones occurs by oxidative aromatic coupling of two pentaketide-derived moieties.25,27 However, this coupling has been proposed to occur in a quite specific way, by prior formation of two tetralone derivative intermediates, which undergo head-to-head or tail-to-head coupling, giving rise to those reported perylenequinones containing biphenyl or dihydroanthracene scaffolds, respectively.25,27 The non-reduced perylenequinone derivative 3 might be produced from a partially reduced perylenequinone, such as alterperylenol (i) (hypothesis 1), or, instead, it would be a pathway intermediate of the biosynthesis of partially reduced perylenequinones (hypothesis 2) (Fig. S31). In order to address this inquiry, we first considered the fact that compound 3 was only detected in fungal cultures extracted with Diaion HP-20 resin. It is known that addition of resin to cultures can enhance secondary metabolites yields by sequestering compounds and thus preventing their degradation, feedback inhibition or cytotoxic effects.34,35 Since resin gradually removes secondary metabolites from culture broth, if pathway intermediates are secreted by the fungus, they are sequestered before undergoing subsequent metabolization, accumulating on the resin. It reinforces hypothesis 2. On the other hand, the transformation of partially reduced perylenequinones, affording compound 3, would be prevented by resin capturing, weakening hypothesis 1. Still considering hypothesis 1, higher amounts of 3 would be observed in the extract from cultures extracted by liquid–liquid partition if compound 3 was derived from partially reduced perylenequinones. However, compound 3 has not even been detected in that extract. Another fact that we considered was the different stability of tautomers T1 and T2, which can be extrapolated to a fully conjugate aromatic product. Taking into account our findings, we hypothesize that the biosynthesis of partially reduced perylenequinones may involve the formation of an early pathway conjugate aromatic intermediate, which reaches tautomeric equilibrium, followed by specific reactions such as reduction, hydroxylation, and/or epoxidation in different positions. In this hypothesis, tautomeric form A is the precursor of all biphenyl perylenequinones while tautomer B gives rise to the dihydroanthracene perylenequinones (Scheme1). Up to now, and also considering compounds 1 and 2, there are eleven partially reduced perylenequinones reported as natural products containing biphenyl framework whereas only four possess dihydroanthracene framework. The hypothesis about formation of a conjugated aromatic biosynthetic intermediate, and the different thermostability of tautomers, may explain the higher number of naturally occurring biphenyl perylenequinones over dihydroanthracene ones, making this inequality beyond a coincidence. By incorporation experiments with sodium [2-13C] acetate, Okuno and co-workers confirmed the polyketide origin of perylenequinones.25 However, experiments aiming to prove that all oxygen atoms are really derivate from acetate precursor have never been performed. Considering our biogenetic proposal, the oxygen atoms at positions 3, 4, 9, and 10 more likely derive from polyketide building blocks, while other oxygen atoms may be incorporated into perylenequinone via oxygenases such as cytochrome P450 (CYP450). The assumption of a common conjugate aromatic intermediate in the biosynthetic pathway of all perylenequinones may seem an
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2013/08166-5, and scholarships FAPESP 2009/17695-6 (FOC) and 150572/2015-8 (FOC) of National Council for Scientific and Technological Development (CNPq). Authors also acknowledge the Coordination for the Improvement of Higher Education Personnel (CAPES) and CNPq for financial support. Supplementary data Supplementary data (structures of known fungal perylenequinones, experimental section, NMR data and NMR, UV and HRMS spectra of isolated compounds, thermostability calculations and biogenic hypothesis for formation of compound 3) associated with this article can be found, in the online version, at http://dx.doi.org/ 10.1016/j.tetlet.2016.06.035. References and notes
Scheme 1. Proposed biogenesis of partially reduced perylenequinones.
