Note Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
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Fusaristatin C, a Cyclic Lipodepsipeptide from Pithomyces sp. RKDO 1698 Logan W. MacIntyre,† Douglas H. Marchbank,‡,§ Hebelin Correa,§ and Russell G. Kerr*,†,‡,§ †
Department of Biomedical Sciences and ‡Department of Chemistry, University of Prince Edward Island, 550 University Avenue, Charlottetown, PE, Canada, C1A 4P3 § Nautilus Biosciences Croda, Regis and Joan Duffy Research Centre, 550 University Avenue, Charlottetown, PE, Canada, C1A 4P3
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S Supporting Information *
ABSTRACT: A new cyclic lipodepsipeptide, fusaristatin C (1), was obtained from the fungus Pithomyces sp. RKDO 1698, which was isolated from the Caribbean octocoral Eunicea f usca. The 2D structure of fusaristatin C was elucidated using NMR spectroscopy and mass spectrometry, while the absolute configuration of the sole chiral amino acid residue (L-serine) was determined using Marfey’s method. 3Hydroxy-2,11-dimethyltetradecanoic acid (HDMT) was cleaved from 1, and the absolute configuration at the C-3 position was determined using Mosher’s ester analysis. Subsequent J-based configuration analysis of 1 allowed for assignment of the C-2 configuration. Fusaristatin C exhibited no antimicrobial activity or cytotoxicity.
D
from E. f usca. It was observed that when fermented in yeast extract sucrose (YES) broth, the EtOAc extract of RKDO 1698 contained predominantly compound 1 in very high abundance. Using UHPLC-HRMS, we observed a protonated molecule (m/z 482.3219 [M + H]+) of 1 that after database queries (AntiBase 2014) yielded no matches of fungal origin within a 10 ppm mass accuracy window.8 This strongly suggested that 1 was an unreported natural product; moreover, its abundance and chromatographic resolution by UHPLC rendered it a good candidate for expedient purification and structure elucidation. Pithomyces sp. RKDO 1698 was fermented at a larger scale in glass culture tubes (total 1.56 L) to obtain higher quantities of 1, and the mycelia and cell-free broth (CFB) were extracted separately with EtOAc. Interestingly, the mycelia extract was highly enriched in 1, whereas this metabolite was barely detected in the CFB extract (Figure S2), suggesting that perhaps RKDO 1698 sequesters 1 in nature. Compound 1 was purified from the mycelia extract using automated reversedphase flash chromatography, and 240 mg of 1 was recovered. The chemical structure of 1 was elucidated using a combination of NMR spectroscopy, mass spectrometry, and chemical derivatization. Purified 1 was obtained as an amorphous, white solid, and ESI+ HRMS analysis supported a molecular formula of C25H43N3O6, which requires six degrees of unsaturation (Figure S3). The 2D structure of 1 (Figure 1) was elucidated using 1H, 13C, COSY, TOCSY, HSQC, HMBC, and ROESY NMR spectra (Figures S4−S10). The peptidic nature of 1 was apparent from three amide proton signals (ranging from 7.06
espite a long-standing recognition that marine environments harbor unique fungal taxa, organisms from these sources have only relatively recently gained widespread attention as sources of natural products with new chemical scaffolds. For example, 1000 new natural products have been isolated from marine-derived fungi during the period spanning from 1970 to 2010.1 In a previous study by our research group investigating the microbial diversity of the octocoral Eunicea f usca, an understudied microbial habitat, we isolated and identified the fungal strain Pithomyces sp. RKDO 1698.2 Reports of secondary metabolites from the genus Pithomyces date as early