Non-geminal Aliphatic Dihalogenation Pattern in Dichlorinated

Note Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

pubs.acs.org/jnp

Non-geminal Aliphatic Dihalogenation Pattern in Dichlorinated Diaporthins from Hamigera fusca NRRL 35721 Celso Almeida,*,†,‡ Ignacio Pérez-Victoria,*,‡ Víctor González-Menéndez, Nuria de Pedro, Jesús Martín, Gloria Crespo, Thomas Mackenzie, Bastien Cautain, Fernando Reyes, Francisca Vicente, and Olga Genilloud Fundación MEDINA, Centro de Excelencia en Investigación de Medicamentos Innovadores en Andalucía, Parque Tecnológico de Ciencias de la Salud, Avenida del Conocimiento 34, 18016 Armilla, Granada, Spain S Supporting Information *

ABSTRACT: Two new epimeric dihalogenated diaporthins, (9R*)-8-methyl-9,11-dichlorodiaporthin (2) and (9S*)-8-methyl9,11-dichlorodiaporthin (3), have been isolated from the soil fungus Hamigera f usca NRRL 35721 alongside the known regioisomeric isocoumarin 8-methyl-11,11-dichlorodiaporthin (1). Their structures were elucidated by high-resolution mass spectrometry and NMR spectroscopy combined with molecular modeling. Compounds 1−3 are the first isocoumarins and the first halogenated metabolites ever reported from the Hamigera genus. The new compounds 2 and 3 display a non-geminal aliphatic dichlorination pattern unprecedented among known fungal dihalogenated aromatic polyketides. A bifunctional methyltransferase/aliphatic halogenase flavoenzyme is proposed to be involved in the biosynthesis of dichlorinated diaporthins 1−3. These metabolites are weakly cytotoxic.

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ore than 5000 naturally occurring organohalogen compounds have been discovered to date from plant, animal, and microbial sources.1,2 Frequently, halogenated secondary metabolites display interesting biological activities, with a few molecules known for their clinical medical value.3 Among fungal halogenated metabolites, chlorinated diaporthins4 (considered as mycotoxins)5 and related chlorinated aromatic polyketides are a striking case which display a geminal dichlorination pattern at the terminal carbon of their aliphatic side chain (see Table S1). In this work we describe the isolation and structural elucidation of the known 8-methyl-11,11dichlorodiaporthin (1) alongside two new epimeric regioisomers which remarkably do not display the usual geminal dichlorination pattern, (9R*)-8-methyl-9,11-dichlorodiaporthin (2) and (9S*)-8-methyl-9,11-dichlorodiaporthin (3), from a fermentation broth of Hamigera f usca NRRL 35721 (Ascomycota, Eurotiales, Trichocomaceae).6,7 Compounds 1−3 are the first isocoumarins and the first halogenated metabolites ever reported from the Hamigera genus.8 A discussion on their hypothetical biosynthesis is also presented herein. Fundación MEDINA continuously investigates its proprietary microbial collection to discover novel bioactive lead structures.9 During one of our screening campaigns, the soil fungus H. f usca NRRL 357216,7 was selected for further investigations based on the cytotoxicity of the acetone extract of its fermentation broth. Analysis by LC-UV-LRMS and dereplication against our in-house library10 revealed the presence of the cyclic pentapeptide avellanin A, the archetypical © XXXX American Chemical Society and American Society of Pharmacognosy

secondary metabolite of Hamigera species.11 Additionally, an abundant dichlorinated component was detected based on its characteristic isotopic pattern easily identifiable by lowresolution MS.12 Remarkably, no halogenated compounds had previously been detected in the Hamigera genus.8,11 Further LC-HRMS analysis was pursued to determine the Received: January 11, 2018

A

DOI: 10.1021/acs.jnatprod.8b00041 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Note

Table 1. 1H (500 MHz) and 13C (125 Hz) NMR Data of 1−3 (DMSO-d6, 24 °C) δC, type

δH, (J in Hz)

no.