energy waste when compounds such as stemphyperylenol (h), and even stemphytriol (f) (Fig. S1), are considered, since the oxidative coupling of properly cyclized pentaketide units, as suggested by Okuno and co-worker,25 directly could lead to referred structures, followed by an additional hydroxylation in the case of stemphytriol. However, regarding other partially reduced perylenequinones such as altertoxin II (e), alterlosin II (g) and alterperylenol (i) (Fig. S1), among others, the existence of the proposed early pathway intermediate is very feasible. The incorporation of sodium [1-13C, 18O2] acetate into partially reduced perylenequinones may assist to prove this hypothesis; but, first, the perylenequinone production by A. tenuissima SS77 needs to be optimized to increase the amounts of target compounds. Our results showed that the isolation of new compounds may bring new insights into the biosynthesis of natural products. Genetic and further biosynthetic studies may help to unravel details of the biosynthesis of partially reduced perylenequinones. Acknowledgements This work was supported by the São Paulo Research Foundation (FAPESP) grants 2008/09540-0, 2013/07600-3 Center for Innovation in Biodiversity and Drug Discovery (CEPID-CIBFar),
1. Weiss, U.; Merlini, L.; Nasini, G. In Progress in the Chemistry of Organic Natural Products; Herz, W., Grisebach, H., Kirby, G. W., Tamm, C., Eds.; Springer-Verlag: Wien New York, 1987; Vol. 52, pp 1–71. 2. Blumer, M. Nature 1960, 188, 1100. 3. Cameron, D.; Cromartie, R.; Todd, L. J. Chem. Soc. 1964, 48. 4. Gao, S.-S.; Li, X.-M.; Wang, B.-G. Nat. Prod. Commun. 2009, 4, 1477. 5. Kishi, T.; Tahara, S.; Taniguchi, N.; Tsuda, M.; Tanaka, C.; Takahashi, S. Planta Med. 1991, 57, 376. 6. De Riccardis, F.; Iorizzi, M.; Minale, L.; Riccio, R.; Richer de Forges, B.; Debitus, C. Org. Chem. 1991, 56, 6781. 7. Wolkenstein, K.; Gross, J. H.; Falk, H.; Schöler, H. F. P. R. Soc. B 2006, 273, 451. 8. Mulrooney, C. A.; Morgan, B. J.; Li, X.; Kozlowski, M. C. J. Org. Chem. 2009, 75, 16. 9. Daub, M. E.; Herrero, S.; Chung, K.-R. FEMS Microbiol. Lett. 2005, 252, 197. 10. Mulrooney, C. A.; O’Brien, E. M.; Morgan, B. J.; Kozlowski, M. C. Eur. J. Org. Chem. 2012, 2012, 3887. 11. Anderson, J.; Murray, J. Chem. Ind. 1956, 376. 12. Kuyama, S.; Tamura, T. J. Am. Chem. Soc. 1957, 79, 5725. 13. Lousberg, R. C.; Weiss, U.; Salemink, C.; Arnone, A.; Merlini, L.; Nasini, G. J. Chem. Soc. D 1971, 1463. 14. Yamazaki, S.; Ogawa, T. Agri. Biol. Chem. Tokyo 1972, 36, 1707. 15. Lou, J.; Fu, L.; Peng, Y.; Zhou, L. Molecules 2013, 18, 5891. 16. Stierle, A. C.; Caddlina, J. H.; Strobel, G. A. J. Nat. Prod. 1989, 52, 42. 17. Hradil, C. M.; Hallock, Y. F.; Clardy, J.; Kenfield, D. S.; Strobel, G. Phytochemistry 1989, 28, 73. 18. Alexander, J.; Benford, D.; Boobis, A.; Ceccatelli, S.; Cottrill, B.; Cravedi, J.; Di Domenico, A.; Doerge, D.; Dogliotti, E.; Edler, L. EFSA J. 2011, 9, 2407. 19. Stack, M. E.; Mazzola, E. P.; Page, S. W.; Pohland, A. E.; Highet, R. J.; Tempesta, M. S.; Corley, D. G. J. Nat. Prod. 1986, 49, 866. 20. Fleck, S. C.; Burkhardt, B.; Pfeiffer, E.; Metzler, M. Toxicol. Lett. 2012, 214, 27. 21. Tiessen, C.; Gehrke, H.; Kropat, C.; Schwarz, C.; Bächler, S.; Fehr, M.; Pahlke, G.; Marko, D. World Mycotoxin J. 2013, 6, 233. 22. Chagas, F. O.; Dias, L. G.; Pupo, M. T. J. Chem. Ecol. 2013, 39, 1335. 23. Geiseler, O.; Müller, M.; Podlech, J. Tetrahedron 2013, 69, 3683. 24. Podlech, J.; Fleck, S. C.; Metzler, M.; Bürck, J.; Ulrich, A. S. Chem.-Eur. J. 2014, 20, 11463. 25. Okuno, T.; Natsume, I.; Sawai, K.; Sawamura, K.; Furusaki, A.; Matsumoto, T. Tetrahedron Lett. 1983, 24, 5653. 26. Krohn, K.; John, M.; Aust, H. J.; Draeger, S.; Schulz, B. Nat. Prod. Lett. 1999, 14, 31. 27. Arnone, A.; Nasini, G.; Merlini, L.; Assante, G. J. Chem. Soc., Perkin Trans. 1 1986, 525. 28. Saha, D.; Fetzner, R.; Burkhardt, B.; Podlech, J.; Metzler, M.; Dang, H.; Lawrence, C.; Fischer, R. PLoS ONE 2012, 7, e40564. 29. Chen, H.; Lee, M. H.; Daub, M. E.; Chung, K. R. Mol. Microbiol. 2007, 64, 755. 30. Mazzini, S.; Merlini, L.; Mondelli, R.; Scaglioni, L. J. Chem. Soc., Perkin Trans. 2 2001, 409. 31. Zhang, H.; Zhang, Z. Sci. China Ser. B 1998, 41, 85. 32. Zhang, H.-Y. J. Photochem. Photobiol. A 1999, 126, 27. 33. Arnone, A.; Merlini, L.; Mondelli, R.; Nasini, G.; Ragg, E.; Scaglioni, L.; Weiss, U. J. Chem. Soc., Perkin Trans. 2 1993, 1447. 34. González-Menéndez, V.; Asensio, F.; Moreno, C.; de Pedro, N.; Monteiro, M. C.; de la Cruz, M.; Vicente, F.; Bills, G. F.; Reyes, F.; Genilloud, O. Mycology 2014, 5, 179. 35. Phillips, T.; Chase, M.; Wagner, S.; Renzi, C.; Powell, M.; DeAngelo, J.; Michels, P. J. Ind. Microbiol. Biotech. 2013, 40, 411.
