Nematicidal Cyclic Lipodepsipeptides and a Xanthocillin Derivative

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

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Nematicidal Cyclic Lipodepsipeptides and a Xanthocillin Derivative from a Phaeosphariaceous Fungus Parasitizing Eggs of the Plant Parasitic Nematode Heterodera f ilipjevi Soleiman E. Helaly,†,‡ Samad Ashrafi,§ Rémy B. Teponno,†,⊥ Steffen Bernecker,† Abdelfattah A. Dababat,∥ Wolfgang Maier,§ and Marc Stadler*,† †

Department of Microbial Drugs, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, 38124 Braunschweig, Germany Department of Chemistry, Faculty of Science, Aswan University, Aswan 81528, Egypt § Institute for Epidemiology and Pathogen Diagnostics, Julius Kühn-Institut (JKI)Federal Research Centre for Cultivated Plants, Braunschweig 38104, Germany ⊥ Department of Chemistry, Faculty of Science, University of Dschang, P.O. Box 67, Dschang, Cameroon ∥ International Maize and Wheat Improvement Center (CIMMYT), P.K. 39 Emek, 06511 Ankara, Turkey

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‡

S Supporting Information *

ABSTRACT: The new cyclic lipodepsipeptide ophiotine (1), two new arthrichitin derivatives named arthrichitins B (4) and C (5), a new xanthocillin-like alkaloid, xanthomide Z (2), and the previously described arthrichitin (3) were isolated from the liquid culture broth of a nematode-associated fungus with affinities to the genus Ophiosphaerella. The structural elucidation and determination of the absolute configuration of the new molecules were accomplished using a combination of spectroscopic and chemical techniques, including 1D and 2D NMR, HRMS, and Marfey’s analysis. Opiotine (1) displayed moderate nematicidal activity against the host nematode (Heterodera filipjevi), while xanthomide Z (2) exhibited very weak activity. Arthrichitin C (5) showed very weak cytotoxic effects on several cancer cell lines, with IC50 values in the range of 24−33 μM. Xanthomide Z is among few xanthocillin derivatives that comprise formamide functions instead of the cyano functions that are usually observed in this class of fungal alkaloids.

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fungal cyclodepsipeptides has recently been provided by Wang et al.5 About 4000 different plant parasitic nematodes are known to cause decreases in yield and health of their host plants. Cyst nematodes are globally important pathogens in agriculture. Their sedentary lifestyle and long-term association with the roots of host plants render cyst nematodes especially good targets for attack by their natural enemies.6 A diverse group of fungi has been described as associates of cyst nematodes.7 Attempts to effectively control nematodes by their natural antagonists including nematophagous fungi have so far been

aturally occurring cyclic lipodepsipeptides have attracted a great deal of attention and are promising candidates for the development of new antibiotics. Some of these natural products are either already marketed or in advanced stages of clinical development.1 For example, the nematicidal cyclodepsipeptide PF-1022A, originally isolated from an endophytic fungus that inhabits the Japanese tea plant, inspired the commercial antiparasitic agent emodepside, which was derived by semisynthesis.2 PF-1022A is the only metabolite from an endophytic fungus from which a marketed drug has hitherto been developed. Beauvericin, a cyclic hexadepsipeptide that belongs to the enniatin antibiotic family, is produced by many fungi, including Beaveria bassiana and Fusarium spp.3 Insecticidal, antimicrobial, antiviral, cytotoxic, and nematicidal activities were reported for beauvericin.4 An overview of other © XXXX American Chemical Society and American Society of Pharmacognosy

Received: June 14, 2018

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DOI: 10.1021/acs.jnatprod.8b00486 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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mostly limited by the complexity of the soil ecosystem.8,9 The destructive parasitism of fungi toward nematodes is in some cases linked with the production of biologically active fungal secondary metabolites,10,11 including nematicidal compounds that display anthelmintic effects. While not enough is known about the ecology of many nematode-associated fungi to develop effective strategies for the biological control of nematodes, more than 180 nematicidal secondary metabolites of fungi have been published to date.12 These molecules show remarkable structural diversity, ranging from low molecular weight organic acids to pyrones, benzoquinones, and cyclic peptides to highly complex macrolides. Many of these compounds have a broad spectrum of activity and can thus serve as potential starting points for the development of new antibiotics, therapeutics, or pesticides. As a part of our program focused on the discovery of bioactive secondary metabolites from nematode-associated fungi,6 we isolated an undescribed pleosporalean fungus with affinities to the genus Ophiosphaerella (Phaerosphaeriaceae) from eggs of the cereal cyst nematode Heterodera f ilipjevi. Subsequently, the fungus was cultivated and extracted, and one known and four novel natural products were obtained. Their structure elucidation and biological activities are discussed below.

