Cysteine-Derived Pleurotin Congeners from the Nematode-Trapping

Jan 22, 2018 - (2) Although currently pleuromutilins constitute the only basidiomycete-derived antibiotics on the market,(3) the rich secondary metabo...
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Article Cite This: J. Nat. Prod. 2018, 81, 286−291

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Cysteine-Derived Pleurotin Congeners from the Nematode-Trapping Basidiomycete Hohenbuehelia grisea Birthe Sandargo,†,‡ Benjarong Thongbai,§ Marc Stadler,†,‡ and Frank Surup*,†,‡ †

Department of Microbial Drugs, Helmholtz Centre for Infection Research GmbH, Inhoffenstraße 7, 38124 Braunschweig, Germany German Centre for Infection Research (DZIF), partner site Hannover-Braunschweig, 38124 Braunschweig, Germany § School of Science, Mae Fah Luang University, Chiang Rai 57100, Thailand ‡

S Supporting Information *

ABSTRACT: The discovery of a Hohenbuehelia grisea specimen during a field trip in Northern Thailand led to the isolation and identification of three novel sulfur-bearing derivatives of dihydropleurotinic acid (4). Thiopleurotinic acid A (1) was established by the interpretation of spectral data (HRESIMS, 2D-NMR) as a 2-hydroxy-3-mercaptopropanoic acid conjugate of dihydropleurotinic acid. Thiopleurotinic acid B (2) was shown to be the N-acetylcysteine conjugate of 4. A third compound (3) was established as a thiazole-containing derivative. Through feeding experiments with [U−13C3, 15N]-L-cysteine the formation of all three metabolites was shown to involve cysteine condensation with 4. The decreased cytotoxicity and antimicrobial activities of the new derivatives 1−3, compared to the parent compound 4, indicate a possible detoxification pathway of filamentous fungi.

F

ungi are excellent producers of terpenes and related terpenoids, a highly diverse group of natural products.1 Basidiomycota are especially good producers of elaborate terpenoids, yet have been studied little in terms of their bioactive metabolites.2 Although currently pleuromutilins constitute the only basidiomycete-derived antibiotics on the market,3 the rich secondary metabolism of fungi constitutes an excellent source for further bioactive entities. The basidiomycete-derived antibiotic of partial terpenoid origin, pleurotin (5), was first isolated in 1947 from cultures of Pleurotus griseus Peck,4 a fungus whose currently accepted name is Hohenbuehelia grisea (Peck) Singer. Pleurotin (5), the dihydro form leucopleurotin, and dihydropleurotinic acid (4) have long been known to show anticancer effects,5,6 as well as to possess activity against Gram-positive bacteria4,5 and pathogenic fungi.7 The total synthesis of (±)-pleurotin (5)8 and its production in multigram scale by fermentation9 have also been reported. More recently, pleurotin (5) regained interest, as it was found to act as a highly potent inhibitor of the thioredoxin (Trx)− thioredoxin reductase (TrxR) system,10 a promising target in cancer treatment and mercury intoxication,11 making pleurotin (5) a potential new drug in the fight against cancer. Herein, we report the isolation, structure elucidation, and biological evaluation of three novel sulfur-bearing dihydropleurotinic acid derivatives, thiopleurotinic acids A (1) and B (2) and pleurothiazole (3). All three metabolites were isolated together with their known parental metabolites dihydropleurotinic acid (4) and pleurotin (5) from crude extracts of submerged cultures of the basidiomycete strain MFLUCC 120451 collected in Thailand. © 2018 American Chemical Society and American Society of Pharmacognosy

Figure 1. Structures of new compounds thiopleurotinic acid A (1), thiopleurotinic acid B (2), and pleurothiazole (3) and parental metabolites dihydropleurotinic acid (4) and pleurotin (5).