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Tetrahedron Letters journal homepage: www.elsevier.com/locate/tetlet
New perylenequinone derivatives from the endophytic fungus Alternaria tenuissima SS77 Fernanda O. Chagas a, Luís G. Dias b, Mônica T. Pupo a,⇑ a Departamento de Ciências Farmacêuticas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Avenida do Café, s/n, 14040-903, Ribeirão Preto, SP, Brazil b Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Avenida Bandeirantes, 3900, 14040-901, Ribeirão Preto, SP, Brazil
a r t i c l e
i n f o
Article history: Received 6 April 2016 Revised 6 June 2016 Accepted 8 June 2016 Available online 9 June 2016 Keywords: Perylenequinones Mycotoxins Phytotoxins Biogenesis Endophyte
a b s t r a c t Three new natural products, 1,4,6b,7,10-pentahydroxy-1,2,6b,7,8,12b-hexahydroperylene-3,9-dione (1), 1,4,9,12a-tetrahydroxy-12-methoxy-1,2,11,12,12a,12b-hexahydroperylene-3,10-dione (2) and 1,4,9-trihydroxy-1,2-dihydroperylene-3,10-dione (3), were obtained from cultures of the endophytic fungus Alternaria tenuissima SS77. Their chemical structures were determined by combined analysis of oneand two-dimensional NMR spectra, UV and ESIHRMS data. Compound 3 is likely an intermediate in the biosynthetic pathway of some perylenequinones produced by this fungus. A new hypothesis for the biosynthesis of partially reduced perylenequinones is suggested. Ó 2016 Elsevier Ltd. All rights reserved.
Introduction Perylenequinones are secondary metabolites characterized by a conjugated aromatic pentacyclic dione; however, natural products with partially reduced perylene core have also been included in this class of compounds.1 Perylenequinone derivatives are found in fungi, plants, insects (aphids), and animals (crinoids).1–10 Different structural features may be attached to the perylenequinone backbone, making perylenequinone derivatives relatively diverse. Most reported perylenequinones have fungal origin, and the simplest naturally occurring perylenequinone known, the 4,9dihydroxyperylene-3,10-quinone (a), was isolated from the fungus Daldinia concentrica in 1956.11 Cercosporin (b) is among the first of isolated mold perylenequinones, which was reported in 1957, even though its structure was only completely determined by 1972.12–14 This compound was deeply studied mainly due its photoactivity.9,10 Cercosporin and other similar compounds, such as hypocrellin A (c) and scutiaquinone A (d) contain carbon substituents attached to perylenequinone backbone1,8–10 (Fig. S1). Another group of mold perylenequinones, in which the parent natural product 4,9-dihydroxyperylene-3,10-quinone is included, does not contain carbon substituents and usually has a partially reduced perylene.1,8 Many mycotoxins produced by Alternaria ⇑ Corresponding author. Tel.: +55 16 3315 4710; fax: +55 16 3315 4178. E-mail addresses: [email protected] (F.O. Chagas), [email protected] (L.G. Dias), [email protected] (M.T. Pupo). http://dx.doi.org/10.1016/j.tetlet.2016.06.035 0040-4039/Ó 2016 Elsevier Ltd. All rights reserved.
species, such as altertoxin II (e), stemphytriol (f), and alterlosin II (g) (Fig. S1), belong to this second group of mold perylenequinones,15 some of which have been reported as phytotoxins16,17 and others have gained importance over the years for causing food and feed contaminations, offering risks to human health due to their mutagenic effects.18–21 In plants, toxic effects of these perylenequinones may occur in the same way that of cercosporin, by generation of free radicals that damage host cells after photoactivation.9 Mutagenicity observed in bacterial and mammal cells have been attributed to various mechanisms of action.20,21 Particular structural features of different perylenequinone derivatives contribute to their photoactive and mutagenic effects, affording compounds with variable levels of bioactivity. As part of our ongoing program for prospecting natural products from endophytic microorganisms, the fungus Alternaria tenuissima SS77 has been cultured in diverse growth conditions, including mixed microbial cultivation. A. tenuissima SS77 has produced polyketides belonging to perylenequinones class, such as stemphyperylenol (h), alterperylenol (i) and altertoxin I (j) (Fig. S1), in single and mixed cultures, and was able to remodel its secondary metabolism in mixed fermentation.22 In this Letter we report the identification of further perylenequinones produced by this fungus. The high structural similarity among those compounds made it possible to undoubtedly characterize them by NMR and UV. Furthermore, we have proposed that the biosynthetic pathways for all those perylenequinones more
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likely share the same precursor in early steps, which later has the perylene core reduced and undergoes peripheral oxidations, affording the final products. Results and discussion Compound 1 was obtained as a pale yellow powder. NMR spectra of 1 were acquired in CD3OD and CDCl3, and the combined analyses allowed the structural determination. Carbons were assigned from gHSQC and gHMBC spectra (CD3OD) (Table S1). The 1H NMR, gHSQC, and gHMBC spectra (Supplementary material) contained resonances for eighteen carbons in a five fused ring structure comprising two aromatic rings with a couple of aromatic ortho coupled hydrogens each [dH 6.99 (1H, d, J = 8.6 Hz, H-5), 8.16 (1H, d, J = 8.6 Hz, H-6), 6.89 (1H, d, J = 8.6 Hz, H-11), 8.20 (1H, d, J = 8.6 Hz, H-12); dC 117.4 (C-5), 136.6 (C-6), 115.7 (C-11), 137.1 (C-12)], two