compounds 1, 2, and 3. To produce additional material of the fermentation products, a 10 L scale culture was grown in a fully controlled 10 L scale bioreactor. From the resulting extract, compounds 4 and 5 were isolated in addition to the abovementioned compounds. Ophiotine (1) was obtained as a white, amorphous powder. The molecular formula was determined as C45H62N8O10 on the basis of the HRMS spectrum. 1H, 13C, and HSQC NMR data (Table 1) revealed the presence of eight characteristic proton signals that were not assigned to corresponding carbons, which, together with the eight nitrogen atoms from the molecular formula and eight carbonyl carbons, suggested that 1 might be a peptide. Comprehensive analysis of the 1D and 2D NMR data of 1 particularly COSY, TOCSY, and HMBC data revealed six isolated spin systems, attributed to six amino acid residues as follows: Phe, Ser, Trp, β-Ala, Thr, and Gln. In addition, COSY and HMBC data showed the presence of a decanoyl chain. Its connectivity to the NH− of Gln was confirmed by HMBC correlations from α-H (H-31) and NH protons of Gln to the carbonyl carbon of the decanoyl chain (C-35) (Figure 1). The lactone linkage of 1 was established by HMBC correlation from Thr-β-H (δH 5.53, H-19) to the carbonyl carbon of Phe (C-21), finally confirming that 1 is a cyclic lipodepsipeptide. The amino acid sequence was determined on the basis of ROESY data, which showed a series of correlations as follows: from Phe-NH to α-H-Ser; SerNH to α-H-Trp; Trp-NH to α-H2-β-Ala; β-Ala-NH to NHThr; and Thr-NH to α-H-Gln (Figure 1). Finally, the absolute configuration of ophiotine (1) was established by using Marfey’s method,13 i.e., acid hydrolysis of 1 followed by derivatization with Marfey’s reagent, 1-fluoro-2,4-dinitrophenyl-5-L-alaninamide (L-FDAA), and comparison with authentic standards using HPLC-MS. The procedure described by Capon et al. (heating at 110 °C in 6 M HCl for 24 h)14 was initially used, but after Marfey’s analysis the peaks corresponding to L-FDAA derivatives of Gln and Trp were not present in the HPLC chromatogram. This could be explained by the fact that Gln and Trp were not liberated in the above reaction conditions. The reaction conditions were then modified by increasing the temperature to 120 °C, and after reaction with L-FDAA the derivatives of the Gln and Trp were detected. Consequently, the interpretation of Marfey’s analysis revealed that in addition to β-alanine identified by NMR ophiotine (1) is composed of L-Ser, L-Thr, D-Gln, D-Trp, and D-Phe. Thus, the structure of ophiotine (1) was determined as shown. A structurally related antifungal lipodepsipeptide, colisporifungin, was previously reported from the fungus Colispora cavincola,15 and the endothelin inhibitors, aselacins, from Acremonium16 are also structurally related. The molecular formula of compound 2 was determined as C20H22N2O5 from the molecular ion peak at m/z 371.1597 [M + H]+. 13C and HSQC NMR data showed 18 carbon resonances, two of which, at δC 129.6 and 114.8, were assigned to four aromatic methines (C-6/C-10 and C-7/C-9, respectively). Furthermore, two protonated carbons at δC 159.9 (C-1) and 160.1 (C-1′), attributed to two formamide functions, were assigned. In addition, three aromatic methines, two methylenes, two methoxy carbons, and seven sp2 carbons were detected (Table 2). Close examination of the 2D NMR data of 2 including COSY and HMBC spectra enabled the structure determination of 2 as follows: COSY correlations between H-6/H-7 and H-9/H-10 together with HMBC of the aromatic ring and HMBC correlations from the hydroxy group

