Halogenated Bianthrones From Penicillium roseopurpureum : a Fungal Endophyte of the Marine Alga Petalonia fascia

Abstract

An extract of the fungus Penicillium roseopurpureum (KP1-135C) isolated from the marine alga Petalonia fascia showed selective antimicrobial activity against Staphylococcus aureus and Mycobacterium tuberculosis H37Ra. Bioassay-guided fractionation revealed that three halogenated bianthrones, neobulgarone D, neobulgarone E, and neobulgarone F, were responsible for the observed activity of the extract. The stereochemistry of the neobulgarones was unambiguously assigned based on polarimetric data and the analysis of ¹H-nuclear magnetic resonance data obtained for the three bianthrones.

Although marine organisms are a prolific source of bioactive natural products, ^{1 -4} endophytes from marine algae represent an underinvestigated source of biodiversity. ⁵ During our investigation of endophytes of marine macroalgae, the extract of a Penicillium roseopurpureum isolate (KP1-135C) obtained from the brown alga Petalonia fascia (false kelp) was selected for bioassay-guided fractionation as it selectively inhibited the growth of Staphylococcus aureus and Mycobacterium tuberculosis H37Ra in our screening assays.

Solid-phase extraction, reversed-phase flash column chromatography, and reversed-phase high-performance liquid chromatography (HPLC) of the ethyl acetate (EtOAc) extract of a 2-week, bench-scale (2 L) fermentation of KP1-135C led to the isolation of 3 optically inactive bianthrones. Analysis of nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HR-MS) data determined the bianthrones to be neobulgarone D (1), neobulgarone E (2), and neobulgarone F (3) (Figure 1), assignments that were consistent with those reported in the literature. ⁶

Figure 1

Structures of neobulgarone D (1), neobulgarone E (2), and neobulgarone F (3).

Neobulgarones D, E, and F were first isolated as moderately cytotoxic inhibitors of appressorium formation from the fungus Neobulgaria pura, ⁶ with neobulgarone E subsequently being reported from an endophytic Penicillium species isolated from Limonium tubiflorum. ⁷ However, elucidating the stereochemistry of bianthrones is challenging due to the occurrence of mesomeric dimers and racemic mixtures, ⁸ presumably formed through non-enzymatic oxidative coupling reactions. Methods that rely on polarimetric data often lead to erroneous stereochemical assignments and are therefore untenable. ⁸ As such, when originally isolated, the configurations of 1, 2, and 3 were not determined and, unfortunately, 2 was later incorrectly reported to have syn (cis) relative stereochemistry on the basis of polarimetric data. ⁷ A procedure to unambiguously distinguish between diastereomeric bianthrones based upon detailed analysis of their molecular conformations and the shielding effects observed in their ¹H-NMR spectra was recently reported. ⁸ Application of these analyses to our data confirmed the relative configurations of neobulgarones D (1), E (2), and F (3) as cis, trans, and cis, respectively, and our polarimetric data were consistent with 1 and 2 being obtained as racemic mixtures of the 10R, 10′S/10S, 10′R, and 10R, 10′R/10S, 10′S diastereomers and 3 as the R, S mesomer. It should be noted that, although it has been reported that some bianthrones can epimerize and undergo photo-oxidation under acidic conditions, ⁸ at no stage of the work described here did we observe any interconversion of 2 and 3. There was also no evidence of neobulgarone C (ie, the diastereomer of 1) being present in the initial fungal extract nor of its formation during the fractionation of the extract. We therefore conclude that compounds 1 -3 are stable under the conditions employed in this study.

In our hands, the limited aqueous solubility of the bianthrones made the acquisition of bioassay data challenging. Despite this, we were able to reveal moderate, selective antibiotic activity for neobulgarones D, E, and F (Table 1) although, cognizant of the limitations of the bioactivity data obtained, the absolute median inhibitory concentrations should be evaluated with some caution. ⁹ Only the cis isomers, neobulgarones D and F, were active against M. tuberculosis whilst they also displayed superior activity against S. aureus. However, the trans isomer neobulgarone E was alone in its ability to inhibit the growth of methicillin-resistant S. aureus. These preliminary structure–activity data are intriguing and may warrant further investigation, particularly if the observed antibiotic activity is enantiospecific.