chelated phenolic hydroxyls [dH 12.09 (s, 4-OH), 12.23 (s, 10-OH); dC 161.1 (C-4), 162.0 (C-10)], five non-hydrogenated aromatic carbons [dC 144.0 (C-3b), 133.3 (C-6a), 115.8 (C-9a), 146.0 (C-9b), 133.9 (C-12a)], a carbonyl carbon [dC 204.4 (C-3)], two methylene groups [dH 3.09 (1H, dd, J = 12.4 Hz, 15.6 Hz, H-2a), 3.20 (1H, dd, J = 4.5 Hz, 15.6 Hz, H-2b), 3.29 (1H, dd, J = 12.4 Hz, 15.6 Hz, H-8a), 3.01 (1H, dd, J = 4.5 Hz, 15.6 Hz, H-8b); dC 48.0 (C-2), 43.9 (C-8)], two hydroxylated methine secondary carbons [dH 4.69 (1H, m, H-1), 2.07 (sl, 1-OH), 4.88 (1H, m, H-7), 2.62 (d, J = 6.3 Hz, 7-OH); dC 68.7 (C-1), 69.5 (C-7)], a methine tertiary carbon [dH 4.42 (1H, d, J = 9.8 Hz, H-12b); dC 45.3 (C-12b)], and a hydroxylated tertiary carbon [dH 2.71 (s, 6bOH)]. Data from gHSQC and gHMBC spectra confirmed that each couple of aromatic hydrogen belongs to different phenol groups (rings A and E). The correlations showed the methylene group at position 2 was connected to hydroxylated methine secondary carbon C-1, which was linked to methine tertiary carbon C-12b, constituting part of the cyclohexenone ring B; additionally, the methylene group at position 8 was linked to the hydroxylated methine secondary carbon C-7, which together were part of ring D. The spectral data of 1 were similar to those of partially reduced perylenequinones previously isolated from A. tenuissima SS77.22 In comparison to other analogues, compound 1 possessed five hydroxyls, including two chelated phenolic ones, and no double bond in any cyclohexenone rings. The positions of hydroxyl groups were determined by gHMBC correlations. The ESIHRMS data confirmed the molecular formula of 1 as C20H16O7 by the presence of pseudo-molecular ion at m/z [M H] 367.08510 (calculated m/z 367.08233). The partially reduced perylenequinones from Alternaria spp., and related genera, are divided into two sub-groups according to their structural scaffolds. These compounds exhibit biphenyl or dihydroanthracene frameworks according to relative position of both phenol groups of their structures23,24 (Fig. 1).
Determining whether compound 1 has a biphenyl or a dihydroanthracene framework was not possible by gHMBC, since no key correlations were observed. However, the UV spectra of compounds presenting one or another scaffold are quite characteristic (Fig. 1). The main difference between UV spectra is the presence of a shoulder (sh) band at 285 nm in perylenequinones displaying dihydroanthracene framework, which is absent in perylenequinones with biphenyl framework. Besides, when compared to biphenyl perylenequinones, dihydroanthracene perylenequinones present a characteristic bathochromic shift in the 300–400 nm band. Also, dihydroanthracene perylenequinones display the maximum molar absorptivity (e) at 258 nm, followed by 214 nm, 285 (sh) and 357 nm, while biphenyl perylenequinones have the maximum e bellow 200 nm, followed by 215 nm, 260 and 342 nm, as shown in the UV spectra of stemphyperylenol (h) and alterperylenol (i) (Fig. 1). Therefore, the relative intensities between bands around 250 and 350 nm can be used to indicate the correct framework. This is the first Letter correlating perylenequinone UV absorptions with their respective frameworks. These characteristic UV spectra were observed in all isolated reduced perylenequinones from A. tenuissima SS77 (Fig. S28) and also in previously reported compounds.16,17,25–27 Thus, based on UV spectra, it was determined that compound 1 possesses a dihydroanthracene framework. Therefore, compound 1 is different from the previously reported stemphytriol (f) (Fig. S1), which possesses a biphenyl framework.26 The relative stereochemistry of 1 was established based on hydrogen coupling constants. The coupling constants of H-2a and H-12b hydrogens enabled presuming the spatial orientation of hydroxyl at C-1 and hydrogen H-12b. The coupling constants of 12.4 Hz and 9.8 Hz indicated that H-2a presents a pseudo-diaxial correlation to H-1, and H-1 presents a pseudo-diaxial correlation to H-12b, respectively. Thus, the spatial orientation of hydroxyl at C-1 and hydrogen H-12b were pseudo-equatorial and pseudo-axial, respectively. In the same way, by the coupling constants between H-7 and H-8a hydrogens (J = 12.4 Hz) that indicated a pseudo-diaxial correlation, the orientation of hydroxyl at C-7 was determined as pseudo-equatorial. Such as found in all structures of partially reduced perylenequinones, the hydroxyl at C-6b is suggested to be in pseudo-axial, likewise hydrogen H-12b, maintaining the relative planarity of the five fused ring structure. The relative configuration between C-12b and C-6b could not be determined. However, considering all isolated reduced perylenequinones, it was assumed that H-12b and OH-6b were in the same side of molecule. Then, the relative configurations of the stereogenic centers at position 1, 12b, 6b and 7 were S⁄, S⁄, R⁄ and S⁄, respectively. Compound 1 is very similar to stemphyperylenol (h) (Fig. S1), of which we have previously determined the absolute configuration of stereogenic centers as 1R, 12bR, 6bR, and 7R, by comparing experimental ECD with theoretical calculations.22 However, due
Figure 1. Basic structures of perylenequinones presenting dihydroanthracene (I) and biphenyl (II) scaffolds, and characteristic experimental UV spectra of both types of perylenequinones.
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to the tiny amount of compound 1 and required further purifications, ECD was not performed and absolute configuration was not determined.