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RESULTS AND DISCUSSION As outlined in the Supporting Information, the producer strain was identified as Hohenbuehelia grisea (Peck) Singer by comparison of its morphological features and its 5.8S/ITS/ LSU rDNA sequences. To the best of our knowledge, this is the first record of the occurrence of this species in Thailand and Asia. Hohenbuehelia is well-known to be the sexual morph of Received: August 21, 2017 Published: January 22, 2018 286

DOI: 10.1021/acs.jnatprod.7b00713 J. Nat. Prod. 2018, 81, 286−291

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Table 1. NMR Spectroscopic Data (1H 500 MHz, 13C 125 MHz, 15N NMR 71 MHz, Acetone-d6) for Thiopleurotinic acid A (1), Thiopleurotinic acid B (2), and (1H 700 MHz, 13C 176 MHz, 15N NMR 71 MHz, Acetonitrile-d3) Pleurothiazole (3) 1 position

δC, type

1

34.7, CH2

2

25.0, CH2

3 4 5 6

46.4, 32.1, 52.0, 21.9,

7

30.6, CH2

8 9 10 11

42.8, 44.4, 46.9, 74.6,

12 13 14

20.7, CH3 175.9, C 24.6, CH2

15 16 17 18 19 20 21 22

71.9, CH 140.4, C 141.3, C 183.2, C 127.0, CH 150.7, C 184.2, C 34.6, CH2

23 24 25 26 N

69.2, CH 173.1, C

CH CH CH CH2

CH CH C CH2

2 δH (J in Hz)

δC/δN, type

2.09, m 1.23, m 1.74, m

35.8, CH2

2.14, 2.20, 1.69, 1.92, 1.76, 1.60, 2.09, 2.18, 1.96,

47.5, 33.2, 53.1, 23.0,

m m m m m dtd (13.0, 12.0, 4.4) m m m

3.97, dd (12.4, 8.6) 3.33, dd (12.4, 6.6) 0.93, d (7.0) 2.54, ddd (19.8, 2, 1.6) 2.47, ddd (19.8, 5.0, 2.8) 4.49, dd (2.8, 1.6)

6.60, s

3.38, dd (13.7, 3.9) 3.20, dd (13.7, 6.7) 4.55, dd (6.7, 3.9)

25.6, CH2 CH CH CH CH2

31.7, CH2 43.8, 45.4, 48.0, 75.7,

CH CH C CH2

21.7, CH3 177.0, C 25.6, CH2 73.0, CH 141.4, C 142.5, C 184.2, C 128.0, CH 151.3, C 185.2, C 32.8, CH2 52.0, CH 172.1, C 171.0, C 23.2, CH3 116.4, NH

3 δH (J in Hz)

δC/δN, type

2.09, m 1.24, m 1.74, m

34.3, CH2

2.14, 2.20, 1.69, 1.92, 1.77, 1.60, 2.08, 2.18, 1.96,

46.5, 34.5, 53.2, 23.1,

m m m m m m m m m

3.97, dd (12.4, 8.5) 3.33, dd (12.4, 6.6) 0.93, d (7.0) 2.54, ddd (19.6, 2.2, 1.5) 2.47, ddd (19.6, 5.0,3.0) 4.49, dd (3.0, 1.5)

6.60, s

3.42, dd (13.3, 4.7) 3.27, dd (13.3, 7.8) 4.79, td (7.8, 4.7)

1.95, s 7.61, d (7.7)

the nematode-trapping hyphomycete genus Nematoctonus, and pleurotins have accordingly also been isolated previously from strains assigned to both aforementioned genera.4 Thiopleurotinic acid A (1) was obtained as an orange powder and showed a molecular ion cluster [M + H]+ at m/z 477.1579, revealing a molecular formula of C24H28O8S, indicating 11 degrees of unsaturation. The 1H NMR data (Table 1) exhibited a characteristic aromatic singlet methine (δH 6.60), as well as signals of seven nonaromatic methines (δH 1.69, 1.96, 2.14, 2.18, 2.20, 4.49, 4.55). Additionally, the 1H NMR data showed signals of seven methylenes (δH 1.23, 1.60, 1.74, 1.76, 1.92, 2.09, 2.47, 2.54, 3.20, 3.33, 3.38, 3.97) and one doublet of a methyl (δH 0.93). Examination of 13C and DEPT data revealed 24 carbons, of which two are typical signals for carboxylic acids (δC 173.1, 175.9), two carbonyls (δC 183.2, 184.2), one olefinic methine (δC 127.0), three sp2 carbons (δC 140.4, 141.3, 150.7), one aliphatic carbon (δC 46.9), seven methylene carbons (δC 21.9, 24.6, 25.0, 30.6, 34.6, 34.7, 74.6), seven methines (δC 32.1, 42.8, 44.4, 46.4, 52.0, 71.9), and one methyl carbon (δC 20.7). The correlation of these 1D data with 1 1 H, H COSY, 1H,13C HMBC (Figure 2), and 1H,1H ROESY (Figure 3) data led to the establishment of the known compound dihydropleurotinic acid (4) as the underlying core