1

2

3

1 3 4 4a 5 6 6-OCH3 7 8 8-OCH3 8a 9

157.6, C 154.4, C 105.1, CH 141.7, C 100.5, CH 165.2, C 55.8, CH3 98.4, CH 162.8, C 56.1, CH3 101.9, C 36.3, CH2

156.8, C 152.2, C 106.0, CH 140.6, C 101.8, CH 165.4, C 55.9, CH3 99.5, CH 162.9, C 56.2, CH3 102.3, C 60.9, CH

156.9, C 151.8, C 107.1, CH 140.5, C 101.6, CH 165.3, C 55.9, CH3 99.4, CH 162.9, C 56.2, CH3 102.6, C 58.6, CH

10 10-OH 11

72.2, CH

71.1, CH

70.2, CH

77.0, CH

46.4, CH2

47.8, CH2

1

2

3

6.45, s

6.79, s

6.78, s

6.62, d (2.3)

6.74, d (2.1)

6.69, d (2.1)

3.88, s 6.59, d (2.3)

3.89, s 6.68, d (2.1)

3.89, s 6.68, d (2.1)

3.86, s

3.88, s

3.88, s

2.82, 2.57, 4.16, 6.06, 6.27,

4.96, d (5.6)

4.77, d (9.1)

4.17, 6.07, 3.74, 3.59,

4.23, 6.07, 3.92, 3.89,

dd (14.5, 3.0) dd (14.5, 9.5) dddd (9.5, 6.4, 3.0, 3.0) d (6.4) d (3.1)

dddd (5.7, 5.7, 5.7, 5.3) br d (6.1) dd (11.4, 4.5) dd (11.4, 6.1)

br m br d (4.1) dd (11.6, 2.5) m

Figure 1. (A) Key COSY (bold bonds) and HMBC (arrows) correlations that determine the connectivity of compounds 2 and 3. (B) Key NOESY correlations (dashed arrows) and coupling constants of 2 and 3.

an R configuration at C-10.13,14 Analysis of the 1H and 2D NMR data of compounds 2 and 3 was straightforward by direct comparison with the data of 1. The key COSY and HMBC correlations observed for 2 and 3 (Figure 1A) showed that the two compounds share identical connectivity (8-methyl-9,11dichlorodiaporthin) being diastereomers of each other and regioisomers of 1 (8-methyl-11,11-dichlorodiaporthin). While compound 1 displays a geminal dichlorination pattern (at C11), 2 and 3 have the two chlorine atoms at different positions (C-9 and C-11). As later discussed, co-occurrence of 1−3 in H. f usca suggests a common biosynthetic precursor, desmethyldiaporthin (4)15 (also known as orthosporin),16 and thus the same configuration for C-10 in 1−4. Since 2 and 3 are epimers at C9, to distinguish the relative configuration of each diastereomer, NOE and J-based configuration analyses were carried out.17 Homonuclear 3JHH coupling constants were derived from the 1 H NMR spectra, while long-range heteronuclear 3JCH values were measured with the J-HMBC experiment18 and interpreted based on their Karplus-type dependence on dihedral angles.19,20 The energy-minimized molecular models of (9R*)-8-methyl9,11-dichlorodiaporthin (2) and (9S*)-8-methyl-9,11-dichlorodiaporthin (3) (Figure 1B) perfectly explained the NOEs and coupling constants observed. Both models display proton H-9 forming a dihedral angle close to zero with C-4, in agreement with the strong NOE observed between H-9 and H-4 and the 3 JCH values of 8.5 Hz (2) and 8.2 Hz (3) measured for the pair C-4/H-9. This orientation of H-9 in the models defines the actual rotamer occurring in solution along the C-3 to C-9 bond.