Compound 2 was obtained as a yellow powder. The carbons were assigned from gHSQC and gHMBC spectra (Table S1). The interpretation of 1H NMR, gHSQC, and gCOSY spectra (Supplementary material) showed compound 2 possesses structural similarities to partially reduced perylenequinones: presence of two aromatic rings with a couple of aromatic ortho coupled hydrogens each [dH 7.04 (1H, d, J = 8.9 Hz, H-5), 7.84 (1H, d, J = 8.9 Hz, H-6), 7.84 (1H, d, J = 8.9 Hz, H-7), 7.11 (1H, d, J = 8.9 Hz, H-8); dC 117.6 (C-5), 132.7 (C-6), 132.7 (C-7), 119.6 (C-8)], two chelated phenolic hydroxyls [dH 12.46 (s, 4-OH), 12.42 (s, 9-OH); dC 162.4 (C-4), 161.6 (C-9)], six non-hydrogenated aromatic carbons [dC 117.3 (C-3a), 135.6 (C-3b), 123.6 (C-6a), 123.2 (C-6b), 113.9 (C-9a), 139.1 (C9b)], two carbonyl carbons [dC 202.4 (C-3), 202.4 (C-10)], two methylene groups [dH 2.91 (1H, dd, J = 12.2 Hz, 16.2 Hz, H-2a), 3.17 (1H, dd, J = 4.6 Hz, 16.2 Hz, H-2b), 3.18 (1H, dd, J = 3.5 Hz, 17.3 Hz, H-11a), 3.39 (1H, dd, J = 2.2 Hz, 17.3 Hz, H-11b); dC 47.7 (C-2), 36.7 (C-11)], a hydroxylated methine secondary carbon [dH 4.68 (1H, m, H-1), 2.83 (d, J = 6.0 Hz, 1-OH); dC 65.9 (C-1)], a methoxylated methine secondary carbon [dH 4.67 (1H, dd, J = 2.2 Hz, 3.5 Hz, H-12), 3.45 (3H, s, 12-OMe); dC 65.9 (C-12), 55.7 (12-OMe)], a methine tertiary carbon [dH 3.56 (1H, d, J = 9.6 Hz, H-12b); dC 48.0 (C-12b)], and a hydroxylated tertiary carbon [dH 1.98 (s, 12a-OH); dC 71.1 (C-12a)]. Differing from 1, compound 2 possesses only four hydroxyls. Instead, a methoxyl group (12-OMe) was attached to carbon C-12. The molecular formula of 2 was confirmed by ESIHRMS as C21H18O7 by the presence of the pseudo-molecular ion at m/z [M H] 381.10081 (calculated m/z 381.09798). The biphenyl framework of compound 2 was determined based on its UV spectrum (Fig. S28). The relative stereochemistry of 2 was established based on hydrogen coupling constants. The coupling constant between hydrogens H-1 and H-2a (J = 12.2 Hz) indicated a pseudo-diaxial correlation between them, and the orientation of hydroxyl attached to C-1 was determined as pseudo-equatorial. A pseudodiaxial correlation was also observed between hydrogens H-1and H-12b (J = 9.6 Hz), indicating H-12b presented pseudo-axial orientation. Similarly, the coupling constants of hydrogen H-11a with H-12 (J = 3.5 Hz) and hydrogen H-11b with H-12 (J = 2.2 Hz) indicated that H-12 had a pseudo-equatorial orientation. Thus, methoxyl group attached to C-12 was in a pseudo-axial orientation. Accordingly to other partially reduced perylenequinones, the hydroxyl attached to C-12a was suggested to have pseudo-axial orientation, and H-12b and 12a-OH were in opposite sides of the molecular plane. Then, the relative configurations of the stereogenic centers at position 1, 12b, 12a, and 12 were determined as S⁄, S⁄, R⁄ and R⁄, respectively. Compound 2 was believed to be a methylated derivative of stemphytriol (f) (Fig. S1) at first, but the opposite relative configuration at C-12a brought doubts about compound 2 being a natural product or, instead, a solvent derivative artifact formed during extraction and fractionation with methanol. Since O-methylation
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process is not expected to change the configuration of the center in which the hydroxyl group is attached, we considered compound 2 might be a solvent artifact. However, an epimer of stemphytriol possessing R⁄-configuration at C-12a has been isolated from an endophytic Alternaria alternate.4 Thus, compound 2 possibly would be a methylated analogue of 12a-epi-stemphytriol. Indeed, enzymes O-methyltransferases are involved in the biosynthesis of some mycotoxins, such as cercosporin and alternariol.28,29 The presence of genes encoding such enzymes in the biosynthetic gene cluster responsible for the biosynthesis of partially reduced perylenequinones could confirm compound 2 is a natural product instead a solvent artifact. Nevertheless, genetic studies on those compounds have never been performed, and the doubt remains. Compound 3 was obtained as a yellow brownish powder. Carbons were assigned from gHSQC and gHMBC spectra (Table S1). The interpretation of 1H NMR, gHSQC, and gCOSY spectra (Supplementary material) showed compound 2 possesses structural similarities to partially reduced perylenequinones and some additional features: presence of two aromatic rings with a couple of aromatic ortho coupled hydrogens each [dH 7.53 (1H, d, J = 9.4 Hz, H-5), 8.86 (1H, d, J = 9.4 Hz, H-6), 8.87 (1H, d, J = 9.4 Hz, H-7), 7.50 (1H, d, J = 9.4 Hz, H-8); dC 122.7 (C-5), 130.9 (C-6), 132.5 (C-7), 120.8 (C8)], two chelated phenolic hydroxyls [dH 13.23 (s, 4-OH), 15.20 (s, 9-OH); dC 162.6 (C-4), 168.0 (C-9)], eight non-hydrogenated aromatic carbons [dC 110.8 (C-3a), 127.7 (C-3b), 121.8 (C-6a), 129.2 (C-6b), 112.6 (C-9a), 125.5 (C-9b), 124.3 (C-12a), 134.9 (C-12b)], a carbonyl carbon [dC 201.1 (C-3)], a conjugated carbonyl carbon [dC 187.9 (C-10)], two olefinic hydrogens [dH 7.14 (1H, d, J = 10.1 Hz, H-11), 8.56 (1H, d, J = 10.1 Hz, H-12); dC 129.3 (C-11), 136.9 (C-12)], a methylene group [dH 3.27 (1H, dd, J = 3.9 Hz, 16.9 Hz, H-2a), 3.42 (1H, dd, J = 2.6 Hz, 16.9 Hz, H-2b); dC 44.8 (C2)], and a hydroxylated methine secondary carbon [dH 6.17 (1H, dd, J = 2.6 Hz, 3.9 Hz, H-1); dC 