Macrocyclic Trichothecenes from Myrothecium roridum Strain M10

Aug 29, 2015 - The cytotoxicity of the extract obtained from Myrothecium roridum M10 and a characteristic 1H signal at δH ∼8 led to the assumption ...
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Macrocyclic Trichothecenes from Myrothecium roridum Strain M10 with Motility Inhibitory and Zoosporicidal Activities against Phytophthora nicotianae Muhammad Abdul Mojid Mondol,† Musrat Zahan Surovy,§ M. Tofazzal Islam,§ Anja Schüffler,# and Hartmut Laatsch*,† †

Institute for Organic and Biomolecular Chemistry, Georg-August-University Göttingen, Tammannstrasse 2, D-37077 Göttingen, Germany § Department of Biotechnology, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur 1706, Bangladesh # Institute of Biotechnology and Drug Research, D-67663 Kaiserslautern, Germany S Supporting Information *

ABSTRACT: The cytotoxicity of the extract obtained from Myrothecium roridum M10 and a characteristic 1H signal at δH ∼8 led to the assumption that verrucarin/roridin-type compounds were present. Upscaling on rice medium led to the isolation of four new metabolites: verrucarins Y (1) and Z (6) (macrocyclic trichothecenes), bilain D (12) (a diketopiperazine derivative), and hamavellone C (14) (an unusual cyclopropyl diketone). In addition, nine known trichothecenes [verrucarin A (3), 16hydroxyverrucarin A (5), verrucarin B (7), 16-hydroxyverrucarin B (8), verrucarin J (2), verrucarin X (4), roridin A (9), roridin L-2 (10), and trichoverritone (11)] and a bicyclic lactone [myrotheciumone A (15)] were identified. Their structures and configurations were determined by spectroscopic methods, published data, Mosher’s method, and considering biosyntheses. Some trichothecenes showed motility inhibition followed by lysis of the zoospores against devastating Phytophthora nicotianae within 5 min. Compounds 2, 3, 7, and 9 also exhibited potent activities against Candida albicans and Mucor miehei. KEYWORDS: antibiotic activity, structure elucidation, Phytophthora, roridin, verrucarin

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INTRODUCTION

Endophytic fungi reside in the tissue of plants, forming a diverse group of organisms ubiquitous in the plant kingdom, and can be either mutualistic or latently pathogenic to their hosts. Endophytic fungi produce secondary metabolites that defend their hosts against pathogens including oomycetes,2 insects, and herbivores3−7 in exchange for carbohydrate energy resources. Datura metel (the thorn-apple), belonging to the Solanaceae family, is one of the most important medicinal plants in Bangladesh. This plant has many medicinal properties and is also known for its pesticidal activity.8 The endophytic fungi isolated from this plant have been reported to produce cytotoxic components.9 As a part of our ongoing search for motility inhibitory and zoosporicidal metabolites, the endophytic fungus Myrothecium roridum M10 was isolated from the leaves of D. metel. This strain was cultured using a rice medium, and the extract subsequently obtained showed signals at δH ∼ 8 in the 1H NMR spectrum characteristic of verrucarin/roridintype compounds.10 As verrucarin and roridin are known for their cytotoxic activity, the culture of this strain was scaled up to isolate sufficient quantities of the active components for structural elucidation and subsequent investigations of their biological activities against P. nicotianae zoospores. Herein, we

Peronosporomycetes (oomycetes) are notorious fungus-like plant pathogens responsible for destroying a broad range of economically important agricultural crops and ornamental plants.1 One of the Peronosporomycetes, Phytophthora nicotianae (previously denominated Phytophthora parasitica), is a devastating pathogen with a wider host range than any other described Phytophthora species, infecting more than 100 genera of plants. Economically important host plants destroyed by P. nicotianae include solanaceous crops, many orchard tree crops, tropical fruits, oilseeds, vegetables, and many ornamental plants,1 resulting in multibillion dollar crop losses globally. In general, plant diseases caused by members of the Phytophthora genus are difficult to control using synthetic fungicides, as they are physiologically different from true fungi. Moreover, the currently available synthetic fungicides are deleterious to human health and the ecosystem, thus warranting the search for safer and environmentally friendly pesticides from natural sources. One of the important characteristics of Phytophthora pathogens is the asexual production of biflagellate motile zoospores in their early life stages. This zoosporic stage is the most sensitive phase in the lifecycle and is used for dispersal and plant infection. Natural products that either inhibit the motility of zoospores or cause lysis would be good candidates in the search for effective pesticides to control Phytophthora and other related phytopathogens. © XXXX American Chemical Society

Received: May 12, 2015 Revised: August 28, 2015 Accepted: August 29, 2015

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DOI: 10.1021/acs.jafc.5b02366 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Figure 1. Structures of compounds 1−15. Sigma-Aldrich, Steinheim, Germany. Ciprofloxacin was obtained from Square Pharmaceutical Co., Bangladesh. Solvents used for extraction and chromatography were distilled prior to use. For HPLC-MS, HPLC grade solvents (LiChrosolv, Merck AG, Darmstadt, Germany) were used. Fungal Material and Identification. Healthy stems (∼4 cm length) of D. metel were collected from the garden of medicinal plants, Department of Pharmacy, Rajshahi University, Bangladesh, and kept in plastic zipper packs at 4 °C for 2 months. The surface sterilization of each stem was done according to a previously described procedure.2 The sterilized stems were air-dried on sterile filter paper, and the bark was removed by a sterile knife. Pieces of the stem (∼1 × 1.5 cm) were then placed on a water-agar Petri dish (18 g agar per L tap water) and incubated at 25 °C. The outgrowing endophytic fungal strain M10 was isolated and maintained on M2 agar medium (10 g of glucose, 4 g of yeast extract, 4 g of malt extract, 18 g of agar, and 1 L of tap water). The strain M10 was identified by its ITS and morphological features. The ITS sequence of M10 showed 99.8% homology to the ITS sequence of M. roridum strain CBS 331.51 (GenBank accession no. HQ115647). The M10 strain is kept in the Microbial Culture Collection at the Institute for Organic and Bimolecular Chemistry,

report the structure determination and biological activities of compounds 1−12, 14, and 15 (Figure 1) isolated from this fungus.