molecular formula of the target dichlorinated component. The accurate mass measured by ESI-TOF assigned the molecular formula C14H14Cl2O5. This formula was not encountered in our in-house dereplication library,10 and one hit was retrieved when querying the Dictionary of Natural Products database,8 corresponding to the fungal dichlorinated isocoumarin 8methyl-11,11-dichlorodiaporthin (1), also known as 8-methyldichlorodiaporthin (or 8-methyldichlorodiaportin).13,14 The UV (DAD) spectrum of the target component was compatible with the reported UV absorption maxima for 1.13 Nevertheless, an isomer of 1 or a closely related structure could not be completely discarded at this stage. We decided to pursue a bioassay-guided isolation to unambiguously establish the identity of the dichlorinated component and confirm its cytotoxicity. Very interestingly, this strategy rendered three compounds, 1−3, eluting at different retention times in semipreparative HPLC but which turned out to be isobaric (ESI-TOF), having thus identical molecular formula, C14H14Cl2O5. They also shared identical UV (DAD) spectra, immediately pointing out that the three compounds were isomers (regio- and/or stereoisomers). The NMR data of 1 (Table 1) perfectly matched those reported for 8-methyl-11,11dichlorodiaporthin, providing an unequivocal dereplication.14 This gem-dichlorinated isocoumarin was first isolated from a lichen mycobiont (Graphis sp.)13 and very recently from Aspergillus oryzae (Koji mold).14 The specific rotation of 1 {[α]25D +36.6 (c 0.05, CHCl3)} also matched the bibliographic value {[α]25D +36.6 (c 0.26, CHCl3)},13 indicating, as expected, B



Note

expression in this H. fusca strain will provide very vaulable information on gene regulation among filamentous fungi.23 The cytotoxicity of compounds 1−3 was evaluated against seven cancer cell lines (see Experimental Section), exhibiting moderate activity against two of them. Compound 1 was active against human fibroblasts (CCD25sk cells CC50 = 27.0 μM), while all three dichlorinated compounds 1−3 were active against neuroblastoma (SHSY5y cells: 1 CC50 = 19.4 μM, 2 CC50 = 39.2 μM, and 3 CC50 = 36.2 μM). These results correlate well with the cytotoxicity against HeLa cells recently reported for dichlorodiaporthin (6)4 (CC50 = 9 μg/mL = 28 μM),14 a compound considered as a mycotoxin.5 In conclusion, two novel epimeric non-geminally dichlorinated diaporthins, 2 and 3, have been isolated from the soil fungus H. f usca NRRL 35721 alongside the known regiosiomeric gem-dichlorinated diaporthin 1. These cytotoxic compounds are the first halogenated metabolites and the first isocoumarins ever reported from the Hamigera genus. Compounds 2 and 3 display an unprecedented non-geminal aliphatic dichlorination pattern among known fungal dihalogenated aromatic polyketides. Their production in a fermentation media not previously employed for Hamigera species (including the one herein studied) is yet another example of the value of surveying different nutritional conditions to awake otherwise silent gene clusters (one encoding a PKS in this case) and produce novel compounds.24−26 Further investigations to unravel the regulation of hexaketide isocoumarin (diaporthin scaffold) biosynthesis in this H. f usca strain are currently underway.