Table 1

Relative and Absolute Median Inhibitory Concentrations ⁹ (IC₅₀ Values; µM) of Neobulgarones D, E, and F Against Staphylococcus aureus ATCC 29213, Methicillin-Resistant S. aureus ATCC 33591 (MRSA), Mycobacterium tuberculosis H37Ra ATCC 25177, Pseudomonas aeruginosa ATCC 10145, and Candida albicans ATCC 14053.

Compound	Relative [absolute] IC50 (95% CI a ), µM
	S. aureus	MRSA	M. tuberculosis	P. aeruginosa	C. albicans
1	36.3 (30.1-41.1) [167 (117-268)]	NA b	46.1 (44.6-47.7) [80.7 (64.1-103)]	NA	NA
2	96.9 (77.0-126) [325 (234-533)]	67.4 (53.9, 78.4) [142 (104, 210)]	NA	NA	NA
3	58.3 (45.5-74.0) [111 (86.1-149)]	NA	31.1 (29.7-32.5) [35.1 (31.8-39.2)]	NA	NA

^a95% confidence interval.

^bNA: not active; less than 50% growth inhibition at 200 µM.

Experimental

General Experimental Procedures

Solvents for extraction and isolation were purchased from Fisher Scientific (Ottawa, ON, Canada), and deuterated solvents for NMR spectroscopy were purchased from Sigma-Aldrich (Oakville, ON, Canada). Solid-phase extraction was performed using Sep-Pak C18 Cartridges (55-105 µm, 125 Å, 2 g; Waters, NA, USA). Flash chromatography was performed using a Biotage Flash+chromatography system fitted with C18 (reversed-phase) SiliaSep cartridges (40-63 µm, 60 Å, 25 g; SiliCycle, QC, Canada). Semi-preparative reversed-phase HPLC was performed on a Phenomenex Luna C18 column (250 × 10 mm, 10 µm, 100 Å) using an Agilent 1100 HPLC system comprising a G1311A quaternary pump and a G1315C diode array detector. Optical rotations were recorded on an Optical Activity Ltd. AA-10 polarimeter at 589 nm. NMR spectra were recorded on an Agilent 400-MR DD2 instrument in deuterated solvents and were calibrated to residual protonated solvent resonances. HR-MS data were recorded on a Thermo LTQ Exactive instrument with an electrospray ionization (ESI) source.

Endophyte Isolation

KP1-135C was isolated from the marine macroalga P. fascia collected from the shores of Green’s Point, L’Etete, NB, Canada (45°02.363′’N, 066°53.483′W) in July 2013. Portions (5 cm in length) of algal tissue were individually sterilized by sequential immersion in 6.0% sodium hypochlorite (10 seconds), sterile distilled water (10 seconds) and 70% ethanol (15 seconds). The tissue was then rinsed with sterile distilled water, blotted dry on an autoclaved paper towel, and cut into pieces (0.60 cm in diameter) that were placed onto 2.0% malt extract agar and incubated at room temperature under ambient light. Endophytic fungi were subcultured onto fresh 2.0% malt extract agar until pure cultures were obtained.

Endophyte Identification

Isolate KP1-135C was identified as P. roseopurpureum through the examination of spore morphology and colonies grown on cornmeal, Czapek-Dox, malt extract, and potato dextrose agar. The taxonomic classification was confirmed by comparison of the internal transcribed spacer (ITS) and 5.8S rRNA gene deoxyribonucleic acid (DNA) regions with corresponding sequences available in the GenBank database (National Center for Biotechnology Information, US National Library of Medicine, Bethesda, MD, USA). The genomic DNA of KP1-135C was isolated using a DNEasy plant mini kit (Qiagen, Toronto, Ontario) as directed by the manufacturer; the ITS gene was amplified by polymerase chain reaction using the ITS1 and ITS4 universal fungal primers (Invitrogen, Burlington, Ontario) as previously described, ¹⁰ and the amplified ITS DNA was sequenced by Genome-Québec (Montreal, Québec). The KP1-135C DNA sequence was checked for ambiguity before being compared with existing GenBank sequence data using the Basic Local Alignment Search Tool. The ITS gene sequence of KP1-135C was found to have >99% homology with numerous conspecific P. roseopurpureum isolates and has been deposited in GenBank (accession number: KY054768).