26.3, CH2 CH CH CH CH2

32.0, CH2 44.8, 46.8, 48.6, 78.0,

CH CH C CH2

21.7, CH3 177.5, C 29.0, CH2 83.1, CH 132.0, C 120.6, C 158.3, C 106.7, CH 135.4, C 147.4, C 152.3, CH

δH (J in Hz) 1.92, 1.44, 1.89, 1.78, 2.31, 2.14, 1.89, 1.96, 1.85, 1.67, 2.11, 2.30, 2.06,

m m m m m m m m m dddd (13.0,12.5,12.0,5.0) m ddd (12.5, 12.0, 4.1) ddd (12.5, 5.8, 1.4)

4.15, dd (12.9, 7.6) 3.97, dd (12.9, 2.2) 1.06, d (7.3) 3.27, br d (18.4) 3.21, dd (18.4, 5.8) 5.14, br s

OH: 9.14, br s 7.24, s

8.83, s

312.7, N

Figure 2. Key COSY (bold lines) and HMBC correlations (arrows) of thiopleurotinic acid A (1).

structure with the same stereochemical configuration.5 The additional signals in our spectra assembled a 2-hydroxy-3mercaptopropanoic acid moiety, which is attached to C-20 via a thioether link, as demonstrated by the HMBC correlation from H-22 to C-20. Feeding of [U−13C3 15N]-L-cysteine to the fungus indicates the 2-hydroxy-3-mercaptopropanoic acid moiety is derived from cysteine. HRMS/MS data of compound 4 after feeding [U−13C3 15N]-L-cysteine show peaks at m/z 477.1616 [M + H]+ belonging to the molecular formula 12 C24H29O8S and m/z 480.1723 [M + H]+ of 12C2113C3H29O8S. The solitary MS/MS fragment at m/z 357.1723 [M + H]+ (indicated in red, Figure 4) belonging to dihydropleurotinic acid shows the enriched 13C carbons being part of the cleaved 287

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HSQC data. 13C NMR data (Table 1) also exhibited an additional signal typical of carboxylic acids at δC 171.0, as well as a signal for an additional methyl group (δC 23.2). 1H,1H COSY and 1H,13C and 1H,15N HMBC (see Supporting Information) revealed an N-acetylcysteine moiety linked to dihydropleurotinic acid via a thioether at position C-20. Feeding of [U−13C3 15N]-L-cysteine also revealed the Nacetylcysteine to be derived from cysteine, and thus, the configuration of C-23 was deduced as R. The molecular formula C22H25NO4S of pleurothiazole (3), a light brown powder, was obtained by HRESIMS, entailing 11 degrees of saturation. NMR data pointed to a structural similarity to metabolites 1 and 2, with variations in the olefinic part of dihydropleurotinic acid (4). The 13C NMR data (Table 1) showed upfield shifts of the olefinic carbons indicating the quinone moiety being present in its leuco form. Further, the 13 C NMR data exhibit an additional peak for one aromatic methine (δC 152.3) characteristic for a C−N double bond. HSQC data show the corresponding proton shift at δH 8.83, also being characteristic for a thiazole moiety. HMBC correlations of H-22 to C-20 and C-21 establish this thiazole moiety is attached to the aromatic part of leuco-dihydropleurotinic acid. The positioning of the sulfur and nitrogen atom in the thiazole ring can be deduced from 13C chemical shifts and is supported by the biosynthesis argument and the absence of an HMBC correlation between N and H-19 in a 1H,15N HMBC experiment, which would be expected if the nitrogen was linked to C-20. Through feeding [U−13C3, 15N]-L-cysteine to the fungal culture, the thiazole structure was shown to be cysteine derived (Figure 4). The simple thiazole heterocycle is a common moiety in nature; it is usually biosynthesized by cyclization of a cysteine side chain onto the preceding carbonyl group in a peptide chain.13 Benzothiazoles, however, are relatively rare natural products. During biosynthesis, a p-benzoquinone moiety serves as the electrophile in a 1,4 Michael addition with cysteine; by this means benzothiazole and its simple derivatives, the well-known firefly luciferin and thioazorifamycins, are formed.13−16 Metabolites 1−4 were tested for their antimicrobial and cytotoxic properties (see Table S1, Supporting Information). Pleurotin (5) and dihydropleurotinic acid (4) are known to exhibit antibacterial and cytotoxic properties.4−6,17 In our study, dihydropleurotinic acid (4) showed antimicrobial activity against fungi and Gram-positive bacteria, as well as cytotoxic effects against the tested cell lines KB3.1 and L929, summarized in Table 2. However, none of the newly isolated metabolites 1− 3 showed any signs of cytotoxic effects against the tested cell lines (Table 2). Thiopleurotinic acid A (1) did not exhibit any antimicrobial activities against the test organisms, while thiopleurotinic acid B (2) and pleurothiazole (3) possessed only a weak activity against yeasts, such as Candida tenuis, Pichia anomala, and Rhodotorula glutinis (Table 2). This can be explained due to pleurotin’s activity being mainly based on the p-benzoquinone moiety, whereas condensation with cysteine to the p-benzoquinone moiety was demonstrated to inactivate pleurotin.17 While cysteine conjugation is a common practice in the detoxification process in plants18,19 and animals, little is known about these detoxification pathways in fungi.20 Recently, with the isolation of cyclothioculvularins, a detoxification via mercaptopyruvate condensation to macrocyclic polyketides by Penicillium species (Ascomycota) was discovered.21 The discovery of metabolites 1 and 2 provides additional under-