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MATERIALS AND METHODS

Safety Information. Trichothecenes are highly toxic inhibitors of the protein biosynthesis. They may penetrate the skin and have been discussed as potential biological warfare agents (“yellow rain”). General Experimental Procedures. Optical rotation values were measured on a PerkinElmer polarimeter, model 241, at the sodium D line (λ = 589 nm). The NMR spectra were recorded on a Mercury-300 (300.141 MHz), a Varian Inova 500 (125.707 MHz for 13C NMR spectra), and a Varian Inova 600 (599.740 MHz) spectrometer. IR spectra were taken on a Jasco FT/IR-4100 type A instrument. Chemical shifts (δ) were referenced to CH3OH at 3.30 for 1H and at 49.0 for 13C and to CHCl3 at 7.24 for 1H and at 77.0 for 13C. ESIHRMS spectra were acquired on a micrOTOF 10237. Size exclusion chromatography was performed on Sephadex LH-20 purchased from Sigma-Aldrich Chemie, Steinheim, Germany. Open column chromatography was done on silica gel 60 (0.063−0.20 mm), and PTLC was performed on silica gel P/UV254 (both obtained from Macherey-Nagel, Düren, Germany). Mosher’s reagent and nystatin were bought from B

DOI: 10.1021/acs.jafc.5b02366 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Table 1. 13C (125 MHz) and 1H (600 MHz) NMR Data for 1 and 6 in CDCl3

Georg-August-University Göttingen, Germany, under the curatorship of Prof. Laatsch. Fermentation and Isolation. For the seed culture, the fungal strain M10 was subcultured on M2 agar medium and incubated at 25 °C for 3 days. For the batch fermentation, 200 g of rice and 150 mL of tap water were added to each of 24 P-flasks (28 × 17 × 7 cm) and then sterilized for 15 min at 121 °C. From the seed culture, the mycelium was scraped out using a sterile loop, spread in 200 mL of sterile water, and then added into each P-flask containing the rice medium. The flasks were incubated at 30 °C for 20 days, and then the whole culture was extracted with EtOAc and filtered through Celite (diatomaceous earth). The filtrate was concentrated to dryness in vacuo using a rotary evaporator at 40 °C to obtain a reddish gum. The concentrated extract was dissolved in MeOH and treated with cyclohexane to remove fats. The methanol was then evaporated, and the extract was partitioned between EtOAc and distilled water. The water phase was evaporated to dryness (1.5 g) and separated into 30 fractions (F1−F30) using Sephadex LH-20 column chromatography, eluting with CH2Cl2/MeOH (1:1). Compounds 3 (6.0 mg from F7− F9) and 4 (4.5 mg from F16−F26) were isolated by PTLC using 10% MeOH in CH2Cl2. Compounds 5 (10.0 mg), 10 (6.0 mg), and 11 (6.5 mg) were obtained from F12 in a similar way, eluting with CHCl3/nhexane/MeOH (4:1:1). The EtOAc phase was also evaporated to dryness (10.0 g) and fractionated by silica gel column chromatography using a stepwise gradient: 2% MeOH in CH2Cl2 (F1−F24), 7% MeOH in CH2Cl2 (F25−F35), and 12% MeOH in CH2Cl2 (F36−F43). Compounds 1 (5.4 mg from F34), 2 (5.0 mg, F25), 6 (4.5 mg, F34), 7 (5.0 mg, F24), 8 (13.0 mg, F24), 9 (15.0 mg, F36−F37), 12 (6.0 mg, F28), 14 (1.0 mg, F10), and 15 (5.0 mg, F29) were isolated by first subfractionating each fraction on Sephadex LH-20 (CH2Cl2/MeOH 4:6) and finally by PTLC (2, 8, 9, and 12 with CHCl3/MeOH/n-hexane 2:0.4:0.6; 1, 6, and 7 with EtOAc/CHCl3/n-hexane 1:1:1; 14 and 15 with CH2Cl2/ MeOH 95:5). Verrucarin Y (1): colorless, amorphous solid; [α]20 D +6 (c 0.54, CHCl3); IR (neat) νmax 3221, 2963, 1711, 1650, 1414, 1359, 1266, 1219, 1184, 1086, 1040 cm−1; 1H and 13C NMR data (CDCl3), see Table 1; (+)-ESI-HRMS m/z 537.1731 [M + Na]+ (calcd for C27H30O10Na, 537.1731). Verrucarin Z (6): colorless, amorphous solid; [α]20 D +22 (c 0.12, CHCl3); IR (neat) νmax 3166, 2927, 1711, 1411, 1198, 1082, 1002, 962 cm−1; 1H and 13C NMR data (CDCl3), see Table 1; (+)-ESI-HRMS m/z 553.1680 [M + Na]+ (calcd for C27H30O11Na, 553.1680). Bilain D (12): colorless crystals; [α]20 D −24 (c 0.47, CHCl3); IR (neat) νmax 3434, 2929, 1652, 1507, 1450, 1399, 1295, 1240, 1173, 1088, 1025 cm−1; 1H and 13C NMR data (CDCl3), see Table 2; (+)-ESI-HRMS m/z 419.1611 [M + Na]+ (calcd for C19H28N2O5SNa, 419.1611). Hamavellone C (14): colorless, amorphous solid; [α]20 D +8 (c 0.05, CHCl3); IR (neat) νmax 3427, 2927, 1697, 1365, 1177, 1070, 981 cm−1; 1H and 13C NMR data (CD3OD), see Table 2; (+)-ESI-HRMS m/z 207.0992 [M + Na]+ (calcd for C10H16O3Na, 207.0991). Preparation of the (S)-MTPA Ester (10a) of 10. Compound 10 (1.6 mg) was dissolved in 300 μL of pyridine, 10 μL of (R)(−)-MTPA-Cl was added, and the mixture was stirred at 20 °C for 16 h. The reaction mixture was dried under air flow and purified by PTLC (CHCl3/n-hexane/MeOH 1:0.8:0.2) to obtain 10a (1.9 mg). Compound 10a: colorless oil; selected 1H NMR data (CDCl3, 600 MHz) δH 1.28 (H3-14′, d, J = 6.5 Hz), 5.16 (H-13′, m), 3.89 (H-6′, t, J = 7.3 Hz), 5.68 (H-7′, dd, J = 15.5, 7.3 Hz), 7.59 (H-8′, dd, J = 15.5, 11.3 Hz), 6.53 (H-9′, t, J = 11.3 Hz), 3.43 (H-5′a, dt, J = 9.2, 5.5 Hz), 3.57 (H-5′b, m), 2.51 (H2-4′, m); (+)-ESI-HRMS m/z 985.3204 [M + Na]+ (calcd for C49H52F6O13Na, 985.3204). Preparation of the (R)-MTPA Ester (10b) of 10. The (R)MTPA ester of compound 10 (1.4 mg) was prepared with (S)(+)-MTPA-Cl and purified in the same way as 10a to obtain 10b (1.3 mg). Compound 10b: colorless oil; selected 1H NMR data (CDCl3, 600 MHz) δH 1.21 (H3-14′, d, J = 6.5 Hz), 5.17 (H-13′, m), 3.95 (H-6′, t, J = 7.0 Hz), 5.78 (H-7′, dd, J = 15.7, 7.0 Hz), 7.63 (H-8′, dd, J = 15.7,

1

6

no.