Next, the coupling constant measured for the pair H-9/H-10 was employed to define the rotamer along the C-9 to C-10 bond. In 2, this coupling constant equals 5.6 Hz, in agreement with the dihedral angle (78°) measured between these two protons in its minimized model. On the other hand, in 3, the corresponding coupling constant equals 9.1 Hz, in agreement with the dihedral angle (175°) measured between these two protons in its model. The absolute configuration in this series is thus 10R (1), 9R*, 10S* (2), and 9S*, 10S* (3). The biosynthesis of 8-methyl-11,11-dichlorodiaporthin (1) in Aspergillus oryzae RIB40 was recently unravelled by Hertweck and co-workers.14 They discovered an unprecedented hybrid flavoenzyme (AoiQ) containing two domains for both halogenation and O-methylation, which catalyzes the regioselective sequential geminal dichlorination of a nonactivated aliphatic carbon atom on the precursor substrate desmethyldiaporthin (4) to yield 1, which carry methoxy groups due to the action of the O-methylation domain of the bifunctional enzyme.14 The halogenation position, a nonactivated methyl group instead of the aromatic ring, is most unusual and suggested a mechanism strikingly different from canonical flavin-dependent halogenases involved in aromatic substitution reactions using a Cl+ equivalent.21 They also revealed by genome mining that, surprisingly, homologous hybrid enzymes are encoded in cryptic gene clusters from diverse ecologically relevant fungi.14 In fact, a thorough literature survey of naturally occurring dichlorinated aromatic polyketides of the diaporthin family already shows their presence in diverse fungi, all of them Ascomycota, from various orders (Table S1). The isolation of 8-methyl-11,11-dichlorodiaporthin (1) from the culture broth of H. fusca NRRL 35721 indicates that an AoiQ homolog must be involved in the biosynthesis of 1 in this fungus, further confirming the spread of the genes encoding these striking enzymes among diverse fungi. We hypothesize that this enzyme could also be involved in the biosynthesis of the new non-geminally dichlorinated diaporthins 2 and 3. Dichlorinated diaporthins 1−3 would all derive from the same precursor, desmethyldiaporthin (4),15,16 via the action of the putative AoiQ homolog of H. f usca. The occurrence of chlorine atoms at two different positions of the aliphatic side chain in the epimeric dichlorinated diaporthins 2 and 3 is compatible with the sequential dichlorination mechanism demonstrated for the diaporthin dihalogenase activity of AoiQ.14 However, this dichlorination pattern at two different positions in 2 and 3 is unique since to date only geminal dichlorination had been observed among halogenated diaporthins and related metabolites (Table S1). Not only are compounds 1−3 the first halogenated metabolites ever reported from Hamigera, but they are also the first isocoumarins found in this fungal genus. 8,11 Interestingly, the fermentation media herein employed, SMK, differs from the six media surveyed by Bills and co-workers in their thorough genus-wide assessment of chemical diversity in Hamigera.11 Since such study included, among others, the same H. f usca strain herein employed,11 it seems clear that our nutritional conditions for the fungus fermentation awake the otherwise silent polyketide synthase (PKS) gene cluster involved in the biosynthesis of the diaporthins scaffold. In fact, only after genetic activation of such a gene cluster had diaporthin (5) and related non-chlorinated congeners been detected in A. oryzae.22 Future investigations to unravel the modulation of the hexaketide isocumarin (diaporthin scaffold)

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured with a Jasco P-2000 polarimeter. IR spectra were registered with a JASCO FT/IR-4100 spectrometer equipped with a PIKE MIRacle single-reflection ATR accesory. NMR spectra were recorded on a Bruker Avance III spectrometer (500 and 125 MHz for 1H and 13 C NMR, respectively) equipped with a 1.7 mm MicroCryoprobe, using the signal of the residual solvent as internal reference (δH 2.50 and δC 39.5 ppm for DMSO-d6). LC-UV-MS and LC-HRESIMS analyses were performed as previously described10,27 on an Agilent 1100 single-quadrupole LC-MS system and a Bruker maXis QTOF mass spectrometer coupled to an Agilent 1200 LC. Flash chromatography was carried out on a CombiFlash Teledyne ISCO Rf400x system. Semipreparative RP-HPLC was performed on a Gilson GX-281 322H2 LC with UV−vis detection. Acetone used for extraction was analytical grade. Solvents employed for isolation were HPLC grade. Molecular models were generated using Chem3D Pro 12.0. The structures were energy-minimized by molecular mechanics with the MM2 force field using as gradient convergence criteria an RMS value of 0.001. The conformational search along the C-3 to C-9 and C-9 to C-10 bonds was carried out using the dihedral driver tool of the software to obtain the lowest-energy conformer for the side chain of compounds 2 and 3. Molecular modeling figures were generated with PyMol. Strain and Fermentation. The strain of Hamigera f usca employed (NRRL 35721) was isolated in 2006 from phenol-treated soil collected at a banana tree orchard in Maouéni, Grande Comore Island (The Comoros), and deposited at the Agricultural Research Service Culture Collection (NRRL) by G. F. Bills.6 A seed culture of the fungal strain was prepared by inoculating an Erlenmeyer flask (250 mL) containing 50 mL of SMYA medium (Bacto neopeptone 10 g, maltose 40 g, yeast extract 10 g, agar 3 g, and distilled H2O 1 L) with 10 mycelia agar plugs followed by incubation for 7 days on a rotary shaker at 220 rpm and 22 °C with 70% relative humidity. Aliquots of 3 mL of the seed culture were used to inoculate 1 L of SMK medium (soluble starch from potato 40 g, Bacto yeast extract 1 g, and Murashige & Skoog salts C