Figure 3. Key ROESY correlations of thiopleurotinic acid A (1).

Figure 4. HRESIMS/MS Profile of [U-13C315N]-L-cysteine feeding to Hohenbuehelia grisea MFLUCC 12-0451. (a) MS/MS profile of pleurothiazole peak, m/z 402.1613 [M + H]+ of 12C2113C1H2615NO4S. (b) MS/MS profile of thiopleurotinic acid B (2) peak, m/z 522.2038 [M + H] + of 12 C 23 13 C 3 H 32 15 NO 8 S. (c) MS/MS profile of thiopleurotinic acid A (1) peak, m/z 477.1616 [M + H]+ of 12 C24H29O8S and m/z 480.1723 [M + H]+ of 12C2113C3H29O8S.

2-hydroxy-3-mercaptopropanoic acid moiety. The absolute configuration of thiopleurotinic acid A (1) was established with Mosher’s method.12 The (S)- and (R)-MTPA esters were generated by treating metabolite 1 with (R)-(−) and (S)-(+) MTPA chloride, respectively. An R-configuration was assigned for C-23 based on ΔδSR = δ(S-MTPA ester) − δ(R-MTPA ester) for neighboring protons (see Table S3, Supporting Information), leading to the 3S,4S,5S,8R,9S,10R,15S,23R absolute configuration of thiopleurotinic acid A (1). A second, structurally related compound, thiopleurotinic acid B (2), was isolated as a brown powder with a molecular formula of C26H31NO8S as deduced from HRESIMS, implying 12 degrees of unsaturation and indicating the presence of one nitrogen atom. The 1H NMR data (Table 1) of compound 2 resembled in large part those of compound 1 with an additional doublet, belonging to a secondary amine, as revealed by 1H,15N 288

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Table 2. Minimal Inhibitory Concentration (MIC for Bacteria and Fungi) and Half-Inhibitory Concentrations (IC50 for Cell Lines) in μg/mLa organism Mucor plumbeus MUCL49355 Candida tenuis MUCL29892 Bacillus subtilis DSM10 Escherichia coli DSM498 Schizosaccharomyces pombe DSM70572 Pichia anomala DSM6766 Candida albicans DSM1665 Rhodotorula glutinis DSM10134 Mucor hiemalis DSM2656 Micrococcus luteus DSM1790 Staphylococcus aureus DSM346 Mycobacterium smegmatis DSM44200

1 −c − − − − − − − − − − −

2 − 100 − − − 66.7 − 33.3 − − − −

MIC [μg/mL] 3 − 100 − − − 66.7 − 33.3 − − − −

4b 100 25 50 − − 66.7 33.3 16.7 8.3 66.7 33.3 −

5b 100 100 100 − − 66.7 − 33.3 16.7 − 66.7 −

7.5 8.5

2.2 2.8

ref (MIC) nystatin nystatin penicillin ciprofloxacin nystatin nystatin nystatin nystatin nystatin oxytetracycline oxytetracycline kanamycin

(12.5) (12.5) (6.3) (3.1) (16.7) (16.7) (16.7) (16.7) (16.7) (0.4) (6.7) (0.4)