δC

type

δH, mult (J in Hz)

δC

type

2 3

79.4 34.9

CH CH2

3.87, d (4.8) 2.18, dt (15.0, 4.8)

79.4 34.9

CH CH2

75.3

CH

49.2 43.9 19.6

C C CH2

21.6

CH2

133.9 134.5 66.3 64.9 47.9

C CH CH C CH2

7.7 63.3

CH3 CH2

170.3 167.6 58.3 61.8 36.9

C C CH C CH2

60.8

CH2

165.3 127.6 138.2

C CH CH

138.8 125.7 166.2 16.0

CH CH C CH3

2.53, m 4

75.0

CH

5 6 7

48.8 43.1 20.2

C C CH2

8

21.7

CH2

9 10 11 12 13

133.6 135.1 66.4 65.2 48.0

C CH CH C CH2

14 15

7.0 62.8

CH3 CH2

16 1′ 2′ 3′ 4′

170.5 165.8 117.8 157.0 40.2

C C CH C CH2

5′

60.4

CH2

6′ 7′ 8′

165.5 127.5 139.0

C CH CH

9′ 10′ 11′ 12′

139.7 125.2 165.7 17.2

CH CH C CH3

5.98, dd (10.0, 5.0)

1.87, dd (12.6, 5.5) 1.92, dd (12.0, 5.5) 2.14, m 2.54, overlapped 6.89, brd (6.0) 3.89, brd (6.0) 2.82, 3.10, 0.82, 3.84, 4.43,

d d s d d

(4.2) (4.2) (13.2) (13.2)

5.79, brs 2.52, m

4.12, ddd (11.5, 9.7, 3.8) 4.43, dt (11.5, 4.2) 5.99, d (15.6) 8.03, dd (15.6, 11.7) 6.60, t (11.7) 6.07, d (11.7) 2.24, s

δH, mult (J in Hz) 3.75, d (4.8) 2.22, dt (15.8, 4.8) 2.52, dd (15.8, 8.2) 5.83, dd (8.2, 4.7)

1.86, td (12.6, 5.4) 1.94, m 2.16, m 2.54, m 6.87, brd (6.0) 3.75, brd (6.0) 2.82, 3.12, 0.88, 4.28, 4.37,

d d s d d

(4.2) (4.2) (13.6) (13.6)

3.37, s 1.69, ddd (15.5, 9.1, 3.8) 2.29, ddd (15.5, 6.0, 2.9) 4.30, m 4.33, m 6.04, d (15.6) 7.87, dd (15.6, 11.4) 6.62, t (11.4) 6.14, d (11.4) 1.53, s

11.3 Hz), 6.58 (H-9′, t, J = 11.3 Hz), 3.51 (H-5′a, m), 3.67 (H-5′b, m), 2.62 (H2-4′, m); (+)-ESI-HRMS m/z 985.3204 [M + Na]+ (calcd for C49H52F6O13Na, 985.3204). Preparation of 11a and 11b from 11. To prepare the (S)- and (R)-MTPA esters 11a and 11b, two separate amounts of compound 11 (1.0 mg and 1.2 mg) were esterified with (R)-(−)- and (S)(+)-MTPA-Cl, respectively, using the procedure described above, to obtain 11a (1.2 mg) and 11b (1.2 mg). Compound 11a: colorless oil; selected 1H NMR data (CDCl3, 600 MHz) δH 1.27 (H3-14′, d, J = 6.5 Hz), 5.14 (H-13′, m), 3.87 (H-6′, dd, J = 8.0, 6.0 Hz), 5.61 (H-7′, dd, J = 15.4, 8.0 Hz), 7.56 (H-8′, dd, J = 15.4, 11.3 Hz), 6.45 (H-9′, t, J = 11.3 Hz), 3.41 (H-5′a, dt, J = 9.2, 5.6 Hz), 3.57 (H-5′b, m), 2.53 (H2-4′, m); (+)-ESI-HRMS m/z 1097.3729 [M + Na]+ (calcd for C55H60F6O15Na, 1097.3728). Compound 11b: colorless oil; selected 1H NMR data (CDCl3, 600 MHz) δH 1.19 (H3-14′, d, J = 6.5 Hz), 5.15 (H-13′, m), 3.93 (H-6′, dd, J = 8.0, 6.8 Hz), 5.73 (H-7′, dd, J = 15.0, 6.8 Hz), 7.62 (H-8′, dd, J = C

DOI: 10.1021/acs.jafc.5b02366 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Table 2. 13C and 1H NMR Data for 12 and 14 12 no.

δ Ca

type

1 2 3

163.7 64.7

C CH

14 δH,b mult (J in Hz)

3.82, s

4 5

165.1

6 7

62.7 36.5

CH CH2

8 9 10 11 12 13 14

126.4 130.8 114.5 158.0 114.5 130.8 69.1

C CH CH C CH CH CH2

15

75.7

CH

16 17 18 19 20 21

71.6 33.0 11.3 32.4 26.4 24.9

C CH3 CH3 CH3 CH3 CH3

Zoospores’ Motility Inhibitory and Lysis Activity Assays. The test pathogen P. nicotianae was isolated from leaves collected in June 2014 from the plant Catharanthus roseus, grown in the nursery beds of Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur, Bangladesh. Infected leaves of C. roseus were placed in water to develop sporangia. Abundant sporangia were produced overnight, and mycelia developed after 2 days. The bioassay was carried out as described previously.11,12 Antimicrobial Test. The antimicrobial test was performed according to a previously described procedure.13

C 4.24, t (3.5) 3.09, dd (14.2, 3.5) 3.22, dd (14.2, 3.5) 6.90, d (8.6) 6.78, d (8.6)

δ Cc

type

30.8 210.0 51.1

CH3 C CH2

70.4

CH

36.1

CH

25.4 32.5

CH CH

208.9 32.0 11.5

C CH3 CH3

δH,d mult (J in Hz) 2.15, s 2.63, dd (12.0, 4.0) 3.62, td (7.3, 4.0) 1.48, td (7.3, 4.9) 1.57, m 2.12, dd (9.2, 4.9)