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RESULTS AND DISCUSSION A five-liter fermentation of the fungal strain DSM106825 was grown in ZM media in Erlenmeyer flasks. The culture was extracted using XAD adsorber resin and ethyl acetate, and the pooled extract was fractionated by preparative HPLC to give B



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Table 1. NMR Spectroscopic Data for Ophiotine (1) and Arthrichitins B (4) and C (5) 1 (500 MHz, acetone-d6) pos.

δC, type

L-Ser

1 2 3 NH

7 8 9 9a 10 11 12 13 13a NH β-Ala 14 15 16 NH L-Thr 17 18 19 20 NH D-Phe

pos.

δC, type

δH (J in Hz)

L-Ser

171.3, C 58.2, CH2 62.0, CH2

4.20, 3.62, 3.75, 8.29,

td (2 × 6, 4) dd (4, 11) dd (11, 4) d (6)

D-Trp

4 5 6

5 (700 MHz, acetone-d6)

δH (J in Hz)

175.6, C 55.9, CH 28.2, CH2

124.8, 110.5, 128.5, 119.3, 119.7, 122.3, 112.3, 137.6,

CH C C CH CH CH CH C

4.58, 3.13, 3.19, 10.5, 7.24,

t (6) dd (6, 15) dd (8, 15) s d (2)

7.61, 7.02, 7.09, 7.38,

d (8) dd, (8, 7) dd (7, 8) d (8)

7.77, d (6) 173.3, C 35.1, CH2 37.7, CH2

2.27, m 2.64, ddd (14, 12, 5) 3.02, m; 3.53, m 7.22, da

169.9, C 57.3, CH 71.7, CH 16.6, CH3

21 22 23 24 25 26 27 28 29 NH D-Gln 30 31 32

170.7, C 56.6, CH 35.8, CH2 140.0, C 130.5, CH 129.1, CH 127.0, CH 129.1, CH 130.5, CH

173.9, C 55.3, CH 27.8, CH2

33

32.4, CH2

34 NH

175.1, C

4.55, 5.53, 1.18, 8.58,

dd (3, 10) dq (3 × 6, 3) d (6) d (10)

3.73, t (7) 3.28, d (8) 7.22, 7.23, 7.17, 7.23, 7.22, 7.76,

4.68, 2.07, 2.11, 2.38, 2.43,

da dda dd (6, 7) dda da d (7)

m m m ddd (7, 8, 15) ddd (15, 8, 7)

8.09, d (6)

1 2 3

169.8, C 55.9, CH 61.2, CH2

β-Oxo-Trp 4 5 6

167.8, C 62.5, CH 184.8, C

7 8 9 9a 10 11 12 13 13a D-Glu 14 15 16

171.4, C 53.8, CH 26.1, CH2

17

30.6, CH2

137.3, 114.2, 126.5, 122.1, 122.9, 123.9, 112.5, 137.1,

CH C C CH CH CH CH C

18 172.9, C OMe 51.8, CH3 Dimethyl-oxy-duodecanoyl 19 175.8, C 20 43.1, CH 21 79.4, CH 22 35.2, CH 23 34.6, CH2 24 25−27 28 29 30 31 32

28.0, ∼30, 23.4, 32.1, 13.6, 16.3, 13.8,

CH2 3 CH2 CH2 CH2 CH3 CH3 CH3

4.19, dd (7, 4) 3.56, dd (7, 12)

5.69, s

8.62, s

8.30, 7.26, 7.29, 7.56,

d (7) dda dda d (7)

4.56, t (7) 2.03, m 2.47, m 2.39, m

L-Ser 170.9, C 57.5, CH 61.2, CH2

Trp 176.2, C 55.9, CH 27.8, CH2

δH (J in Hz)

4.21, m 3.71, dd (7, 10)

4.62,dd (10, 5) 3.30, dd (4, 15) 3.13, dd (15, 10)