IC50 [μg/mL] cell line L929 KB3.1

− −

− −

− −

a For MICs, a total of 20 μL of either 1 mg/mL stock solution (67 μg/mL) or 1.5 mg/mL (100 μg/mL) of 1−5 was tested. Cell density was adjusted to 6.7 × 105 cells/mL; 20 μL of ethanol was tested as negative control and showed no activity against the selected test organisms. For IC50 values, 6 × 103 cells/well were seeded in 96-well microtiter plates and treated with 1−5 for 5 days. bPleurotin (4) and dihydropleurotinic acid (5) were used for comparison. c−, no inhibition observed under test conditions.

standing of detoxification mechanisms in filamentous fungi. Pleurothiazole (3) also appears to be a detoxification product based on its missing bioactivity. Further research is essential to elucidate this detoxification pathway in general.

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DNA extraction was performed using the EZ-10 Spin Column Genomic DNA Miniprep kit (Bio Basic Canada Inc., Markham, Ontario, Canada) and performed as reported previously.22 A Precellys 24 homogenizer (Bertin Technologies, France) was used for cell disruption at a speed of 6000 rpm for 2 × 40 s. The DNA regions were amplified using standard primers ITS 1f23 and NL424 following a protocol described by Otto et al.25 For a detailed description of the producer strain as well as the morphological characterization, see the Supporting Information. Fermentation and Isolation of Metabolites 1−3. A well-grown culture of H. grisea strain MFLUCC 12-0451 on BAF (DSMZ 392) agar was used to inoculate 200 mL of BAF media in a 500 mL Erlenmeyer flask, incubated on a rotary shaker at 24 °C and 140 rpm. After 14 days, 30 mL of this primary culture was transferred into 6 × 2 L Erlenmeyer flasks with 800 mL of medium each. These cultures were incubated at 24 °C and 140 rpm on rotary shakers until depletion of free glucose after 21 days. For extraction, the supernatant was separated from the mycelia using a Sorvall RC-5B centrifuge (DuPont Instruments, Wilmington, DE, USA). The supernatant was extracted by adding 5% Amberlite XAD 16N absorbent (Rohm & Haas Deutschland GmbH, Frankfurt am Main, Germany) and stirring for 2 h. After sieving, the XAD absorbent was extracted using acetone. The acetone was evaporated (30 °C), and the residue diluted in water and extracted with ethyl acetate. The organic phase was dried over sodium sulfate and evaporated to dryness (30 °C), leading to 4 g of crude extract. The crude extract was filtered using an SPME Strata-X 33 u Polymeric RP cartridge (Phenomenex, Inc., Aschaffenburg, Germany) and prefractionated using an RP-MPLC [Kronlab ODS-AQ 120 16 C18, 48 × 3 cm column (YMC Europe GmbH, Dinslaken, Germany), solvent A: water (Milli-Q), solvent B: acetonitrile; gradient: 10% B increasing to 100% B in 100 min and staying at 100% B for 20 min; flow rate 20 mL/min, UV detection at 220 nm]. The isolation of compounds 1 and 2 from an MPLC fraction (tR 43−49 min) was performed using preparative NP-HPLC [Orbit 100 Diol column, 250 × 20 mm, 5 μm (MZ-Analysentechnik, Mainz, Germany); solvent A: 75% n-heptane−25% tert-butyl methyl ether, solvent B: 67% tert-butyl methyl ether−23% n-heptane−10% acetonitrile, gradient: 100% A for 15 min, then increasing to 40% B within 35 min, afterward increasing to 100% B within 20 min, staying at 100% B for another 20 min; UV detection at 215 and 248 nm].