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RESULTS The structure determinations of the new compounds 1, 6, 12, and 14 were achieved using selected NMR experiments and by comparison of these data with published values. Using AntiBase,10 the structures of the known compounds were identified as verrucarin A (3),14 16-hydroxyverrucarin A (5),15 verrucarin B (7),16 16-hydroxyverrucarin B (8),17 verrucarin J (2),18 verrucarin X (4),15 roridin A (9),19 roridin L-2 (10),20 trichoverritone (11),21 and myrotheciumone A (15).22 Trichothecenes are sesquiterpene alcohols or esters that have a tricyclic skeleton in common, the trichothecane, typically with a double bond between C-9 and C-10 and a 12,13-epoxide ring.23 There are two classes of trichothecenes: those that have only a central sesquiterpenoid structure and those that have an additional macrocyclic or opened ring (simple and macrocyclic trichothecenes, respectively).24 Macrocyclic trichothecenes are further subdivided as either verrucarins (generally C 27 compounds) or roridins (generally C29 compounds).23 The ester groups involved in forming the macrocyclic ring or open chain are attached at positions 4β and 15 of the trichothecane core. Compound 1 was isolated as a colorless, optically active, amorphous solid by PTLC. The 1H NMR spectrum showed similarities to that of verrucarin J (2), and the (+)-ESI-HR mass spectrum showed a molecular ion peak at m/z 537.1731 [M + Na]+ corresponding to the molecular formula C27H30O10 (13 DBEs), indicating a new verrucarin-like trichothecene derivative. The 13C NMR spectrum displayed 27 carbon resonances that were attributed, with the help of a phase-sensitive HSQC spectrum, to 2 methyl, 7 methylene, 9 methine (including 6 olefinic), and 9 quaternary carbons (Table 1). Because analysis of the NMR data suggested that the molecule contains four C,C double bonds and 4 carbonyl groups, 5 rings were expected. The basic trichothecene moiety was identified by the HMBC spectrum, with signals nearly overlapping with those of verrucarin J (2), and confirmed by a detailed NMR analysis (Figure 2). Besides the trichothecene skeleton, two further structural subunits were observed. Using a COSY correlation between H2-4′ and H2-5′ and HMBC correlations of H-2′ to C1′, -3′, -4′, and -12′ (Figure 2), the first of these was established as a 5-hydroxy-3-methylpent-2-enoic acid, a subunit also found in verrucarin J (2). The second subunit was identified as hexa-2,4-dienedioic acid by COSY correlations from H-7′ to H-10′ and HMBC correlations of H-8′ to C-6′ and H-10′ to C-11′. Finally, these subunits were connected via an ester linkage to each other and to the trichothecene moiety. The resulting structure 1 was confirmed by additional HMBC correlations (H-4/C-11′, H2-15/C-1′, and H2-5′/C-6′, Figure 2) and was named verrucarin Y. The configuration of 1 was elucidated by the analysis of coupling constants and 1H chemical shifts and by considering its biosynthesis. The coupling constants of H-7′ (J = 15.6 Hz)

2.22, s 1.07, d (6.2)

6.78, d (8.6) 6.90, d (8.6) 3.94, dd (9.6, 7.5) 4.06, dd (9.6, 3.0) 3.76, dd (7.5, 3.0) 3.09, 1.89, 2.81, 1.28, 1.24,

s s s s s

a

Measured at 125 MHz in CDCl3. bRecorded at 300 MHz in CDCl3. Measured at 125 MHz in CD3OD. dRecorded at 600 MHz in CD3OD.

c

15.0, 11.3 Hz), 6.54 (H-9′, t, J = 11.3 Hz), 3.63 (H-5′a, m), 3.69 (H5′b, m), 2.60 (H2-4′, m); (+)-ESI-HRMS m/z 1097.3729 [M + Na]+ (calcd for C55H60F6O15Na, 1097.3728). Preparation of 12a and 12b. The (S)-MTPA (12a) and (R)MTPA (12b) esters were prepared by derivatization of 12 (1.5 mg each case) with (R)-(−)- and (S)-(+)-MTPA-Cl, following the procedure described earlier, to obtain 12a (1.8 mg) and 12b (1.7 mg), respectively. Compound 12a: colorless oil; 1H NMR (CDCl3, 600 MHz) δH 3.79 (H-3, s), 4.24 (H-6, t, J = 3.5 Hz), 3.10 (H-7a, dd, J = 14.2, 3.5 Hz), 3.22 (H-7b, dd, J = 14.2, 3.5 Hz), 6.90 (H-9, d, J = 8.5 Hz), 6.70 (H10, d, J = 8.5 Hz), 6.70 (H-12, d, J = 8.5 Hz), 6.90 (H-13, d, J = 8.5 Hz), 4.04 (H-14a, dd, J = 10.5, 8.1 Hz), 4.23 (H-14b, dd, J = 10.5, 3.0 Hz), 5.38 (H-15, dd, J = 8.0, 3.0 Hz), 3.10 (H3-17, s), 1.90 (H3-18, s), 2.79 (H3-19, s), 1.29 (H3-20, s), 1.29 (H3-21, s), 3.49 (OMe, s), 7.36 (H3, m), 7.55 (H2, dd, J = 7.1, 1.8); (+)-ESI-HRMS m/z 635.2011 [M + Na]+ (calcd for C29H35N2F3SO7Na, 635.2009). Compound 12b: colorless oil; 1H NMR (CDCl3, 600 MHz) δH 3.85 (H-3, s), 4.26 (H-6, t, J = 3.6 Hz), 3.13 (H-7a, dd, J = 14.2, 3.6 Hz), 3.24 (H-7b, dd, J = 14.2, 3.6 Hz), 6.94 (H-9, d, J = 8.6 Hz), 6.77 (H10, d, J = 8.6 Hz), 6.77 (H-12, d, J = 8.6 Hz), 6.94 (H-13, d, J = 8.6 Hz), 4.13 (H-14a, dd, J = 10.6, 8.3 Hz), 4.32 (H-14b, dd, J = 10.6, 2.7 Hz), 5.40 (H-15, dd, J = 8.3, 2.7 Hz), 3.11 (H3-17, s), 1.92 (H3-18, s), 2.81 (H3-19, s), 1.21 (H3-20, s), 1.24 (H3-21, s), 3.54 (OMe, s), 7.32− 7.27 (H2, m), 7.35 (H1, d, J = 7.4 Hz), 7.58 (H2, d, J = 7.8 Hz); (+)-ESI-HRMS m/z 635.2011 [M + Na] + (calcd for C29H35N2F3SO7Na, 635.2009). D

DOI: 10.1021/acs.jafc.5b02366 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

The configurations of the double bonds at Δ7′ and Δ9′ were determined as E and Z, respectively, on the basis of the observed coupling constants (Table 1). The chemical shifts of H-2′ and H3-12′ in 6 were δH 3.37 and 1.53, respectively, indicating the epoxide ring was trans substituted26 as in verrucarin B16 (δH 3.41 and 1.56). This was also supported by an NOE correlation between H-2′β and H2-4′. For the same reasons as in 1, an identical core configuration can be assumed, resulting in (2R,4R,5S,6R,11R,12S,2′S*,3′R*)-6.19,24 In addition to the verrucarins, roridins were also isolated from strain M10. Whereas the verrucarins contain a hexadienedicarboxylic acid unit in the macrolactone ring, in all of the approximately 40 currently known roridins,10 the dicarboxylic acid is substituted by a chiral 6,7-dihydroxyoctadienoic acid.27 1 H and 13C resonances of the three roridins isolated here were identical to those published for roridin A (9), roridin L-2 (10), and trichoverritone (11); however, the configuration at C-6′ and C-13′ had been reported only for compound 9.20,21,27 Consequently, the modified Mosher’s method was applied to determine the configuration of C-13′ in 10 and 11.28 A positive ΔδH value for H3-14′ and negative values for the rest of the selected protons (Figure 3) indicated that the 13′R configuration was present in both 10 and 11, as in 9. Bilain D (12) was isolated as an optically active, colorless crystals. The molecular formula C19H28N2O5S (7 DBEs) was inferred on the basis of (+)-ESI-HRMS data. The 1H NMR spectrum revealed 5 methyl singlets, 2 methylene ABX signals, and 3 sp3 methine resonances. Two aromatic 2H doublets at δH 6.78 and 6.90 indicated a 1,4-disubstituted benzene ring in the molecule (Table 2). With the help of an HSQC spectrum, the 19 carbon resonances were established to be 5 methyl, 2 methylene, 3 aliphatic oxymethine, 4 methine (2 signals of double intensity), and 5 quaternary carbons (Table 2). COSY and HMBC correlations revealed three structural fragments: a phenol unit, an oxidized isopentane, and a diketopiperazine fragment (Figure 2). The typical A2B2 pattern and the shift values indicated a para-substituted phenol derivative. The phenolic carbon showed an HMBC correlation with an aliphatic methylene group, which must be connected via an ether bond. A COSY correlation indicated that C-14 was connected to an oxymethine group and then to a dimethylcarbinol (indicated by HMBC correlations; Figure 2), resulting in the presence of a dihydroxylated isoprene phenol ether. Among the remaining signals, two N-methyl groups were identified as a result of their C and H shifts. Both showed HMBC correlations with CH groups and amide carbonyls (e.g., Me-17 with C-2 and C-6); further HMBC and COSY correlations resulted in an N,N-dimethyl diketopiperazine skeleton. The open bond at C-3 was determined to be