as 1960 when P. chartarum collected from pastures in New Zealand was shown to produce sporidesmin, a hepatotoxic metabolite that causes facial eczema in grazing sheep.3 This discovery stimulated additional prospecting efforts that afforded numerous depsipeptide metabolites including the sporidesmolides, pithomycolide, pimaydolide, and angolide (Figure S1).4−7 Herein we report the purification and chemical structure elucidation of a new cyclic lipodepsipeptide from RKDO 1698, fusaristatin C (1), which we categorized as a new member of the fusaristatin family of natural products (Chart 1). It is accompanied by fusaristatin A (2; Fusarium sp. YG-45), fusaristatin B (3; Fusarium sp. YG45), and congeners topostatin (4; Thermomonospora alba) and YM-17032 (5; unidentified fungus). Pithomyces sp. RKDO 1698 was isolated from the tissue of E. f usca, which was collected on a reef in Crab Cove, south Florida, USA, in June 2009. Nucleotide BLAST search results of rDNA genes (ITS and nLSU) placed this isolate within the genus Pithomyces.2 RKDO 1698 was fermented under various media conditions as part of a broader unpublished study examining the secondary metabolic potential of fungi isolated © XXXX American Chemical Society and American Society of Pharmacognosy
Received: September 14, 2018
A
DOI: 10.1021/acs.jnatprod.8b00787 J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
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Chart 1
correlations involving only the protons indicated in Figure 1 were discernible due to overlapping signals. HDMT is connected to (Dha-β-Ala-Ser) through amide and ester linkages at the N-terminus and C-terminus, respectively; therefore, 1 may be classified as a cyclic lipodepsipeptide. The position of the C-11 methyl branch within HDMT was assigned on the basis of 3J HMBC correlations between both CH3-11/C-14 and C-12. Four degrees of unsaturation were accounted for within (Dha-β-Ala-Ser), one by the HDMT amide and another by cyclization of (Dha-β-Ala-Ser) with HDMT. In addition to the discussed NMR evidence, MS/MS fragmentation (Figure S14) supports the proposed 2D planar structure of 1. It was expected that with the exception of C-11 the configurations of all stereogenic carbons within 1 could be readily determined. The Ser residue was first addressed using Marfey’s method.9 The acid hydrolysate of 1 in addition to LSer and DL-Ser was derivatized with 1-fluoro-2,4-dinitrophenyl5-L-alanine amide (L-FDAA) and then analyzed by UHPLCHRMS. By comparing the retention time of Ser-L-FDAA derived from 1 to that of the derivatized amino acid standards, 1 was found to contain L-Ser (Figure S15). Insoluble material generated during the hydrolysis of 1 was extracted from the acidified hydrolysate with hexanes and found to contain HDMT (6) by UHPLC-HRMS (m/z 271.2274 [M − H]−), which we suspected would be suitable for configuration analysis of C-3 (Figure S16). Given the potential for epimerization of 6 at C-2, the acid hydrolysis was repeated on a larger scale using DCl in D2O/CD3OD to introduce a deuterium label into any epimerized product. Fortunately, a deuterium-labeled 6 (m/z 272.2341 [M − H]−) was not observed by UHPLC-HRMS, indicating that epimerization did not occur and that cleaved 6 retained the same C-2 configuration as 1 (Figure 2). Compound 6 was purified using automated normal-phase flash chromatography, and its structure was elucidated using 1D and 2D NMR spectroscopy (Figures S17−S21). Mosher’s ester analysis was carried out on a sub-milligram quantity of 6 in order to determine the C-3 configuration of 1.
Figure 1. Key COSY, HMBC, and ROESY NMR correlations for fusaristatin C (1).