124.4, CH 111.1, C 128.6, C 119.3, CH 120.0, CH 122.7, CH 112.5, CH 138.2, C D-Glu 174.4, C 54.1, CH 26.1, CH2

7.15, s

28.1, CH2

2.19, t (7)

7.59, 7.03, 7.09, 7.33,

d (8) br t (7) br t (7) d (8)

4.33, t (8) 2.04, m

174.6, C 3.61, s

2.92, m 5.06, dd (9, 3) 1.83, m 1.41, m 1.15, m 1.38, m ∼1.30, m 1.30, m 1.26, m 0.87, t (7) 1.20, d (7) 0.89, d (7)

Decanoyl moiety 35 175.5, C 36 36.2, CH2 37 26.3, CH2 38−41 ∼30, 4CH2 42 32.7, CH2 43 23.4, CH2

2.27, ma 1.58, m ∼1.25, ma 1.25, ma 1.26, ma

44

0.84, t (7.0)

14.3, CH3

4 (700 MHz, methanol-d4) δC, type

Dimethyl-oxy-duodecanoyl 177.7, C 43.6, CH 2.81, dd (9, 7) 80.6, CH 5.06, dd (9, 3) 35.9, CH 1.91, m 35.1, CH2 1.41, m 1.15, m 28.6, CH2 1.38, m ∼30, 3 CH2 ∼1.30, m 23.9, CH2 1.30, m 33.2, CH2 1.28, m 14.2, CH3 0.87,a t (7) 16.9, CH3 1.17, d (7) 14.6, CH3 0.88,a d (7)

a

Overlapping signals.

(8-OH) to C-7/C-8/C-9 allowed the construction of a monosubstituted phenol ring. Similarly, a trisubstituted phenyl

ring was determined by HMBC, of which two correlations from the methoxy groups to C-6′/C-9′ revealed the positions C



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Figure 1. COSY (bold bonds), HMBC (red arrows), and ROESY (green arrows) correlations of ophiotine (1) and xanthomide Z (2).

as arthrichitin. Close examination of the NMR data of 3 showed that they are in good accordance with the literature, indicating that compound 3 is arthrichitin, also known as LL15G256γ, a cell wall active antifungal depsipeptide isolated from the fermentation broth of the fungus Arthrinium phaeospermum HIL Y-903022 (Xylariales)19 and the marine fungus Hypoxylon oceanicum (current name Halorosellinia oceanica)20 NRRL 21363.21,22 The optical rotation of 3 ([α]20D +38, c 0.2, MeOH), compared with that of LL15G256γ ([α]25D +24, c 0.5, MeOH), indicated that both compounds have the same absolute configuration, which was further confirmed by comparison of the CD spectrum of 3 (Figure 2) with that of LL15G256γ reported in the literature.

Table 2. NMR Spectroscopic Data for Xanthomide Z (2) 2 (700 MHz, DMSO-d6) position

δC, type

δH (J in Hz)

1 2 3 4 5 6 7 8 9 10 8-OH 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 6′-OMe 9′-OMe

159.9, CH

7.91, d (2) 8.99, br s

130.7, C 33.4, CH2 128.5, C 129.6, CH 114.8, CH 155.6, C 114.8, CH 129.6, CH 160.1, CH 125.9, C 29.2, CH2 127.0, C 151.3, C 111.3, CH 111.4, CH 152.9, C 115.3, CH 55.7, CH3 55.1, CH3

