Natural Products of Picea Endophytes from the Acadian Forest

Article pubs.acs.org/jnp

Natural Products of Picea Endophytes from the Acadian Forest David R. McMullin, Blake D. Green, Natasha C. Prince, Joey B. Tanney, and J. David Miller* Ottawa Carleton Institute of Chemistry, Carleton University, 1125 Colonel By Drive, Ottawa, Ontario, Canada, K1S 5B6 S Supporting Information *

ABSTRACT: Endophytes of healthy needles were collected from Picea rubens (red spruce) and P. mariana (black spruce) in a survey of southeastern New Brunswick, Canada. Four endophyte strains were selected for further investigation based on the production of biologically active extracts from culture filtrates during screening as well as phylogenetic relationship to species known to produce natural products or taxonomic novelty. A novel endophyte within the family Rhytismataceae produced two new dihydropyrones (1 and 2) as major metabolites together with phthalides (3 and 4), isocoumarins (5 and 6), and tyrosol (7). Lachnum cf. pygmaeum synthesized a new chlorinated para-quinone, chloromycorrhizinone A (8), and the nematicidal compounds (1′Z)-dechloromycorrhizin A (9), mycorrhizin A (10), and chloromycorrhizin A (11). A new isocoumarin (12) and four related structures (13−16) were isolated from an undescribed taxon in the Mycosphaerellaceae. The known antifungal metabolites cryptosporiopsin (17), 5-hydroxycryptosporiopsin (18), (+)-cryptosporiopsinol (19), and mellein (20) were produced by Pezicula sporulosa. Phylogenetically diverse conifer endophytes from the Acadian forest continue to be a productive source of new biologically active natural products.

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characterized from a P. strobus endophyte from the family Massarinaceae.13 As part of our ongoing survey of the mycological and chemical diversity of foliar endophytes from the Acadian forest, we report metabolites from endophytes of Picea rubens (red spruce) and P. mariana (black spruce). Needle endophytes were isolated from tree branches of various age classes in both managed and natural stands to capture a representative phylogenetic and chemotaxonomic diversity across the landscape. From the more than 300 endophyte strains screened for biologically active natural products from this collection, four additional strains were selected for larger scale fermentations. We discuss the characterization of the major natural products and their potential ecological relevance.

oliar endophytes of conifers cause asymptomatic infections that can improve the fitness of the host plant. Some endophytes improve host tolerance to insect pests and/or diseases. Sometimes called defensive symbiosis, this is associated with the accumulation of effective concentrations of fungal secondary metabolites primarily in leaf tissues.1,2 Metabolites toxic to herbivorous insects have been characterized from phylogenetically diverse foliar endophytes of conifers.3−6 When inoculated in seedlings, some endophytes have been shown to persist for at least a decade, producing their respective metabolites in the needles.7 The best understood example of the impact of toxigenic foliar endophytes of white spruce concerns the needle herbivore Choristoneura f umiferana (eastern spruce budworm). The rugulosin-producing endophyte Phialocephala scopiformis delays the development of this destructive pest. This exposes C. f umiferana larvae for an extended period to birds, parasitoids, and pathogens prior to moth formation.2,8 We have extended our studies of needle endophytes to look for antifungal compounds that may reduce needle diseases such as Cronartium ribicola (white pint blister rust). Potently antifungal macrolides and racemic sesquiterpenoids were characterized from Lophodermium nitens (Rhytismataceae), an endophyte of Pinus strobus (eastern white pine).9,10 The homodimeric macrolide pyrenophorol inhibited the growth of Cronartium ribicola, the causative agent of the disease white pine blister rust, at low micromolar concentrations.11 The potent antifungal compound griseofulvin was identified from a Xylaria endophyte of P. strobus foliage and low bush blueberry (Vaccinium angustifolium) stems collected in Eastern Canada.12 Unusual xanthenes and chlorinated dihydrobenzofurans were © 2017 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The four endophyte strains included in this study were selected based on their production of biologically active culture filtrate extracts and phylogenetic relationships with species known to produce bioactive secondary metabolites or taxonomic novelty. The internal transcribed spacer (ITS) barcode and partial 28S nuclear ribosomal large subunit (LSU) rRNA gene region were used for strain identification through comparison with reference sequences from NCBI GenBank and a sequence database (J.B. Tanney, Carleton University, unpublished data) using the Basic Local Alignment Search Tool (BLAST) algorithm. The ITS barcode and partial 28S nuclear ribosomal LSU rRNA gene region were sequenced for each strain and deposited in the Received: December 22, 2016 Published: April 11, 2017 1475