63.9 (C-1)]. Data from gHSQC and gHMBC spectra confirmed that each couple of aromatic hydrogens belongs to different phenol groups (rings A and D) and the olefinic hydrogens are part of a conjugated carbonyl system (ring E). These NMR data strongly suggested compound 3 was a perylenequinone. However, all mentioned hydrogens were deshielded compared to those of similar compounds, such as 1 and 2, and other partially reduced perylenequinones isolated from A. tenussima,22 indicating compound 3 exhibited a particular structural feature. Besides, compound 3 possessed two additional olefinic/aromatic carbons (C-12a, C12b). The methylene group at position 2 was connected to the hydroxylated methine secondary carbon C-1, constituting part of the cyclohexenone ring B. A hydroxyl attached to C-1, which is common in perylenequinones, was partially responsible for the carbon chemical shift of C-1; however, the hydrogen chemical shift of H-1 was more deshielded than expected, since the chemical shift of the referred hydrogen in other perylenequinones is about dH 4.50–4.80. The gHMBC correlation from methylene hydrogen H2b to the carbonyl C-3 confirmed their vicinity, and also the ring B features, whereas the correlation from the hydrogen H-2b to the carbon C-12b indicated the presence of a double bond connecting rings B and E. Thus, differing from other perylenequinones isolated from A. tenuissima SS77, compound 3 possesses a planar aromatic ring C, such as in cercosporin (b) (Fig. S1). The anisotropic effects of ring C on hydrogen H-1 were likely responsible for the additional shift to downfield observed in this hydrogen. The ESIHRMS data confirmed the molecular formula of 3 as C20H12O5 by the presence of pseudo-molecular ion at m/z [M H] 331.0612 (calculated m/z 331.06120). The gHMBC correlations from hydrogens H-7 and H-12 to carbon C-9b confirmed the rings D and E were fused. However, based on NMR data, it was not possible to determine whether compound
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3 presents similarity to biphenyl or dihydroanthracene scaffolds, since additional gHMBC correlations connecting rings A/B to D/E were not observed. Actually, in the case of perylenequinones presenting a non-reduced ring C, such as compound 3, the scaffolds are called phenanthrene or anthracene, more specifically (Fig. 2). We performed electronic structure calculations to estimate carbon and hydrogen chemical shifts of the two possible isomers and to determine the correct framework of compound 3 by comparison to experimental NMR data. However, the isomers were not distinguished (data not shown). Differing from partially reduced perylenequinones, both isomeric forms of non-reduced perylenequinones are in a tautomeric equilibrium in solution. Tautomerism was studied in natural perylenequinones such as hypocrellin A (c), cercosporin (b) and analogues30–33 (Fig. S1). Arnone and co-workers found that tautomeric equilibrium seems to be affected by substituent effects, strength of the intramolecular phenol–quinone hydrogen bond, distortion from the planarity of the perylenequinone system, solvation and aggregation effects.33 We also performed computational statistical thermodynamics studies to determine the stability of both tautomers T1 and T2 in chloroform (Fig. 2). Differing from other studied non-reduced perylenequinones, compound 3 possesses only one substituent in ring B, a hydroxyl group. Additionally, this ring is partially reduced resulting in the absence of tautomerism between rings A and B. The absence of electronic and steric effects of substituents in rings D and E makes tautomeric equilibrium being very effective in this part of molecule. The planarity of naphthalene moiety favors the formation of a strong intramolecular hydrogen bond, which is confirmed by the hydrogen chemical shift of phenolic hydroxyl at position 9 (dH 15.20). After energy calculations, considering solvation effects and no aggregation effects (see Supplementary material for detail), we found tautomer T1 a little more stable then T2, with a free energy difference of 2 kcal mol 1 between them. This result is in agreement with calculations performed to 4,9-dihydroxyperylene3,10-quinone (a) (Fig. S1), showing this tautomer, displaying phenanthrene framework, is more stable than 3,9-dihydroxyperylene-4,10-quinone, which possesses anthracene framework, for about 3 kcal mol 133. Based on the difference of energy, we could predict that both tautomers T1 and T2 co-exist in solution, but the relative abundance of each one has not been calculated. Furthermore, polar solvents, such as chloroform, favor tautomeric equilibrium and may contribute to the mixture of compounds observed in the 1H NMR spectrum. Therefore, based on thermodynamic analyses, compound 3 preferably assumes a phenanthrene framework (T1) in solution. Although compound 3 possesses only one stereogenic center, its absolute configuration was not determined since the inability to purify this minority compound precluded ECD analyses. Nevertheless, by coupling constant of H-1 with hydrogens H-2a and H-2b (J = 3.9 Hz, 2.6 Hz, respectively), we determined the hydrogen H1 was in a pseudo-equatorial orientation. Thus, hydroxyl attached
Figure 2. Non-reduced perylenequinone scaffolds and tautomeric forms of compound 3.