to 8.91 ppm) in the 1 H NMR spectrum and their corresponding carbonyl signals in the 13C spectrum (ranging from 163.6 to 170.6 ppm; Table 1). It was revealed in the COSY spectrum that only two of these protons belong to distinct 1H−1H spin systems, which suggested the presence of one α-unsaturated amino acid residue within a tripeptide moiety. Amino acid side chains were assigned using COSY, TOCSY, HSQC, and HMBC spectra, and the tripeptide was found to contain a dehydroalanine (Dha), β-alanine (β-Ala), and serine residue. Dha, an uncommon and nonproteinogenic amino acid, was identified based on unusually high 1H and 13C resonances at the α-position (137.0 ppm) and β-position (5.40/5.61 and 111.7 ppm), which are characteristic of an exocyclic methylene group. The order of amino acid residues was assigned using HMBC and ROESY correlations involving amide protons; it was determined to be (Dha-β-Ala-Ser). The nonpeptidic portion of 1 exhibited a molecular formula of C16H30O2 and was identified as a 3-hydroxy-2,11-dimethyltetradecanoic acid (HDMT). Although this group is expected to contain one continuous 1H−1H spin system, COSY B
DOI: 10.1021/acs.jnatprod.8b00787 J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
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Table 1. NMR Spectroscopic Data (1H 600 MHz, 13C 151 MHz, DMSO-d6) for Fusaristatin C (1) position Ser
β-Alaa
CO NH α β β-OH CO NH α β
Dhab
HDMTc
δC, type 169.9, 53.8, 60.9,
170.6,
δH, (J in Hz) C CH CH2
8.25, 4.48, 3.51, 3.58, 4.96,
d (8.7) m m m t (5.5)
7.06, 2.12, 2.30, 3.19, 3.62,
dd (8.0, 4.8) dt (13.7, 3.5) m m m
C
36.0,
CH2
35.4,
CH2
CO NH α β
163.6,
C
137.0, 111.7,
C CH2
1 2 2-CH3 3 4
172.2, 43.4, 14.6, 75.5, 28.8,
C CH CH3 CH CH2
5
31.0,
CH2
6d 7d 8d 9d 10
29.4, 29.0, 28.7, 26.3, 36.3,
CH2 CH2 CH2 CH2 CH2
11 11-CH3 12 13 14
31.9, 19.3, 31.3, 22.1, 14.0,
CH CH3 CH2 CH2 CH3
8.91, s 5.40, s 5.61, s 2.68, 1.07, 4.91, 1.49, 1.62, 1.09, 1.27, 1.22, 1.24, 1.24, 1.22, 1.06, 1.22, 1.34, 0.81, 1.23, 1.25, 0.85,
Figure 2. Determination of C-2 and C-3 configurations in fusaristatin C (1). (A) Mosher’s ester analysis of 3-hydroxy-2,11-dimethyltetradecanoic acid (HDMT; 6) cleaved from 1 with ΔδSR displayed in ppm. Configuration analysis (J-based) of eight possible gauche rotamers of (B) 2S,3S-1 and (C) 2R,3S-1. The magnitudes of relevant 2 J and 3J values (S = small and L = large) were predicted for each rotamer and compared to experimental values obtained using 1H and HETLOC NMR [3JH2H3 = 4.4 Hz (S), 2JH2C3 = −4.0 Hz (L), 3JH3CH3 = 1.8 Hz (S)]. Rotamer iv agrees best with experimental coupling constants and led to the assignment of 2R,3S-1. Predicted NOEs denoted with black arrows (for iv only) agree with correlations observed in a 1D selective-gradient ROESY spectrum of 1.