Figure 2. 1H−1H COSY (bold line) and HMBC (H→C) correlations for 1, 6, 12, and 14.

and H-10′ (J = 11.7 Hz) indicated Δ7′-(E) and Δ9′-(Z) geometries for 1. The Δ2′ geometry was derived from the influence on the protons in the neighborhood. In the case of macrocyclic trichothecenes, the proton shifts, especially of H215, H2-4′, and H2-5′, are strongly dependent on the geometry of the Δ2′ double bond,18 as shown for 2′-(Z)- and 2′-(E)verrucarin J (2) in Table S1 (Supporting Information).18,25 Analysis of the current data clearly indicated a Δ2′-(E) configuration for 1 (Table S1). Because the trichothecene ring in the various derivatives can be assumed to originate from common biosynthetic pathways, it is plausible that the six chiral centers in the core of 1 are (2R,4R,5S,6R,11R,12S), as is found in other trichothecenes.24 NOE correlations between H-2β/H2-13, H-2β/H-3β, H-13b/ H3-14β, H-4α/H2-15, and H-4α/H-11α supported these configurations. The configurations were further confirmed by the closely related shifts for the trichothecene core in 1 compared to other verrucarins and also by the simultaneous formation of 1 and 2 by the same strain. The molecular formula of verrucarin Z (6) was deduced as C27H30O11 by ESI-HRMS, indicating 13 DBEs. 1D and 2D NMR data of 6 were nearly identical to those of 1. The only differences were at C-2′ and C-3′, where an epoxide group in 6 replaced the double bond in 1. The presence of a 2′,3′-epoxide was confirmed by the appearance of two signals of oxygenated carbons (δC 58.3, 61.8) in the 13C NMR spectrum of 6 [replacing the two olefinic signals observed in 1 (Table 1)] and by the HMBC correlations of H2-4′ to C-2′ and -3′ (Figure 2).

Figure 3. Selected ΔδH (ΔδH = δS − δR) values for MTPA esters 10a/10b, 11a/11b, and for MTPA esters 12a/12b. E

DOI: 10.1021/acs.jafc.5b02366 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

respect to H-6 and H-7 (δH 2.12, dd, J = 9.2, 4.9 Hz), as indicated by the NOESY correlations between H-6 and H-7, between H-5 and Me-10, and between Me-9 and Me-10, but not between H-5 and H-6/7 (Figure 4). This was confirmed by

occupied by a thiomethyl (S-Me) group, and the diketopiperazine ring was established to be connected to the phenol unit via a methylene group (CH2-7), as indicated by COSY and HMBC correlations, resulting in structure 12 (Figure 2). This is a new bilain, assigned the letter D. The configuration of C-15 in 12 was determined by using the modified Mosher’s method.28 All proton signals of the resultant two ester derivatives, 12a and 12b, were assigned by 1H−1H COSY experiments and through comparison of their chemical shifts with those from the original 1H NMR data of 12; the shift differences (ΔδH = δS − δR) are shown in Figure 3. The positive ΔδH values of H3-20 and H3-21, but negative ΔδH values of all other protons, indicated the R configuration at C-15.28 The relative configurations of the further two stereocenters in 12 were established by comparing the chemical shifts with those of the synthetic thiodiketopiperazine 13 and via NOE correlations. The chemical shifts of H-3 and H-6 in the cisdiketopiperazine (3S,6S)-methylthiosilvatin (13) were δH 4.58 and 4.22,29 but those in 12 were δH 3.82 and 4.24, respectively, indicating that the substituents at C-3 and C-6 are transoriented in 12. This was confirmed by the observance of the expected NOE correlation between the S-Me group at δH 1.89 and H-6, and by signals between H-3 and the aromatic protons. Therefore, a (3R*,6S*) configuration can be assigned for 12. Calculation of the optical rotations (see Supporting Information) indicated that this is also the absolute configurations, so that finally (3R,6S,15R) resulted for 12. Among the >200 known microbial diketopiperazines, some from fungal sources carry characteristic 3- or 6-thiomethyl or 3,6-di(thiomethyl) groups, with or without N-methyl and N,N′dimethyl groups in the ring. These compounds are mostly derived from phenylalanine; however, fewer than 20 derivatives (including the bilains) contain the O-prenylated tyrosine unit.30−36 In all previously described Phe-derived monothiomethyl diketopiperazines, the S-Me group is present at C-6; bilain D (12), isolated here, is the only example with an S-Me group at C-3.36 It can be speculated that the bilains function as signaling compounds, as do the related benzylidene-diketopiperazines.37 Hamavellone C (14), an unusual cyclopropyl diketone,38 was obtained as an amorphous, colorless solid, for which the molecular formula was determined as C10H16O3 by ESI-HRMS, indicating 3 DBEs. The IR spectrum exhibited a broad absorption band centered at 3427 cm−1 and a sharp band at 1697 cm−1, indicating hydroxy and carbonyl functionalities, respectively. Analysis of 1H, 13C, and HSQC spectra revealed two keto carbonyls (δC 210.0, 208.9). An oxymethine proton resonated at δH 3.62 (δC 70.4), methylene protons appeared at δH 2.63 (δC 51.1), three methine protons were observed at δH 1.48, 1.57, and 2.12 (δC 36.1, 25.4, and 32.5), and three methyl signals appeared at δH 1.07, 2.22, and 2.15 (δC 11.5, 30.8, and 32.0). Analysis of the 1D and 2D NMR spectra of 14 revealed the spin system C3H2−C4H−C5H(C7H)−C6H(C10H3)−C7H and two independent methyl singlets (Figure 2). Besides the two carbonyl groups, the 13C NMR spectrum showed no further double bonds; compound 14 must therefore be monocyclic. The HMBC and COSY correlations of a methyl doublet (C10) with C-5, C-6, and C-7 established a cyclopropane moiety (Figure 2). HMBC correlations of the methyl protons H3-1 and H3-9 with the keto carbons C-2 and C-8, respectively, indicated their terminal positions. The methine H-5 (δH 1.48, td, J = 7.3, 4.9 Hz) of the cyclopropane ring was trans-oriented with