3.62, s 6.94, d (9) 6.63, d (9) 6.63, 6.94, 9.14, 7.96, 9.16,

d (9) d (9) s d (2) s

3.59, s

6.84, d (9) 6.71, d (9) 6.67, s 3.68, s 3.66, s

of the methoxy groups at C-6′ and C-9′ of the phenyl ring (Figure 1). In addition, an N,N′-but-2-ene-2,3-diyldiformamide moiety was determined as the linkage between the two aromatic moieties on the basis of HMBC correlations. Finally, COSY correlations between the NHs and the adjacent aldehyde protons together with HMBC correlations from NH-2/NH-2′ to the methylene carbons C-4/C-4′, respectively, and from the aldehyde protons (H-1/H-1′) to C-3/C-3′ allowed the construction of two formamide functions attached to the double bond of the butene chain. NOE correlations between NH-2 and the methylene H2-4 and between NH-2′ and H2-4′/H-10′ indicated the E-configuration of the double bond C-3/C-3′. Thus, compound 2 was determined as a new xantocillin-type natural product, for which we proposed the name xanthomide Z. Interestingly, xanthomide Z is only the second example of a xanthocillin derivative aside from cordyformamide17 that possesses two formamide groups instead of the isonitrile functionalities commonly found in the xanthocillin family of fungal antibiotics.18 Compound 3 was isolated as a white powder possessing a molecular formula of C33H46N4O9 as determined by HRMS. A database search revealed that 3 has the same molecular formula

Figure 2. ECD spectra of arthrichitin (3) and arthrichitins B (4) and C (5) in ethanol.

Since the data were found to be identical, the absolute configuration of 3 is L-Ser, D-Glu, β-Oxo-D-Trp, and 2S,4Rdimethyl-3S-oxydodecanoic acid. The new derivative of arthrichitin, compound 4, was isolated from the same culture. The molecular formula of C33H48N4O8 was determined by HRESIMS. The molecular formula showed a difference of one oxygen atom less and two additional hydrogens in 4 compared to 3 and therefore suggested very similar structures for both compounds. Comprehensive analysis of the NMR data revealed high similarity to those of arthrichitin. Nevertheless, slight differences were observed in the signals of the β-OxoTrp moiety. Arthrichitin (3) comprises β-keto-tryptophan in which a keto group (δC 185.7) is substituted at the β-carbon instead of the methylene group found in compound 4 (where the tryptophan signals were fully assigned to an unsubstituted tryptophan moiety with the signals of the methylene group assigned at δC 27.8 and δH 3.33, 3.20). COSY and HMBC correlations further confirmed the structure of 4 as a new arthrichitin derivative, named D