to C-1 had a pseudo-axial orientation, differing from all other partially reduced perylenequinones isolated from A. tenuissima SS77, including 1 and 2, which had pseudo-equatorial hydroxyl groups. The isolation of 3 from culture broth of A. tenuissima SS77 raised questions about its biosynthesis and biosynthesis of partially reduced perylenequinones. Based on the literature, the biosynthesis of partially reduced perylenequinones occurs by oxidative aromatic coupling of two pentaketide-derived moieties.25,27 However, this coupling has been proposed to occur in a quite specific way, by prior formation of two tetralone derivative intermediates, which undergo head-to-head or tail-to-head coupling, giving rise to those reported perylenequinones containing biphenyl or dihydroanthracene scaffolds, respectively.25,27 The non-reduced perylenequinone derivative 3 might be produced from a partially reduced perylenequinone, such as alterperylenol (i) (hypothesis 1), or, instead, it would be a pathway intermediate of the biosynthesis of partially reduced perylenequinones (hypothesis 2) (Fig. S31). In order to address this inquiry, we first considered the fact that compound 3 was only detected in fungal cultures extracted with Diaion HP-20 resin. It is known that addition of resin to cultures can enhance secondary metabolites yields by sequestering compounds and thus preventing their degradation, feedback inhibition or cytotoxic effects.34,35 Since resin gradually removes secondary metabolites from culture broth, if pathway intermediates are secreted by the fungus, they are sequestered before undergoing subsequent metabolization, accumulating on the resin. It reinforces hypothesis 2. On the other hand, the transformation of partially reduced perylenequinones, affording compound 3, would be prevented by resin capturing, weakening hypothesis 1. Still considering hypothesis 1, higher amounts of 3 would be observed in the extract from cultures extracted by liquid–liquid partition if compound 3 was derived from partially reduced perylenequinones. However, compound 3 has not even been detected in that extract. Another fact that we considered was the different stability of tautomers T1 and T2, which can be extrapolated to a fully conjugate aromatic product. Taking into account our findings, we hypothesize that the biosynthesis of partially reduced perylenequinones may involve the formation of an early pathway conjugate aromatic intermediate, which reaches tautomeric equilibrium, followed by specific reactions such as reduction, hydroxylation, and/or epoxidation in different positions. In this hypothesis, tautomeric form A is the precursor of all biphenyl perylenequinones while tautomer B gives rise to the dihydroanthracene perylenequinones (Scheme1). Up to now, and also considering compounds 1 and 2, there are eleven partially reduced perylenequinones reported as natural products containing biphenyl framework whereas only four possess dihydroanthracene framework. The hypothesis about formation of a conjugated aromatic biosynthetic intermediate, and the different thermostability of tautomers, may explain the higher number of naturally occurring biphenyl perylenequinones over dihydroanthracene ones, making this inequality beyond a coincidence. By incorporation experiments with sodium [2-13C] acetate, Okuno and co-workers confirmed the polyketide origin of perylenequinones.25 However, experiments aiming to prove that all oxygen atoms are really derivate from acetate precursor have never been performed. Considering our biogenetic proposal, the oxygen atoms at positions 3, 4, 9, and 10 more likely derive from polyketide building blocks, while other oxygen atoms may be incorporated into perylenequinone via oxygenases such as cytochrome P450 (CYP450). The assumption of a common conjugate aromatic intermediate in the biosynthetic pathway of all perylenequinones may seem an
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2013/08166-5, and scholarships FAPESP 2009/17695-6 (FOC) and 150572/2015-8 (FOC) of National Council for Scientific and Technological Development (CNPq). Authors also acknowledge the Coordination for the Improvement of Higher Education Personnel (CAPES) and CNPq for financial support. Supplementary data Supplementary data (structures of known fungal perylenequinones, experimental section, NMR data and NMR, UV and HRMS spectra of isolated compounds, thermostability calculations and biogenic hypothesis for formation of compound 3) associated with this article can be found, in the online version, at http://dx.doi.org/ 10.1016/j.tetlet.2016.06.035. References and notes
Scheme 1. Proposed biogenesis of partially reduced perylenequinones.