m (7.1) d (7.1) td (7.5, 4.4) m m m m m m m m m m m d (6.7) m m t (7.1)
extracted from the HETLOC spectrum on account of insufficient signal-to-noise, and thus our relative configuration assignment may be subject to interpretation. Compound 1 bears strong structural resemblance to the fusaristatins, topostatin, and YM-170320. The name fusaristatin C was chosen in an effort to provide consistent naming for 1−5.13−17 Compounds 1−5 are comprised of a tripeptide core containing an N-terminal Dha residue that is cyclized via an α-methyl-β-hydroxy acyl moiety, but each contains a different C-terminal amino acid. Fusaristatin C is structurally unique in that it lacks the characteristic arachidic acid skeleton and 3-aminoisobutyric acid residue that 2−5 possess. Compound 1 instead contains an HDMT moiety and β-Ala residue, respectively. It is worth noting that although YM170320 was not reported as a cyclic structure, we speculate that perhaps it exists as such in its natural state and that it was methanolyzed during purification because analogous degradation was observed by UHPLC-HRMS during the purification of 1 with acidic mobile phases. Compound 1 was tested for antimicrobial activity in microbroth dilution assays against methicillin-resistant Staphylococcus aureus (MRSA), S. warneri, vancomycin-resistant Enterococcus faecium (VRE), Pseudomonas aeruginosa, Proteus vulgaris, Mycobacterium tuberculosis, and Candida albicans. It was also tested for cytotoxicity against three healthy cell lines (Vero, HEKa, and BJ) and three cancer cell lines (HTB-22, HTB-26, and HCT-116). Compound 1 exhibited no antimicrobial activity at the highest concentration tested (128 μg/mL or 265 μM) and was not cytotoxic toward any of the six cell lines tested. Dose−response curves and IC50
a β-Ala: β-alanine. bDha: dehydroalanine. cHDMT: 3-hydroxy-2,11dimethyltetradecanoyl group. dOverlapping signals may be interchanged.
Values for ΔδSR are displayed in Figure 2A and are consistent with the S configuration at C-3 (Figure S22). The relative configuration of C-2 and C-3 was deduced using J-based configuration analysis (Figure 2B and C).10−12 The magnitudes of 3JHH in addition to various 2JCH and 3JCH values were predicted for rotamers of 2R,3S-1/2S,3S-1 and then compared to experimentally determined values for 1 obtained using 1H and HETLOC NMR experiments (Figure S13). Since 3JH2H3 was measured as 4.4 Hz, rotamers i, iii, iv, and v represent plausible configurations of 1. Rotamer iv is most consistent with the measured values of 2JH2C3 (−4.0 Hz) and 3JH3CH3 (1.8 Hz), leading to the assignment of 2R,3S-1. The 2R,3S configuration is also supported by comparison of predicted NOEs to data obtained using a 1D selective-gradient ROESY experiment. As predicted for rotamer iv, we observed a ROESY correlation between H-3/CH3-2 and H-3/H-2, whereas strong correlations between H-4a/H-2 and H-4b/H-2 were not observed (Figures S11 and S12). It should be noted that additional JCH values pertinent to this analysis could not be C
DOI: 10.1021/acs.jnatprod.8b00787 J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
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streptomycin sulfate, and 100 μg/mL chloramphenicol in filtered seawater), a white filamentous fungal strain (RKDO 1698) was isolated. The fungus was taxonomically identified using sequence homology of rDNA genes, including the ITS1-5.8S-ITS2 region and the nLSU region. A seed culture of Pithomyces sp. RKDO 1698 was prepared by inoculating 13 mL of SMY broth (10 g/L peptone, 40 g/ L maltose, 10 g/L yeast extract, and 18 g/L Instant Ocean in deionized H2O) contained in a 60 mL glass culture tube with cryopreserved mycelia. The seed culture was fermented for 7 days on a rotary shaker (200 rpm at 22.0 °C), after which it was homogenized with glass beads using a vortex mixer. The homogenized seed culture was used to inoculate 120 glass culture tubes containing 13 mL of YES broth (20 g/L yeast extract,150 g/L sucrose, and 0.5 g/L MgSO4·7H2O in deionized H2O) each with approximately 50 μL. The culture tubes were