Note

4.3 g; 1 L of distilled H2O) distributed among 100 × 10 mL in 500 mL Erlenmeyer flasks. The flasks were incubated for 14 days on a rotary shaker at 220 rpm, 22 °C, and 70% relative humidity. Extraction and Isolation. The scaled-up fermentation broth was extracted with acetone (1 L) under continuous shaking at 220 rpm for 1 h. The biomass was separated by centrifugation, and the supernatant (ca. 2 L) was concentrated to 1 L under a stream of nitrogen. The solution was loaded (with continuous 1:1 water dilution) on a column packed with SP207SS resin (brominated styrenic polymer, 65 g) previously equilibrated with water. The loaded column was further washed with H2O (1 L) and afterward eluted at 8 mL/min on an automatic flash-chromatography system using a gradient from 10% to 100% acetone in water for 30 min with a final 100% acetone wash step for 15 min, collecting 19 fractions of 20 mL. Fractions were concentrated to dryness on a centrifugal evaporator. Bioassays revealed cytotoxicity in fractions 10−12. They were pooled and further fractionated by reversed-phase semipreparative HPLC (Agilent Zorbax Rx-C8, 9.4 × 250 mm, 5 μm; 3.6 mL/min, UV detection at 210 nm) with isocratic elution using 30% CH3CN in water (1% TFA) to yield three bioactive fractions (1.8 mL each), namely, impure fraction A (tR 23.2 min), fraction B corresponding to the pure compound 1 (17.0 mg, tR 24 min), and fraction C corresponding to the pure compound 3 (1.0 mg, tR 26 min). Impure fraction A was repurified eluting with 25% CH3CN in water (1% TFA) to yield pure compound 2 (tR 30 min, 300 μg). 8-Methyl-11-dichlorodiaporthin (1): pale yellow, amorphous solid; [α]25D +36.6 (c 0.05, CHCl3); UV (DAD) λmax 242, 283 (sh), 293 (sh), 328 nm; IR (ATR) νmax 3407, 2968, 2927, 1698, 1600, 1206, 1165 cm−1; 1H and 13C NMR, Table 1; HRESIMS m/z 333.0296 [M + H]+ (calcd for C14H15Cl2O5, 333.0291). (9R*)-8-Methyl-9,11-dichlorodiaporthin (2): pale yellow, amorphous solid; [α]25D −29 (c 0.05, CHCl3); UV (DAD) λmax 242, 283 (sh), 293 (sh), 328 nm; IR (ATR) νmax 3429, 3013, 2956, 1710, 1599, 1214, 1164 cm−1; 1H and 13C NMR, Table 1; HRESIMS m/z 333.0298 [M + H]+ (calcd for C14H15Cl2O5, 333.0291). (9S*)-8-Methyl-9,11-dichlorodiaporthin (3): pale yellow, amorphous solid; [α]25D +2 (c 0.05, CHCl3); UV (DAD) λmax 242, 283 (sh), 293 (sh), 328 nm; IR (ATR) νmax 3425, 3014, 2940, 1704, 1598, 1214, 1163 cm−1; 1H and 13C NMR, Table 1; HRESIMS m/z 333.0295 [M + H]+ (calcd for C14H15Cl2O5, 333.0291). Cytotoxicity Assay. The human-derived cell lines CCD25sk (human fibroblasts), SHSY5 (neuroblastoma), MiaPaca-2 (epithelial pancreas carcinoma), MCF-7 (breast adenocarcinoma), HepG2 (hepatocellular carcinoma), A2058 (epithelial melanoma), and A549 (lung carcinoma) were employed for the cytotoxicity MTT colorimetric assay, which was performed as previously described.28,29 Doxorubicin was selected as positive control rendering the following CC50 values: 0.85 μM for CCD25sk, 0.70 μM for SHSY5, 2.97 μM for MiaPaca-2, 7.0 μM for MCF-7, 1.95 μM for HepG2, 1.79 μM for A2058, and 10.5 μM for A549.