Biological Assays

Antifungal activity against C. albicans (ATCC 14053), antibacterial activity against S. aureus (ATCC 29213) and Pseudomonas aeruginosa (ATCC 10145) methicillin-resistant S. aureus (ATCC 33591), and antimycobacterial activity against M. tuberculosis H37Ra (ATCC 25177) was evaluated as previously described. ^10,11

Fermentation and Extraction

KP1-135C was fermented in 2.0% malt extract broth at room temperature with shaking (150 rpm) for 2 weeks (2 L; 20 × 100 mL batches in 250 mL Erlenmeyer flasks stoppered with foam baffles). Fermentation cultures were then sonicated for 30 seconds. Next, the fungal material was removed by filtration before the spent broth was extracted with EtOAc (3 × 660 mL). The organic fractions were combined and concentrated in vacuo (193 mg).

Fractionation

The MEB extract was divided into 6 equal masses and each loaded onto a separate 2 g C-18 Sep-Pak Cartridge and eluted first with water (H₂O) (14 mL) then methanol (MeOH) (14 mL) then EtOAc (14 mL). The MeOH fraction (150 mg) was subjected to C-18 flash chromatography (stepwise gradient from 100% H₂O to 100% acetonitrile [CH₃CN] in 10% increments) to give 11 fractions. The fractions that eluted from the flash column in 1:1 H₂O/CH₃CN (25 mg) 2:3 H₂O/CH₃CN (17 mg) were subjected to reverse-phase HPLC (1:1 H₂O/CH₃CN) to give 1 (2 mg), 2 (3 mg), and 3 (10 mg).

Neobulgarone D (1)

$[\alpha {]}_{\mathrm{D}}^{22}$ : 0 (c 0.05, MeOH) and 0 (c 0.05, acetone); ¹H and ¹³C NMR data were consistent with literature values, ⁶ see supplemental table S1 and figures S1 and S2; HRESIMS: m/z 573.1307 [M + H]⁺ (calcd for C₃₂H₂₆ClO₈ ⁺, 573.1316).

Neobulgarone E (2)

$[\alpha {]}_{\mathrm{D}}^{22}$ : 0 (c 0.05, MeOH) and 0 (c 0.05, acetone); ¹H and ¹³C NMR data were consistent with literature values, ⁶ see supplemental table S2 and figures S3 and S4; HRESIMS: m/z 607.0930 [M + H]⁺ (calcd for C₃₂H₂₅Cl₂O₈ ⁺, 607.0926).

Neobulgarone F (3)

$[\alpha {]}_{\mathrm{D}}^{22}$ : 0 (c 0.05, MeOH) and 0 (c 0.05, acetone); ¹H and ¹³C NMR data were consistent with literature values, ⁶ see supplemental table S3 and figures S5 and S6; HRESIMS: m/z 607.0936 [M + H]⁺ (calcd for C₃₂H₂₅Cl₂O₈ ⁺, 607.0926).

Supplemental Material

Supplementary material - Supplemental material for Halogenated Bianthrones From Penicillium roseopurpureum: a Fungal Endophyte of the Marine Alga Petalonia fascia

Supplemental material, Supplementary material, for Halogenated Bianthrones From Penicillium roseopurpureum: a Fungal Endophyte of the Marine Alga Petalonia fascia by Nicholas J. Morehouse, Andrew J. Flewelling, John A. Johnson and Christopher A. Gray in Natural Product Communications