distributed between two racks that were adjusted such that the tubes lay at 45° with respect to the horizontal and were fermented without agitation at 22.0 °C for 14 days. Extraction and Purification. After 14 days of fermentation, all culture tubes contained a mat of mycelia on the broth surface. The mats were removed from their respective tubes, pooled, and then rinsed with deionized H2O. The remaining broth from each tube was also pooled. Both the mycelia and broth were extracted separately with 1.5 L of EtOAc in 3 L Fernbach flasks. The extracts were dried in vacuo to yield 2.91 g (mycelia) and 0.60 g (broth) of dried material, which was analyzed by UHPLC-HRMS. As 1 was contained almost entirely in the mycelia extract, this extract was chosen for further purification. The mycelia extract was fractionated by automated reversed-phase flash chromatography (Teledyne ISCO CombiFlash Rf) using a 120 g C18 column (Redisep Rf High Performance GOLD) and eluted using the following elution method: 95% H2O (solvent A)/5% CH3OH (solvent B) from 0 to 5 min, linear gradient from 5% solvent B to 100% B from 5 to 40 min and 100% solvent B from 40 to 50 min (flow rate = 60 mL/min). The eluent was detected by UV (227 nm), and 1 eluted between 38.9 and 40.7 min. All fractions within this window were dried in vacuo and analyzed by UHPLCHRMS and those that were deemed pure were pooled to afford 240 mg of 1. Fusaristatin C (1): amorphous, white solid; [α]24.4D = +1.4 (c 0.28, CH3OH); UV (CH3CN/H2O) λmax 230 nm; IR νmax = 1623, 1658, 1730, 2850, 2920, 2954, 3278, 3322, 3346, and 3457 cm−1; see Table 1 for 1H and 13C NMR data; ESI+ HRMS m/z 482.3219 [M + H]+ (calcd for C25H44N3O6+, 482.3225). Amino Acid Configuration Using Marfey’s Method. To a vial containing dried 1 (5 mg, 0.01 mmol) was added 6 M HCl (1 mL), and then the mixture heated to 80 °C for 1 h with stirring. The reaction mixture was dried in vacuo, and a portion of the resulting hydrolysate (1 mg) was transferred to a separate vial, to which 150 μL of deionized H2O, 300 μL of L-FDAA (10 mg/mL in acetone), and 70 μL of aqueous NaHCO3 (1 M) were added. The reaction mixture was heated to 37.0 °C for 2 h, quenched with 70 μL of HCl (1 M), and then dried in vacuo. The same process was carried out with L-serine (1 mg, 0.01 mmol) and DL-serine (1 mg, 0.01 mmol) to afford L-FDAAderivatized serine standards. L-FDAA-derivatized hydrolysate and standards were suspended in CH3OH (500 μg/mL) for UHPLCHRMS analysis. The following chromatographic conditions were used: Thermo Hypersil Gold 100 Å UHPLC column (1.9 μm C18, 50 mm × 2.1 mm), 95% H2O with 0.1% (v/v) formic acid (solvent A): 5% CH3CN with 0.1% (v/v) formic acid (solvent B) from 0 to 55 min followed by a linear gradient from 5% B to 40% B from 55 to 57 min and then 40% solvent B from 57 to 60 min, flow rate of 400 μL min−1 and injection volume of 10 μL. Configuration Analysis of 3-Hydroxy-2,11-dimethyltetradecanoic Acid. Compound 1 (50 mg, 0.1 mmol) was dissolved in 5 mL of DCl (3 M in a 3:1 mixture of D2O/CD3OD) and stirred at 80.0 °C for 1 h. The reaction mixture was extracted three times with 5 mL of hexanes, and the pooled extracts were dried in vacuo to afford 39 mg of material. The entire extract was fractionated by automated flash chromatography using a 12 g silica column (InnoFlash, Canadian Life Sciences) and eluted with 100% EtOAc for 10 min (flow rate = 30 mL/min). All fractions were dried in vacuo, and those containing 6
values are provided in Figure S23. Screening protocols are also provided in the Supporting Information. In conclusion, we report a new Pithomyces cyclic lipodepsipeptide, fusaristatin C, which shares structural features with several natural products of both fungal and bacterial origin. With the exception of the C-11 position, we assigned the absolute configuration using chemical derivatization and Jbased configuration analysis. Fusaristatin C did not exhibit biological activity against the microbial pathogens or cell lines tested. This is the first report of a natural product from a coralderived Pithomyces sp.