Figure 4. Key NOESY correlations for 14.

the small coupling constant between H-5/H-6 (J = 4.9 Hz) and H-5/H-7 (J = 4.9 Hz), indicating a trans orientation, and by the high coupling value between H-7 and H-6 (Jcis = 9.2 Hz) (Table 2). Typically, trans-oriented protons of the cyclopropyl ring show a coupling of 4.2−5.9 Hz, whereas 8.4−9.7 Hz is indicative of a cis orientation.39 It was not possible to address the absolute configuration of C-4 in 14 due to the limited amount of compound available. Open chain alkyl-cyclopropyl ketones are very unusual in natural sources. To the best of our knowledge, hamavellones A and B, isolated from a soil fungus, are the only two microbial compounds known that are similar to 14, which we therefore named hamavellone C.38 Other known cyclopropyl ketones are cyclic sesquiterpenes and are mainly produced by mushrooms.40,41 The compounds 1−12, 14, and 15 were tested against zoospores of P. nicotianae and a few other microorganisms; results are presented in Tables 3 and 4, respectively. Motility inhibitory and lytic activities against zoospores clearly increased to the maximum applied dose (Table 3). None of the macrocyclic trichothecenes tested here showed motility inhibitory and lytic activities, except for those with certain distinct structural features. Compounds 6, 7, 9, and 14 exhibited IC50 values against zoospores at a concentration of 50 μg/mL after 30, 45, 45, and 30 min, whereas double the concentration (IC50 = 100 μg/mL) was required for 3 and 5 to produce the same effect after 45 and 60 min of treatment, respectively (Table 3). Compound 8 showed a 50% inhibitory concentration (IC50) of 250 μg/mL after 5 min of treatment. On treatment with higher concentrations (100−900 μg/mL), all of these motility inhibitors caused lysis of zoospores (zoosporicidal activity) (Table 3). Compounds 1, 2, 4, 10, 11, 12, and 15 did not show any motility inhibitory or lytic activities against zoospores within 60 min up to 900 μg/mL. In the case of compounds 3−5, the presence of a methyl or hydroxymethyl group at C-9 increased the motility halting activity, whereas the presence of a carboxy group (4) at the same position led to a complete loss of activity (Table 3). Verrucarins or roridins with double bonds at C-2′ became inactive, irrespective of their functionalities at C-9 (1 and 2). For the verrucarins, either a CH3, COOH, or CH2OH moiety at C-9 in the presence of an epoxide group at C-2′,3′ (6−8) resulted in motility inhibitory activity followed by lysis of zoospores (Figure 1 and Table 3). When the macrocyclic ring was opened (10 and 11), no activity was observed against the zoospores. F

DOI: 10.1021/acs.jafc.5b02366 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

0 9 6 5 3 4 4 2 0 0

0 0 0 0 0 0 0 4 0 0

0 0 0 0 7 4 5 4

± ± ± ± ± ± ± ± ± ±

± ± ± ± ± ± ± ± ± ±

± ± ± ± ± ± ± ±

100 80 75 60 30 25 20 10 0 0

100 100 100 100 100 100 100 60 0 0

100 100 100 100 80 70 50 50

900 800 700 600 500 400 300 250 100 50

900 800 700 600 500 400 300 250 100 50

900 800 700 600 500 400 300 250

5

6

G

7

100 80 50 0 0 0 0 0

100 80 60 10 5 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0

± ± ± ± ± ± ± ±

± ± ± ± ± ± ± ± ± ±

± ± ± ± ± ± ± ± ± ±

± ± ± ± ± ± ± ± ±

0 0 9 6 5 7 4 0 0

± ± ± ± ± ± ± ± ±

100 100 80 70 50 30 10 0 0

900 800 700 600 500 400 300 250 100

3

0 0 0 0 0 0 0 0 0

lysed

inhibited motility

dose (μg/mL)