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was performed using an amaZon Speed ETD ion trap mass spectrometer (Bruker Daltonics), HR-ESIMS spectra were recorded on a maXis ESI TOF mass spectrometer (Bruker Daltonics), and preparative HPLC purification was performed at room temperature on an Agilent 1100 series preparative HPLC system (Agilent Technologies) according to our previous report.22 NMR spectra were recorded on a Bruker 500 MHz Avance III spectrometer with a BBFO (plus) SmartProbe (1H 500 MHz, 13C 125 MHz) and a Bruker 700 MHz Avance III spectrometer with a 5 mm TCI cryoprobe (1H 700 MHz, 13C 175 MHz). Strain Origin and Identification. A survey was carried out in the experimental wheat fields of CIMMYT (the International Maize and Wheat Improvement Centre) located in Yozgat (39°08′ N, 34°10′ E; altitude, 985 m) in the Central Anatolian Plateau in Turkey. The rhizosphere of wheat plants was sampled at the end of the growing season for nematode cysts of Heterodera f ilipjevi. Soil samples were processed to extract nematode cysts as previously described.6 A culture of the strain (cf. Supporting Information) producing the compounds described here was deposited in the open collection of the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen and Zellkulturen under the accession number DSM106825. As outlined in the Supporting Information, the strain could not be confidentially assigned to a known genus, due to ambiguity of the results of its molecular phylogeny in comparison with literature data. We are concurrently trying to determine whether it represents a new genus or merely a new Ophiosphaerella species. Small-Scale Fermentation and Extraction. Small culture plugs (5 mm diam.) of strain DSM106825 excised from growing cultures on YM 6.3 (malt extract 10 g/L, D-glucose 4 g/L, yeast extract 4 g/L, agar 20 g/L, pH 6.3, before autoclaving) were used to inoculate 5 L of ZM/2 medium in 12 1 L Erlenmeyer flasks each containing 400 mL of the culture medium. The submerged cultures were incubated at 23 °C at 140 rpm on a rotary shaker for 12 days until complete depletion of free glucose. Mycelia and supernatant were separated by filtration and were extracted following previously described protocols.22 Fermentation in a 10 L Bioreactor. For the scale-up, a seed culture of the strain with a total volume of 1000 mL was prepared in ZM medium incubated at 23 °C and 140 rpm for 5 days and homogenized with a Heidolph Silent Crusher. A 15 L xCUBIO in situ bioreactor (bbi biotech, Berlin, Germany) was prepared with 10 L of ZM medium and inoculated with 1 L of the seed culture at a starting pH of 7.2. The incubation temperature was 23 °C, agitation with a Rushton impeller was 150 rpm, and the aeration rate was 1.5 L/min (0.15 vvm) and remained constant during the fermentation. The culture was harvested after 7 days when the glucose was depleted, and a stagnation of secondary metabolite production was observed by analytical HPLC. The extraction process was performed according to our previous report.23 Isolation. The mycelial crude extract was purified over several runs using preparative HPLC using a Kromasil RP C18 (AkzoNobel, Mainz, Germany), particle size 7 μm, dimensions 250 × 25 mm, mobile phase: acetonitrile (B) and water (A), flow rate of 20 mL min−1 with a gradient from 20% to 30% B in 10 min, 30% to 45% B in 5 min, 45% to 80% B in 40 min, 80% to 100% B in 5 min. Compound 1 (20.9 mg) was eluted at a retention time of tR = 27.8 min; compound 2 (6.1 mg) was obtained at tR = 14.1 min. A fraction (35 mg) containing a mixture of compounds 3, 4, and 5 was eluted at tR = 35−36 min. This fraction was further purified over the same column but using different gradient: 50% to 55% B in 10 min, 55% to 60% B in 40 min, 60% to 100% B in 10 min followed by 10 min isocratic conditions at 100% B. Compounds 3 (5.4 mg), 4 (8.9 mg), and 5 (1.4 mg) were eluted at tR = 22.2, tR = 21.2, and tR = 36.7 min, respectively. The fractions were combined according to UV adsorption at 220 and 280 nm and concurrent HPLC-MS analyses. Ophiotine (1): white powder; [α]20D +120 (c 0.5, MeOH); LCMS m/z 875 [M + H]+ (100), 897 [M + Na]+ (22), 873 [M − H]− (100); HRESIMS m/z 875.4652 [M + H]+ (calcd for C45H63N8O10+, 875.4662); 1H NMR and 13C NMR see Table 1. Xanthomide Z (2): white powder; [α]20D +15 (c 0.6, MeOH); LCMS m/z 371 [M + H]+ (38), 393 [M + Na]+ (44), 741 [2M + H]+

arthrichitin B (Figure 3). Arthrichitin B (4) showed a similar ECD spectrum to that of arthrichitin (see Figure 2), and thus

Figure 3. Key HMBC correlations of arthrichitins B (4) and C (5).

Table 3. Nematicidal Activities of Compounds 1−4 against H. filipjevi nematode mortality %a compound

10 μg/ 1 mL

20 μg/ 1 mL

50 μg/ 1 mL

100 μg/ 1 mL

ophiotine (1) xanthomide Z (2) arthrichitin (3) arthrichitin (4)

59 36 15 16

55 33 15 16

79 41 10 10

78 43 10 10

Positive control = ivermectin 100% mortality at 100 μg/mL.

a

the absolute configuration of 4 was determined as L-Ser, D-Glu, D-Trp, and 2S,4R-dimethyl-3S-oxydodecanoic acid. Compound 5 showed the molecular formula C34H48N4O9, indicating a molecular weight of 14 Da more than 3, suggesting the presence of an additional methyl group. Close examination of the NMR data revealed similarities to arthrichitin and arthrichitin B (Table 1). The HSQC spectrum showed an additional signal at δH 3.61, δC 51.8 attributed to a methoxy group (Figure 3). HMBC correlation from the methoxy group to the carbonyl carbon of the carboxylic function in the Glu moiety allowed the construction of 5 as a new derivative of the arthrichitin family incorporating δ-OMe-Glu and named arthricitin C. In a similar manner arthricitin C (5) showed an electronic circular dichroism (ECD) spectrum in good accordance with that of the other arthrichitins (Figure 2). Consequently, the absolute configuration was determined as LSer, δ-OMe-D-Glu, β-oxo-D-Trp, and 2S,4R-dimethyl-3S-oxydodecanoic acid. Compound 3 was active against the yeasts Schizosaccharomyces pombe and Rhodotorula glutinis, whereas the remaining compounds were inactive (Table S1). Furthermore, only the new compound arthrichitin C (5) showed weak cytotoxic activity against HeLa cells KB3.1 with an IC50 value of 28 μM. Arthrichitin C was active against all the tested cell lines, with IC50 values in the range of 26 μM (Table S2 in the Supporting Information). Moreover, ophiotine (1) showed moderate nematicidal activity, while the other examined compounds showed only weak effects against H. filipjevi, the host of the producing fungal strain (Table 3).