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EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotation was measured on a Rudolph Autopol III polarimeter using a 50 mm microcell (1.2 mL). Infrared (IR) spectra were recorded using attenuated total reflectance on a Thermo Nicolet 6700 FT-IR spectrometer. All NMR spectra (unless specified) were acquired on a Bruker Avance III NMR spectrometer (1H: 600 MHz, 13C: 151 MHz) equipped with a 5 mm cryoprobe and located at Agriculture and AgriFood Canada (AAFC) in Charlottetown, PE, Canada. All chemical shifts are reported in ppm and referenced to residual solvent signals [1H (DMSO-d6): 2.50 ppm, 13C (DMSO-d6): 39.51 ppm, 1H (CD3OD): 3.31 ppm, and 13C (CD3OD): 49.00 ppm]. The HETLOC and 1D-ROESY NMR experiments were acquired on a 400 MHz NMR with the dipsi2etgpjcsix1 pulse program. Default acquisition parameters were used except for the following modifications: td (f2) = 6144; td (f1) = 512; ns = 128; sw (f2 and f1) = 4.80 ppm; o1p (f2 and f1) = 2.9 ppm; cnst2 = 145.0; cnst16 = 5.0; gpz1 = 13.0%; gpz2 = 19.0%; gpz3 = 30.0%. The HETLOC spectrum was zero-filled to 8192 and 2048 in the f2 and f1 dimensions, respectively. Tandem mass spectra were acquired by direct infusion on a Thermo Velos Orbitrap mass spectrometer using a collision-induced dissociation energy of 30 eV at a rate of 2 μL/min. UHPLC-HRMS analyses (unless specified) were carried out using a Thermo Accela chromatograph equipped with HRMS-ELSD-UV detection: a Thermo LTQ Exactive fitted with an ESI source, Sedex 80 LT-ELSD, and Thermo PDA, respectively. The following chromatographic conditions were used: Kinetex core−shell 100 Å UHPLC column (2.1 × 50 mm, 1.7 μm, Phenomenex), mobile-phase flow rate of 0.5 mL/min, injection volume of 10 μL (all samples were prepared in CH3OH), and a linear gradient from H2O/CH3CN (95:5, 0.1% formic acid) at 0.2 min to 100% CH3CN (0.1% formic acid) at 4.8 min, which was held until 8.0 min before returning to H2O/ CH3CN (95:5, 0.1% formic acid) for a 1.5 min equilibration. The following HRMS parameters were used: positive ionization mode, mass resolution of 30 000, mass range of m/z 190 to 2000, spray voltage of 2.0 kV, capillary temperature of 300 °C, S-lens RF voltage of 60.0%, maximum injection time of 10 ms, and 1 microscan. The system was controlled by Thermo Xcalibur software modules. All reagents were purchased from commercial sources and used without further purification. All solvents used for purification were of HPLC grade or higher. Isolation and Fermentation of Pithomyces sp. RKDO 1698. E. fusca samples were collected by hand using scuba at a depth of 12.0 m from a reef in Crab Cove, south Florida, USA (26°18.736′ N, 80°03.583′ W) on June 2, 2009. Healthy branches were aseptically removed from a coral colony, brought to the ocean surface, and stored at 18−22 °C (ocean temperature). Approximately 4 h postcollection, the branches were aseptically cut into 0.5−1.0 cm sections, transferred to separate sterile 50 mL conical tubes, and then washed three times with filter-sterilized seawater (0.22 μm cellulose acetate filters from Corning) to remove loosely associated surface bacteria. Branch samples were homogenized in filter-sterilized seawater and then separated according to particle size (≥500, ≥213, ≥104, ≥51, and