energy waste when compounds such as stemphyperylenol (h), and even stemphytriol (f) (Fig. S1), are considered, since the oxidative coupling of properly cyclized pentaketide units, as suggested by Okuno and co-worker,25 directly could lead to referred structures, followed by an additional hydroxylation in the case of stemphytriol. However, regarding other partially reduced perylenequinones such as altertoxin II (e), alterlosin II (g) and alterperylenol (i) (Fig. S1), among others, the existence of the proposed early pathway intermediate is very feasible. The incorporation of sodium [1-13C, 18O2] acetate into partially reduced perylenequinones may assist to prove this hypothesis; but, first, the perylenequinone production by A. tenuissima SS77 needs to be optimized to increase the amounts of target compounds. Our results showed that the isolation of new compounds may bring new insights into the biosynthesis of natural products. Genetic and further biosynthetic studies may help to unravel details of the biosynthesis of partially reduced perylenequinones. Acknowledgements This work was supported by the São Paulo Research Foundation (FAPESP) grants 2008/09540-0, 2013/07600-3 Center for Innovation in Biodiversity and Drug Discovery (CEPID-CIBFar),
1. Weiss, U.; Merlini, L.; Nasini, G. In Progress in the Chemistry of Organic Natural Products; Herz, W., Grisebach, H., Kirby, G. W., Tamm, C., Eds.; Springer-Verlag: Wien New York, 1987; Vol. 52, pp 1–71. 2. Blumer, M. Nature 1960, 188, 1100. 3. Cameron, D.; Cromartie, R.; Todd, L. J. Chem. Soc. 1964, 48. 4. Gao, S.-S.; Li, X.-M.; Wang, B.-G. Nat. Prod. Commun. 2009, 4, 1477. 5. Kishi, T.; Tahara, S.; Taniguchi, N.; Tsuda, M.; Tanaka, C.; Takahashi, S. Planta Med. 1991, 57, 376. 6. De Riccardis, F.; Iorizzi, M.; Minale, L.; Riccio, R.; Richer de Forges, B.; Debitus, C. Org. Chem. 1991, 56, 6781. 7. Wolkenstein, K.; Gross, J. H.; Falk, H.; Schöler, H. F. P. R. Soc. B 2006, 273, 451. 8. Mulrooney, C. A.; Morgan, B. J.; Li, X.; Kozlowski, M. C. J. Org. Chem. 2009, 75, 16. 9. Daub, M. E.; Herrero, S.; Chung, K.-R. FEMS Microbiol. Lett. 2005, 252, 197. 10. Mulrooney, C. A.; O’Brien, E. M.; Morgan, B. J.; Kozlowski, M. C. Eur. J. Org. Chem. 2012, 2012, 3887. 11. Anderson, J.; Murray, J. Chem. Ind. 1956, 376. 12. Kuyama, S.; Tamura, T. J. Am. Chem. Soc. 1957, 79, 5725. 13. Lousberg, R. C.; Weiss, U.; Salemink, C.; Arnone, A.; Merlini, L.; Nasini, G. J. Chem. Soc. D 1971, 1463. 14. Yamazaki, S.; Ogawa, T. Agri. Biol. Chem. Tokyo 1972, 36, 1707. 15. Lou, J.; Fu, L.; Peng, Y.; Zhou, L. Molecules 2013, 18, 5891. 16. Stierle, A. C.; Caddlina, J. H.; Strobel, G. A. J. Nat. Prod. 1989, 52, 42. 17. Hradil, C. M.; Hallock, Y. F.; Clardy, J.; Kenfield, D. S.; Strobel, G. Phytochemistry 1989, 28, 73. 18. Alexander, J.; Benford, D.; Boobis, A.; Ceccatelli, S.; Cottrill, B.; Cravedi, J.; Di Domenico, A.; Doerge, D.; Dogliotti, E.; Edler, L. EFSA J. 2011, 9, 2407. 19. Stack, M. E.; Mazzola, E. P.; Page, S. W.; Pohland, A. E.; Highet, R. J.; Tempesta, M. S.; Corley, D. G. J. Nat. Prod. 1986, 49, 866. 20. Fleck, S. C.; Burkhardt, B.; Pfeiffer, E.; Metzler, M. Toxicol. Lett. 2012, 214, 27. 21. Tiessen, C.; Gehrke, H.; Kropat, C.; Schwarz, C.; Bächler, S.; Fehr, M.; Pahlke, G.; Marko, D. World Mycotoxin J. 2013, 6, 233. 22. Chagas, F. O.; Dias, L. G.; Pupo, M. T. J. Chem. Ecol. 2013, 39, 1335. 23. Geiseler, O.; Müller, M.; Podlech, J. Tetrahedron 2013, 69, 3683. 24. Podlech, J.; Fleck, S. C.; Metzler, M.; Bürck, J.; Ulrich, A. S. Chem.-Eur. J. 2014, 20, 11463. 25. Okuno, T.; Natsume, I.; Sawai, K.; Sawamura, K.; Furusaki, A.; Matsumoto, T. Tetrahedron Lett. 1983, 24, 5653. 26. Krohn, K.; John, M.; Aust, H. J.; Draeger, S.; Schulz, B. Nat. Prod. Lett. 1999, 14, 31. 27. Arnone, A.; Nasini, G.; Merlini, L.; Assante, G. J. Chem. Soc., Perkin Trans. 1 1986, 525. 28. Saha, D.; Fetzner, R.; Burkhardt, B.; Podlech, J.; Metzler, M.; Dang, H.; Lawrence, C.; Fischer, R. PLoS ONE 2012, 7, e40564. 29. Chen, H.; Lee, M. H.; Daub, M. E.; Chung, K. R. Mol. Microbiol. 2007, 64, 755. 30. Mazzini, S.; Merlini, L.; Mondelli, R.; Scaglioni, L. J. Chem. Soc., Perkin Trans. 2 2001, 409. 31. Zhang, H.; Zhang, Z. Sci. China Ser. B 1998, 41, 85. 32. Zhang, H.-Y. J. Photochem. Photobiol. A 1999, 126, 27. 33. Arnone, A.; Merlini, L.; Mondelli, R.; Nasini, G.; Ragg, E.; Scaglioni, L.; Weiss, U. J. Chem. Soc., Perkin Trans. 2 1993, 1447. 34. González-Menéndez, V.; Asensio, F.; Moreno, C.; de Pedro, N.; Monteiro, M. C.; de la Cruz, M.; Vicente, F.; Bills, G. F.; Reyes, F.; Genilloud, O. Mycology 2014, 5, 179. 35. Phillips, T.; Chase, M.; Wagner, S.; Renzi, C.; Powell, M.; DeAngelo, J.; Michels, P. J. Ind. Microbiol. Biotech. 2013, 40, 411.