EXPERIMENTAL SECTION

General Experimental Procedures. HRESIMS mass spectra were measured using the Agilent 1200 series HPLC-UV system in combination with an ESI-TOF-MS (Maxis, Bruker) [column 2.1 × 50 mm, 1.7 μm, C18 Acquity UPLC BEH (Waters), solvent A: 95% 5 mM ammonium acetate buffer (pH 5.5, adjusted with 1 M acetic acid) with 5% acetonitrile, solvent B: 95% acetonitrile with 5% 5 mM ammonium acetate buffer]. The separation was achieved using a gradient from 10% solvent B increasing to 100% solvent B within 30 min, maintaining 100% B for another 10 min, Rf = 0.3 mL min−1, UV detection 200−600 nm]. NMR spectra were recorded on a Bruker Avance III 500 MHz spectrometer with a BBFO(plus) SmartProbe (1H 500 MHz, 13C 126 MHz) and a Bruker Avance III 700 MHz spectrometer with a 5 mm TCI cryoprobe (1H 700 MHz, 13C 175 MHz, 15N 71 MHz). Chemical shifts δ were referenced to the solvents: acetone-d6 (1H, δ = 2.05 ppm; 13C, δ = 29.3 ppm), acetonitrile-d3 (1H, δ = 1.94 ppm; 13C, δ = 1.9 ppm), chloroform-d (1H, δ = 7.27 ppm; 13 C, δ = 77.0 ppm), pyridine-d5 (1H, δ = 7.22 ppm; 13C, δ = 123.9 ppm). UV spectra were recorded using a Shimadzu UV-2450 UV−vis spectrophotometer. Optical rotation was determined using a PerkinElmer 241 polarimeter. Isolation and Identification of Hohenbuehelia grisea (Peck) Singer MFLUCC 12-0451. The fungal specimen was collected and isolated by B.T. from decaying wood in the tropical rainforest near the Mushroom Research Centre, Chiang Mai Province, Thailand (http:// www.mushroomresearchcentre.com/), in August of 2012. The species was identified based on morphology and rDNA sequence comparison [5.8S gene region, the internal transcribed spacer 1 and 2 (ITS) and part of the large subunit (LSU)]. The sequence data are deposited with GenBank, accession number MF150036. The dried specimen and corresponding cultures were deposited at the mycological herbarium of Mae Fah Luang University, Chiang Rai, Thailand, under the accession number MFLUCC 12-0451. 289