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were determined with a PerkinElmer 241 MC polarimeter (using the sodium D line and a quartz cuvette with 10 cm path length and 1 mL volume); CD spectra were recorded on a JASCO spectropolarimeter, model J-815, using a 1 mm quartz cuvette. HPLC-DAD/MS analysis E



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(100), 369 [M − H]− (100), 739 [2M − H]− (85); HRESIMS m/z 371.1597 [M + H]+ (calcd for C20H23N2O5+, 371.1601); 1H NMR and 13C NMR see Table 2. Arthrichitin (3): white powder; [α]20D +38 (c 0.2, MeOH); CD (c 1 mg/mL, EtOH) λmax nm (θ) 318 (+ 7.1), 286 (− 2.2), 258 (− 1.5), 240 (− 5.6), 216 (− 1.1), 200 (− 3.2); LCMS m/z 643 [M + H]+ (61), 1285 [2M + H]+ (56), 641 [M − H]− (44), 1283 [2M − H]− (89); HRESIMS m/z 643.3323 [M + H]+ (calcd for C33H47N4O9+, 643.3338); 1H NMR (700 MHz, methanol-d4) δH Ser: 4.25 (H-2, dd, J = 8 and 4 Hz), 3.59 (H-3a, dd, J = 8 and 10.7 Hz), 3.54 (H-3b, dd, J = 10.7 and 4.7 Hz), β-Oxo-Trp: 5.77 (H-5, s), 8.41 (H-8, s), 8.26 (H10, d, J = 6.9 Hz), 7.24 (H-11, dd, overlapping), 7.29 (H-12, dd, overlapping), 7.47 (H-13, d, J = 7.3 Hz), Glu: 4.54 (H-15, m), 2.03 (H-16, m), 2.37 (H-17, m), dimethyl-oxy-dodecanoic acid: 2.86 (H20, m), 5.12 (H-21, J = 9 and 3 Hz), 1.8 (H-22, m), 1.40 (H-23a, m), 1.15 (H-23b, m), 1.39 (H-24, m), 1.29 (H-25−27, m), 1.31 (H-28, m), 1.28 (H-29, m), 0.89 (H-30, t, J = 7 Hz), 1.18 (H-31, d, J = 7 Hz) 0.92 (H-32, d, J = 6.5 Hz); 13C NMR (175 MHz, methanol-d4) δC Ser: 170.8 (C, C-1), 56.0 (CH, C-2), 61.2 (CH2, C-3), β-Oxo-Trp: 170.1 (C, C-4), 63.9 (CH, C-5), 185.7 (C, C-6), 137.5 (CH, C-8), 115.4 (C, C-9), 127.3 (C, C-9a), 122.9 (CH, C-10), 123.9 (CH, C11), 124.9 (CH, C-12), 113.3 (CH, C-13), 138.6 (C, C-13a), Glu: 173.9 (C, C-14), 53.9 (CH, C-15), 26.6 (CH2, C-16), 31.1 (CH2, C17), 176.3 (C, C-18), dimethyl-oxy-dodecanoic acid: 177.7 (C, C19), 43.8 (CH, C-20), 80.7 (CH, C-21), 35.8 (CH, C-22), 35.2 (CH2, C-23), 28.6 (CH2, C-24), 30.6-21.0 (3CH2, C-25−27), 23.9 (CH2, C28), 33.2 (CH2, C-29), 14.6 (CH3, C-30), 14.1 (CH3, C-31), 16.6 (CH3, C-32). Arthrichitin B (4): pale yellow powder, [α]25D +33 (c 0.1, MeOH); CD (c 1 mg/mL, EtOH) λmax nm (θ) 318 (+5.4), 286 (−2.2), 258 (−1.5), 240 (−9.3), 216 (−1.1), 200 (−5.7); LCMS m/z 629 [M + H]+ (100), 1257 [2M + H]+ (82), 1279 [2M + Na]+, 627 [M − H]− (16), 1255 [2M − H]− (100); HRESIMS m/z 629.3536 [M + H]+ (calcd for C33H49N4O8+, 629.3545); 1H NMR and 13C NMR see Table 1. Arthrichitin C (5): pale yellow powder, [α]25D +22 (c 0.1, MeOH); CD (c 1 mg/mL, EtOH) λmax nm (θ) 318 (+14.7), 286 (−6.4), 258 (−4.0), 240 (−20.1), 216 (−1.7), 203 (−15.5); LCMS m/z 657 [M + H]+ (88), 679 [M + Na]+ (12), 1313 [2M + H]+ (100), 1335 [2M − Na]+ (35), 655 [M − H]− (92), 1311 [2M − H]−; HRESIMS m/z 657.3492 [M + H]+ (calcd for C34H49N4O9+, 657.3494); 1H NMR and 13C NMR see Table 1. Acid Hydrolysis and Marfey’s Analysis of Ophiotine (1). The hydrolysis and Marfey analysis were carried out according to Fujii et al. and Capon et al. with slight modification.13,14 Briefly, to 500 