DOI: 10.1021/acs.jnatprod.6b01157 J. Nat. Prod. 2017, 80, 1475−1483

Journal of Natural Products

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fragment ion for compound 1 at m/z 127.1117 [M + H]+ suggested a C8 side-chain bearing a single oxygen atom. The remaining COSY cross-peaks were between H-5 (δ 2.76 and 2.96) and H-6 (δ 4.90). HMBC correlations were observed from both H-5 and H-6 to C-4 (δ 197.5) and C-7 (δ 172.6), where H-6 showed an additional correlation to C-2 (δ 174.1). COSY and key HMBC correlations for compound 2 were virtually identical to those of compound 1 (Figure 1). These

National Center for Biotechnology Information (NCBI) GenBank sequence database for reference (Supporting Information Table S1). All strains were sterile on culture media tested and under various growth conditions. Additional information regarding the endophytes studied for natural products can be found in the Supporting Information, Appendix 1. Metabolites of Rhytismataceae sp. DAOMC 251461. An unknown fungal species with an uncertain position within the family Rhytismataceae (Rhytismatales) was isolated as an endophyte of healthy P. mariana needles (Supporting Information Figure S1). The culture filtrate from fermentations of DAOMC 251461 yielded a major metabolite (1) obtained as a light yellow solid with the molecular formula C14H20O6 determined by HRMS. The 1H and 13C data revealed 18 protons and 14 carbons, respectively (Table 1). The UV Table 1. 1H (400 MHz) and 13C (100 MHz) NMR Data in CD3OD for the New Dihydropyrones Rhytismatones A (1) and B (2) from the Rhytismataceae Endophyte DAOMC 251461 1 position

δC, type

2 3 4 5

174.1, C 99.6, C 197.5, C 36.6, CH2

6 7 8 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′

79.1, CH 172.6, C

a

195.3, C 36.5, CH2 26.7, CH2 30.1, CH2 30.4, CH2 32.8, CH2 23.6, CH2 14.4, CH3

2

δH (J in Hz)

2.96, dd (17.2, 4.0) 2.76, dd (17.2, 6.6) 4.90, brsa

2.85, 1.65, 1.33, 1.33, 1.28, 1.31, 0.89,

Figure 1. Observed COSY (bold lines) and key HMBC (arrows) correlations for rhytismatone B (2), chloromycorrhizinone A (8), and isocoumarin 12 reported as new structures.

m p (7.4) m m m m t (7.0)

δC, type 174.1, C 99.6, C 197.6, C 36.5, CH2 78.9, CH 171.4, C 52.5, CH3 195.8, C 36.5, CH2 26.6, CH2 30.1, CH2 30.4, CH2 32.8, CH2 23.6, CH2 14.4, CH3

data established the presence of a trisubstituted 5,6dihydropyrone-2-one core structure with a carboxylic acid functionality bound to C-6. The chemical shifts of C-3 and C-4 and the fact that compound 1 undergoes a slow keto/enol tautomerization when left in CD3OD indicated the 1-octanal moiety is attached at C-3. This type of chemical equilibrium was observed for podoblastin A, a metabolite with a similar 3acyl-4-hydroxy-5,6-dihydropyrone core structure.14 The absolute configuration of compound 1 at C-6 was determined to be R based on comparison of its experimentally determined specific rotation, [α]25D −30 (c 0.4, CHCl3), to structurally similar compounds with a single chiral center. The

δH (J in Hz)

3.00, dd (17.1, 3.9) 2.85, m 4.93, brsa 3.67, s 2.85, 1.65, 1.33, 1.33, 1.28, 1.31, 0.89,

m p (7.4) m m m m t (7.0)

Chart 1

4.90, brs in CD3CN.