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Compound 1 (22.6 mg) eluted at 60−63 min, and 2 (90.7 mg) at 66− 69 min. Compound 3 was obtained from an MPLC fraction eluting at tR 82−120 min via RP-HPLC [VP Nucleodur 100-5 C18 ec column, 250 × 40, 7 μm (Macherey-Nagel, Düren, Germany), solvent A: water (Milli-Q), solvent B: acetonitrile; gradient: 30% B for 5 min, then increasing to 100% B in 45 min, staying at 100% B for 10 min; flow rate 30 mL/min; UV detection at 215 and 222 nm]. The fraction at tR 35−38.5 min was reinjected to RP-HPLC [VP Nucleodur 100-5 C18 ec column, 250 × 21, 5 μm (Macherey-Nagel, Düren, Germany), solvent A: water (Milli-Q), solvent B: acetonitrile; gradient: 60% B for 5 min, increasing to 80% B within 25 min; flow rate 15 mL/min; UV detection at 222 and 272 nm] for final purification, providing 7 mg of 3, eluting at 16−18 min. Thiopleurotinic acid A (1): orange powder; [α]21D +42 (c 1, ACN); UV (CHCl3) λmax (log ε) 226 (164.8), 294 (10.4), 431 (4.1) nm; 1H NMR and 13C NMR (Table 1); HRESIMS m/z 477.1579 [M + H]+ (calcd for C24H29O8S 477.1578). Thiopleurotinic acid B (2): brown powder; [α]21D +22 (c 1, ACN); UV (CHCl3) λmax (log ε) 226 (160.2), 298 (4.3), 430 (1.8) nm; 1H NMR, 13C NMR, and 15N NMR (Table 1); HRESIMS m/z 518.1844 [M + H]+ (calcd for C26H32NO8S 518.1843). Pleurothiazole (3): light brown powder; [α]21D +78 (c 1, ACN); UV (CHCl3) λmax (log ε) 236 (160.2), 277 (31.9), 303 (ε = 20.6) nm; 1 H NMR, 13C NMR, and 15N NMR (Table 1); HRESIMS m/z 400.1585 [M + H]+ (calcd for C22H26NO4S 400.1577). Preparation of the (R)- and (S)-MTPA Ester Derivatives of 1. Compound 1 (2.0 mg) dissolved in deuterated pyridine (1 mL) was transferred into two NMR tubes (0.5 mL each). (R)-(−)-α-Methoxyα-(trifluoromethyl)phenylacetyl chloride (2 μL) was added to one NMR tube, and (S)-(+)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride (2 μL) was added into the other NMR tube, immediately before 1H NMR measurement, as previous attempts have shown a decomposition of compound 1 after 15 min following the addition of Mosher chloride. The reaction was monitored by 1H NMR followed by a selective 1D TOCSY to extract coupling constants from overlapping multiplets. (S)-MTPA ester of thiopleurotinic acid A (1a): 1H NMR (700 MHz, pyridine-d5) δ 6.92 (1H, s), 6.03 (1H, dd, J = 4.6 Hz, 3.4 Hz), 4.72 (1H, s), 4.19 (1H, dd, J = 6.8 Hz, 2.5 Hz), 4.30 (1H, dd, J = 15.0 Hz, 3.4 Hz), 3.65, dd, J = 14.8 Hz, 4.5 Hz), 3.54 (1H, m), 3.10 (1H, m), 2.64 (1H, m), 2.53 (1H, m), 2.27 (2H, m), 2.23 (1H, m), 2.21 (1H, m), 2.06 (1H, m), 1.98 (1H, m), 1.86 (1H, m), 1.71 (1H, m), 1.67 (1H, m), 1.60 (1H, m), 1.54 (1H, ddd, J = 12.8 Hz, 6.6 Hz, 3.6 Hz), 1.16, (1H, m), 0.85 (3H, d, J = 7.3 Hz). (R)-MTPA ester of thiopleurotinic acid A (1b): 1H NMR (700 MHz, pyridine-d5) δ 6.92 (1H, s), 6.06 (1H, dd, J = 8.2 Hz, 3.9 Hz), 4.72 (1H, s), 4.19 (1H, dd, J = 6.8 Hz, 2.5 Hz), 3.73 (1H, dd, J = 14.2 Hz, 3.0 Hz), 3.64, dd, J = 13.3 Hz, 8.6 Hz), 3.53 (1H, m), 3.10 (1H, m), 2.64 (1H, m), 2.53 (1H, m), 2.27 (2H, m), 2.23 (1H, m), 2.21 (1H, m), 2.06 (1H, m), 1.98 (1H, m), 1.86 (1H, m), 1.71 (1H, m), 1.67 (1H, m), 1.60 (1H, m), 1.54 (1H, ddd, J = 12.6 Hz, 6.6 Hz, 3.6 Hz), 1.16, (1H, m), 0.85 (3H, d, J = 7.3 Hz). [U−13C3, 15N]-L-Cysteine Feeding. Feeding experiments with [U−13C3, 15N]-L-cysteine were conducted in 2 × 20 mL of culture media, supplemented with [U−13C3, 15N]-L-cysteine (0.1 mg/mL) in 60 mL glass tubes. These tubes were each inoculated with three H. grisea MFLUCC 12-0451 mycelial balls of 3−5 mm diameter and incubated at 24 °C and 140 rpm until depletion of free glucose levels after 21 days. Mycelia and culture broth were extracted in one with ethyl acetate, evaporated to dryness (30 °C), and subjected to HPLCMS to detect the prevailing secondary metabolites. Antibacterial Evaluation. Minimum inhibitory concentrations (MIC) were determined in a serial dilution assay in 96-well microtiter plates in YMG medium for yeasts and filamentous fungi and EBS medium (0.5% casein peptone, 0.5% glucose, 0.1% meat extract, 0.1% yeast extract, 50 mM HEPES [11.9 g/L] and pH = 7.0) for bacteria, as described earlier.26 Cytotoxicity Assay. The in vitro cytotoxicity assay was carried out against the mouse fibroblast cell line L929 and HeLa (KB3.1) cells according to a previously described protocol.27

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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.7b00713. 1 13 H, C, 1H,13C, and 1H,15N HSQC, 1H,13C and 1H,15N HMBC, COSY, TOCSY, and ROESY spectra of 1−4; HRESIMS data of 1−4; Mosher data analysis; morphological analyses of H. grisea MFLUCC 14-051 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel (F. Surup): +49 531 6181 4256. E-mail: frank.surup@ helmholtz-hzi.de. ORCID

Marc Stadler: 0000-0002-7284-8671 Frank Surup: 0000-0001-5234-8525 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to W. Collisi for assisting with the bioassays, to C. Kakoschke and C. Bergmann for recording NMR and HPLC-MS data, respectively, and to C. Chepkirui for valuable scientific discussions. Financial support by the German Academic Exchange Service (DAAD) and the Thai Royal Golden Jubilee-Industry Program (RGJ) for a joint TRFDAAD PPP (2014-2015) academic exchange grant to M.S. and the RGJ for a personal grant to B.T. (No. Ph.D/0138/2553 in 4.S.MF/53/A.3) is gratefully acknowledged.

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REFERENCES

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