μg of ophiotine (1) was added 1 mL of 6 M HCl, and the resulting solution was stirred under reflux at 120 °C for 24 h. The reaction mixture was dried under reduced pressure, dissolved in water (250 μL), and subjected to analytical LC-MS. To 50 μL of this hydrolysate were added 20 μL of sodium bicarbonate (1 M) and 100 μL of L-FDAA (1%) in acetone. The resulting solution was stirred at 37 °C for 60 min, then neutralized with 20 μL of HCl 1 M and diluted with MeCN (800 μL). Authentic standards of L-Gln, L-Phe, L-Ser, L-Thr, and L-Trp (1 mg each) were dissolved in water (500 μL), and 50 μL of each was treated with L-FDAA as described above for the acid hydrolysate to yield L-FDAA standards. HPLC-DAD-MS analysis of Marfey’s derivatives of compound 1 and the above L-FDAA standards was performed using an amaZon Speed ETD ion trap mass spectrometer (Bruker Daltonics) in positive and negative ionization modes. Nematode Bioassays. The isolated compounds were assessed against H. filipjevi for nematicidal activity. The second stage juveniles (J2) were obtained as described previously6 and were used for nematode bioassays. The assays were performed in 24-well microtiter plates at concentrations of 100, 50, 20, and 10 μg/mL in MeOH. An aliquot of each compound was transferred to each well and incubated in a laminar flow hood to dry the material. Then an aliquot of nematode suspension was added to each well. Ivermectin was used as a positive control. For the negative control, an aliquot of pure methanol was transferred to a well, left to dry, and then added with an

aliquot of nematode suspension. All treatments were tested in triplicate. Determination of Antimicrobial and Cytotoxicity Effects. The antimicrobial activities of the isolated compounds toward bacteria, yeasts, and filamentous fungi were determined in a serial dilution assay using 96-well microtiter plates.23 The in vitro cytotoxicity (IC50) of all compounds was determined against a panel of six mammalian cell lines including murine fibroblasts L929, human cervix carcinoma KB-3-1, human lung carcinoma A549, ovarian carcinoma SKOV-3, human prostate cancer PC-3, and breast cancer cell line MCF-7 by using the MTT assay according to established procedures.24,25

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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.8b00486.

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Experimental procedures, 1D and 2D NMR data, LCMS data, and morphological and phylogenetic details of the producing organism (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Marc Stadler: 0000-0002-7284-8671 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS S.E.H. and R.BT. are grateful to the Alexander von Humboldt Foundation for postdoctoral fellowships. We are grateful to W. Collisi for conducting the bioassays, C. Bergmann and S. Karwehl for recoding mass spectra, and C. Kakoschke for recording NMR data.

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REFERENCES

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