compd

5 min

0 8 4 0 0 0 0 0

0 5 6 2 3 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0

nt 100 100 100 100 90 70 60

nt 100 100 100 100 100 100 80 50 0

100 100 100 85 50 45 30 20 0 0

100 100 100 80 70 50 25 15 0

± ± ± ± ± ± ±

± ± ± ± ± ± ± ± ±

± ± ± ± ± ± ± ± ± ±

± ± ± ± ± ± ± ± ±

0 0 0 0 8 8 5

0 0 0 0 0 0 3 8 0

0 0 0 8 7 4 3 2 0 0

0 0 0 4 8 9 5 5 0

inhibited motility

10 min

nt 100 60 10 0 0 0 0

nt 100 75 30 15 10 0 0 0 0

0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0

± ± ± ± ± ± ±

± ± ± ± ± ± ± ± ±

± ± ± ± ± ± ± ± ± ±

± ± ± ± ± ± ± ± ±

lysed

0 5 2 0 0 0 0

0 5 2 8 5 0 0 0 0

0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0

nt nt 100 100 100 100 80 70

nt nt 100 100 100 100 100 100 65 25

100 100 100 100 100 60 50 35 5 0

100 100 100 100 80 60 40 30 20

± ± ± ± ± ±

± ± ± ± ± ± ± ±

± ± ± ± ± ± ± ± ± ±

± ± ± ± ± ± ± ± ±

0 0 0 0 7 4

0 0 0 0 0 0 3 2

0 0 0 0 0 5 8 3 2 0

0 0 0 0 6 6 5 7 4

inhibited motility

15 min

nt nt 70 30 0 0 0 0

nt nt 80 50 25 20 2 0 0 0

0 0 0 0 0 0 0 0 0 0

± ± ± ± ± ±

± ± ± ± ± ± ± ±

± ± ± ± ± ± ± ± ± ±

± ± ± ± ± ± ± ± ±

lysed 10 0 0 0 0 0 0 0 0

5 3 0 0 0 0

9 6 7 2 2 0 0 0

0 0 0 0 0 0 0 0 0 0

4 0 0 0 0 0 0 0 0

nt nt 100 100 100 10 0 100 100

nt nt 100 100 100 100 100 100 80 50

100 100 100 100 100 100 80 55 15 0

100 100 100 100 100 80 70 50 30

± ± ± ± ± ±

± ± ± ± ± ± ± ±

± ± ± ± ± ± ± ± ± ±

± ± ± ± ± ± ± ± ±

0 0 0 0 0 0

0 0 0 0 0 0 6 2

0 0 0 0 0 0 8 4 7 0

0 0 0 0 0 7 8 5 4

inhibited motility

30 min

nt nt 90 50 20 10 0 0

nt nt 100 60 40 30 10 5 0 0

5 5 0 0 0 0 0 0 0 0

25 5 0 0 0 0 0 0 0

± ± ± ± ± ±

± ± ± ± ± ± ± ±

± ± ± ± ± ± ± ± ± ±

± ± ± ± ± ± ± ± ±

lysed

9 6 3 4 0 0

0 6 5 2 9 4 0 0

2 3 0 0 0 0 0 0 0 0

2 2 0 0 0 0 0 0 0

45 min

nt nt 100 100 100 10 0 10 0 100

nt nt nt 100 100 100 100 100 100 70

100 100 100 100 100 100 100 70 35 5

100 100 100 100 100 100 90 70 50

± ± ± ± ± ±

± ± ± ± ± ± ±

± ± ± ± ± ± ± ± ± ±

± ± ± ± ± ± ± ± ±

0 0 0 0 0 0

0 0 0 0 0 0 2

0 0 0 0 0 0 0 4 3 3

0 0 0 0 0 0 7 6 8

inhibited motility

motility inhibitory and zoosporicidal activities over controlb (% ± SE)

Table 3. Motility Inhibitory and Zoosporicidal Activities of 1−12, 14, and 15 against the Late Blight Pathogen P. nicotianaea

nt nt 100 70 50 20 0 0

nt nt nt 90 65 50 25 20 0 0

15 7 0 0 0 0 0 0 0 0

± ± ± ± ± ±

± ± ± ± ± ± ±

± ± ± ± ± ± ± ± ± ±

± ± ± ± ± ± ± ± ±

lysed 30 0 0 0 0 0 0 0 0

0 5 7 5 0 0

8 7 2 8 2 0 0

5 3 0 0 0 0 0 0 0 0

6 0 0 0 0 0 0 0 0

nt nt nt 100 100 100 100 100

nt nt nt 100 100 100 100 100 100 80

100 100 100 100 100 100 100 80 50 10

100 100 100 100 100 100 100 80 60

± ± ± ± ±

± ± ± ± ± ± ±

± ± ± ± ± ± ± ± ± ±

± ± ± ± ± ± ± ± ±

0 0 0 0 0

0 0 0 0 0 0 7

0 0 0 0 0 0 0 8 5 3

0 0 0 0 0 0 0 8 5

inhibited motility

60 min

nt nt nt 100 70 30 5 0

nt nt nt 100 90 70 50 30 5 0

25 10 0 0 0 0 0 0 0 0

± ± ± ± ±

± ± ± ± ± ± ±

± ± ± ± ± ± ± ± ± ±

± ± ± ± ± ± ± ± ±

lysed 55 10 0 0 0 0 0 0 0

0 6 4 2 0

0 9 4 8 3 2 0

6 3 0 0 0 0 0 0 0 0

7 3 0 0 0 0 0 0 0

Journal of Agricultural and Food Chemistry Article

DOI: 10.1021/acs.jafc.5b02366 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

0 7 4 6 2 3 0 0 0 0 0

0 0 0 0 0 3 7 5 5 0 0

± ± ± ± ± ± ± ± ± ± ±

± ± ± ± ± ± ± ± ± ± ±

100 50 30 25 15 15 0 0 0 0 0

100 100 100 100 100 80 70 35 30 0 0

900 800 700 600 500 400 300 250 100 50 25

900 800 700 600 500 400 300 250 100 50 25

9

14

H

± ± ± ± ± ± ± ± ± ± ± 7 5 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 100 100 100 100 100 100 90 50 40 10 0

100 80 60 60 35 25 20 20 0 0 0

100 100 100 100 80 70 50 55 0

± ± ± ± ± ± ± ± ± ± ±

± ± ± ± ± ± ± ± ± ± ±

± ± ± ± ± ± ± ± ±

0 0 0 0 0 0 5 4 7 3 0

0 8 5 7 5 3 5 5 0 0 0

0 0 0 0 9 6 7 5 0

30 ± 2 0±0 0±0

inhibited motility

10 min

100 100 80 50 0 0 0 0 0 0 0

0 50 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0

± ± ± ± ± ± ± ± ± ± ±

± ± ± ± ± ± ± ± ± ± ±

± ± ± ± ± ± ± ± ±

0 0 5 4 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0

0±0 0±0 0±0

lysed

nt nt 100 100 100 100 100 80 70 20 0

100 100 75 70 50 35 30 25 15 10 0

100 100 100 100 100 80 65 60 5

± ± ± ± ± ± ± ± ±

± ± ± ± ± ± ± ± ± ± ±

± ± ± ± ± ± ± ± ±

0 0 0 0 0 4 7 3 0

0 0 2 9 6 3 9 5 8 9 0

0 0 0 0 0 5 7 4 2

50 ± 3 10 ± 2 5±2

inhibited motility

15 min

nt nt 100 65 5 0 0 0 0 0 0

15 80 0 0 0 0 0 0 0 0 0

30 10 0 0 0 0 0 0 0

± ± ± ± ± ± ± ± ±

± ± ± ± ± ± ± ± ± ± ±

± ± ± ± ± ± ± ± ±

0 3 7 0 0 0 0 0 0

4 0 0 0 0 0 0 0 0 0 0

3 3 0 0 0 0 0 0 0

0±0 0±0 0±0

lysed

nt nt nt 100 100 100 100 100 100 50 20

100 100 100 100 80 65 55 50 35 25 0

100 100 100 100 100 90 80 70 10

± ± ± ± ± ± ± ±

± ± ± ± ± ± ± ± ± ± ±

± ± ± ± ± ± ± ± ±

0 0 0 0 0 0 3 9

0 0 0 0 7 3 8 5 9 2 0

0 0 0 0 0 7 8 5 3

60 ± 4 25 ± 2 30 ± 3

inhibited motility

30 min

nt nt nt 90 30 15 5 0 0 0 0

100 100 70 20 5 5 0 0 0 0 0

50 20 0 0 0 0 0 0 0

± ± ± ± ± ± ± ±

± ± ± ± ± ± ± ± ± ± ±

± ± ± ± ± ± ± ± ±

5 4 9 2 0 0 0 0

0 0 0 0 0 3 0 0 0 0 0

7 5 0 0 0 0 0 0 0

0±0 0±0 0±0

lysed

45 min

nt nt nt 100 100 100 100 100 100 75 30

nt nt 100 100 100 80 70 70 60 50 10

100 100 100 100 100 100 100 80 25

± ± ± ± ± ± ± ±

± ± ± ± ± ± ± ± ±

± ± ± ± ± ± ± ± ±

0 0 0 0 0 0 9 3

0 0 0 5 8 3 6 4 4

0 0 0 0 0 0 0 6 2

80 ± 5 50 ± 6 45 ± 4

inhibited motility

motility inhibitory and zoosporicidal activities over controlb (% ± SE)