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ORCID

Ignacio Pérez-Victoria: 0000-0002-4556-688X Fernando Reyes: 0000-0003-1607-5106 Present Address

́ Instituto de Tecnologia Quimica e Biológica António Xavier, Universidade Nova de Lisboa, ITQB NOVA, Avenida da República, 2780-157 Oeiras, Portugal.

†

Author Contributions ‡

C. Almeida and I. Perez-Victoria contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS C.A. acknowledges funding from Fundaçaõ para a Ciência e Tecnologia, FCT, Portugal (fellowship SFRH/BPD/77720/ 2011). The polarimeter, FTIR spectrometer, HPLC, NMR spectrometer, and plate reader used in this work were purchased via grants for scientific and technological infrastructures from the Ministerio de Ciencia e Innovación [Grant Nos. PCT-010000-2010-4 (NMR), INP-2011-0016-PCT010000 ACT6 (polarimeter, HPLC, and FTIR), and PCT01000-ACT7, 2011-13 (plate reader)].

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00041. Description of the MTT cytotoxicity assay, table with known dichlorinated diaporthins and related metabolites, UV (DAD), HRESIMS, and NMR spectra of compounds 1−3 (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. (C. Almeida). *E-mail: [email protected]. Tel: +34 958993965, ext 7017. Fax: +34 958846710 (I. Pérez-Victoria). D



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(21) Agarwal, V.; Miles, Z. D.; Winter, J. M.; Eustáquio, A. S.; El Gamal, A. A.; Moore, B. S. Chem. Rev. 2017, 117, 5619−5674. (22) Nakazawa, T.; Ishiuchi, K. i.; Praseuth, A.; Noguchi, H.; Hotta, K.; Watanabe, K. ChemBioChem 2012, 13, 855−861. (23) Brakhage, A. A. Nat. Rev. Microbiol. 2013, 11, 21−32. (24) Bode, H. B.; Bethe, B.; Höfs, R.; Zeeck, A. ChemBioChem 2002, 3, 619−627. (25) Bills, G. F.; Platas, G.; Fillola, A.; Jiménez, M. R.; Collado, J.; Vicente, F.; Martín, J.; González, A.; Bur-Zimmermann, J.; Tormo, J. R.; Peláez, F. J. Appl. Microbiol. 2008, 104, 1644−1658. (26) Wiemann, P.; Keller, N. P. J. Ind. Microbiol. Biotechnol. 2014, 41, 301−313. (27) Martín, J.; Crespo, G.; González-Menéndez, V.; Pérez-Moreno, G.; Sánchez-Carrasco, P.; Pérez-Victoria, I.; Ruiz-Pérez, L. M.; González-Pacanowska, D.; Vicente, F.; Genilloud, O.; Bills, G. F.; Reyes, F. J. Nat. Prod. 2014, 77, 2118−2123. (28) Mosmann, T. J. Immunol. Methods 1983, 65, 55−63. (29) Cautain, B.; de Pedro, N.; Schulz, C.; Pascual, J.; Sousa, T. da S.; Martin, J.; Pérez-Victoria, I.; Asensio, F.; González, I.; Bills, G. F.; Reyes, F.; Genilloud, O.; Vicente, F. PLoS One 2015, 10, e0125221.

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Non-geminal Aliphatic Dihalogenation Pattern in Dichlorinated

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