nt nt nt 100 50 35 35 10 5 0 0

nt nt 100 50 30 45 20 10 0 0 0

80 50 10 0 0 0 0 0 0

± ± ± ± ± ± ± ±

± ± ± ± ± ± ± ± ±

± ± ± ± ± ± ± ± ±

0 3 8 4 3 2 0 0

0 4 3 7 6 3 0 0 0

6 8 3 0 0 0 0 0 0

0±0 0±0 0±0

lysed

nt nt nt nt 100 100 100 100 100 100 60

nt nt nt 100 100 100 100 100 100 75 35

100 100 100 100 100 100 100 90 40

± ± ± ± ± ± ±

± ± ± ± ± ± ± ±

± ± ± ± ± ± ± ± ±

0 0 0 0 0 0 7

0 0 0 0 0 0 5 4

0 0 0 0 0 0 0 7 6

100 ± 0 70 ± 7 50 ± 5

inhibited motility

60 min

nt nt nt nt 65 50 45 25 10 0 0

nt nt nt 100 80 60 50 30 5 0 0

100 60 15 5 0 0 0 0 0

± ± ± ± ± ± ±

± ± ± ± ± ± ± ±

± ± ± ± ± ± ± ± ±

9 7 2 5 3 0 0

0 8 4 8 5 3 0 0

0 6 3 2 0 0 0 0 0

0±0 0±0 0±0

lysed

a Compounds 1, 2, 4, 10, 11, 12, and 15 did not show inhibitory and lytic activities up to 900 μg/mL. bData represented here are average values ± SE (standard error) of at least three replicates of each dose of compound. nt, not tested.

90 75 50 30 0 0 0 0 0 0 0

0 25 0 0 0 0 0 0 0 0 0

± ± ± ± ± ± ± ± ± ± ±

± ± ± ± ± ± ± ± ±

0 0 7 4 8 6 5 7 0

± ± ± ± ± ± ± ± ±

100 100 90 80 70 65 60 50 0

900 800 700 600 500 400 300 250 100

8

0 0 0 0 0 0 0 0 0

0±0 0±0 0±0

15 ± 3 0±0 0±0 0 0 0 0 0 0 0 0 0

lysed

inhibited motility

100 50 25

5 min

dose (μg/mL)

compd

Table 3. continued

Journal of Agricultural and Food Chemistry Article

DOI: 10.1021/acs.jafc.5b02366 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

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Table 4. Antimicrobial Activity of 1−12, 14, and 15 C. albicans

M. miehei

B. subtilis

E. coli

S. aureus

1 2 3 4 5 6 7 8 9 10 11 12 14 15 nystatin ciprofloxacin

−b 12 16 − − − 20 − 16 − − − − − 17 −

− 36 38 − − 24 46 20 32 − 16 − − 11 21 −

− − − − − − − − − − − − − − − 28

− − − − − − − − − − − − − − − 22

− − − − − − − − − − − − − − − 24

ASSOCIATED CONTENT

S Supporting Information *

diameter of inhibition zone in mm at 50 μg/disk compda

Article

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b02366. 1 H and 13C NMR spectra of compounds 1, 6, 12, and 14; 1 H NMR spectra of 10a, 10b, 11a, 11b, 12a, and 12b; selected 1H chemical shifts for verrucarin J (2) isomers and verrucarin Y (1); calculation of ORD data to determine the absolute configuration of bilain D (12) (PDF)

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AUTHOR INFORMATION

Corresponding Author

*(H.L.) Phone: +49 551 3933211. Fax: +49 551 399660. Email: [email protected]. Funding

This work was financially supported by the Alexander von Humboldt Foundation through a Georg Forster Fellowship to M.A.M.M. and was also supported by the World Bank (CP 2071 to MTI).

a Nystatin and ciprofloxacin were used as positive control. b−, not active at 50 μg/disk.

Notes

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The authors declare no competing financial interest.

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DISCUSSION Motility inhibition followed by lytic activities against phytopathogenic Peronosporomycete zoospores has been reported previously for several plant and microbial metabolites, including polyflavonoid tannins from Lannea coromandelica,11 polyketides from an endophytic fungus Cryptosporiopsis sp.,42 lipopeptides from Bacillus subtilis,12 indolocarbazole alkaloids from Streptomyces sp.,43 and phloroglucinols from Pseudomonas fluorescens,44 among others. However, motility inhibition and subsequent lysis of P. nicotianae zoospores by trichothecenes, as reported here, were previously unknown. In our experiments with Peronosporomycetes, we observed a distinct relationship between structural features and activity in trichothecenes, as described previously by others.45 In the agar diffusion assay for antifungal activity, among the macrocyclic trichothecenes tested here, only the 9-methyl trichothecenes (2, 3, 7, and 9) showed noteworthy activities against Candida albicans and Mucor miehei at a concentration of 50 μg/disk when compared with nystatin (Table 4). The most notable activity against C. albicans and M. miehei was exhibited by compound 7. It gave inhibition zones of 20 and 46 mm in diameter against C. albicans and M. miehei, whereas nystatin, a clinically used antifungal drug, gave 17 and 21 mm zones, respectively. The presence of the COOH or CH2OH group at C-9 rendered the trichothecenes inactive against fungi, whereas among the verrucarins, the 2′,3′-epoxide ring was required for zoosporicidal activity. The mechanisms by which trichothecenes produce cytotoxic effects in eukaryotic cells primarily involve inhibition of the macromolecule biosynthesis:46 verrucarin A is 10 times more active than actinomycin D, one of the most active chemotherapeutic drugs. There is, however, no activity against prokaryotes (see Table 4), as also observed by others.15 In conclusion, trichothecenes are a large family of biologically active mycotoxins, with pronounced differential toxicity for eukaryotes47 and strong structure-activity relationships. On the basis of these differential toxicities, trichothecenes may well be useful compounds for the development of effective agrochemicals and also as therapeutic leads.

ACKNOWLEDGMENTS We are indebted to Dr. H. Frauendorf and Dr. M. John for MS and NMR measurements, respectively. We thank F. Lissy for technical assistance and Dr. S. Hickford for careful linguistic corrections of the manuscript.

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ABBREVIATIONS USED COSY, correlation spectroscopy; DBEs, double-bond equivalents; ESI-HRMS, electrospray ionization high-resolution mass spectrometry; EtOAc, ethyl acetate; HMBC, heteronuclear multiple-bond correlation; HSQC, heteronuclear single-quantum coherence; IC50, 50% motility inhibitory concentration in μg/mL; IR, infrared; ITS, internal transcribed spacer sequence; Sephadex LH-20, type of lipophilic hydrophilic Sephadex for size exclusion chromatography; MTPA-Cl, α-methoxy-α(trifluoromethyl)phenylacetyl chloride; NMR, nuclear magnetic resonance; NOE, nuclear Overhauser effect; P-flasks, Penicillium flasks; PTLC, preparative thin layer chromatography

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REFERENCES

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