spectrum showed absorption maxima at 205, 230, and 270 nm. The 1H NMR spectrum displayed a single terminal methyl at δ 0.89 (t, J = 7.0), five aliphatic methylene signals between δ 1.28 and 1.65, an inequivalent methylene at δ 2.76 (dd, J = 17.2, 6.6) and 2.96 (dd, J = 17.2, 4.0), a methylene adjacent to a carbonyl at δ 2.85 (m), and an oxygenated methine at δ 4.90 (brs). Examination of the 13C and HSQC spectra revealed five of the 14 carbon signals were nonprotonated. These five signals are attributed to a ketone at δ 195.3, a lactone at δ 174.1, a carboxylic acid at δ 172.6, and two additional sp2 carbons at δ 99.6 and 197.5. Examination of the mass spectrometry and spectroscopic data suggested compound 1 possessed a trisubstituted dihydropyrone-2-one core structure with five units of unsaturation attributed to one ring and four double bonds. COSY cross-peaks were observed sequentially from the triplet methyl H-8′ (δ 0.89) to H-2′ (δ 2.85), where five of the methylene signals (δ 0.89−1.65) appeared in the aliphatic region of the 1H NMR spectrum. The chemical shift of H-2′ and an HMBC correlation from H-2′ to C1′ (δ 195.3) place H2′ vicinal to a carbonyl, supporting the presence of a 1-octanal moiety in compound 1. Moreover, the dominant MS/MS 1476



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oryzae, the causative agent of rice blast, also in the low μg mL−1 range.14 Besides dihydropyrones (1 and 2), other metabolites were produced by the Rhytismataceae species black spruce endophyte examined including phthalides (3 and 4), isocoumarins (5 and 6), and the amino acid derived compound tyrosol (7). The two characterized phthalides (3 and 4) were previously isolated together with other antibacterial phthalides and chromanols from an Aspergillus species.20 5-Hydroxy-7methoxy-4,6-dimethylphthalide (3) has previously been isolated from a Microsphaeropsis endophyte obtained from the foliage of Garcinia hombroniana (seashore mangosteen)21 and a wood-associated Lachnum sp. strain.22,23 5-Hydroxy-4-(hydroxymethyl)-7-methoxy-6-methylphthalide (4) was prepared as a reduced derivative of cyclopaldic acid for a structure−activity relationship study examining mosquito bite deterrents and showed activity.24 Pthalides (3 and 4) had modest antifungal activity toward the rust fungus M. violaceum, and 4 showed activity toward both test bacteria species (Table 4). The isocoumarin 8-hydroxy-6-methoxy-3-methyisocoumarin (5) was previously produced by endophytes of Smallanthus sonchifolius (common name, yacón)25 and together with indole3-acetic acid, tyrosol (7), and several other para-hydroxyphenolic metabolites from Ceratocystis f imbriata.26 (R)-8-Hydroxy6-methoxy-3-propylisochromanone (6) was isolated from a Sporormia species with chlorinated dihydroisocoumarin analogues27 and an unidentified P. glauca endophyte, also from the family Rhytismataceae (CBS 120380).4 The dihydroisocoumarin 6 inhibited the growth of E. coli, M. violaceum, and S. cerevisiae more effectively compared to 5, which was not active in these assays (Table 4). Tyrosol (7) has been reported from many fungal genera including Diaporthe, Xylaria, and Epichloë.28−30 It was also identified from three P. glauca (white spruce) endophytes (CBS 120379−120381).4 The P. glauca endophytes CBS 120379 and 120380 are closely related to an unidentified P. mariana endophyte isolated in Quebec, Canada, based on ITS sequence comparisons.31 A phylogenetic analysis of Rhytismataceae endophytes obtained from the Acadian forest determined that DAOMC 251461 studied here and CBS 120380 are distinct species, with CBS 120380 being an undescribed Tryblidiopsis species (Supporting Information Figure S1). Their secondary metabolite profiles are similar, as the dihydroisocoumarin (6) and tyrosol (7) were characterized from both extracts. A phthalide, 5,7-dimethoxy-3-methylphthalide, structurally similar to compounds 3 and 4 was also characterized previously; however, no dihydropyrones related to 1 and 2 were identified from CBS 120380.4 Incorporation of this strain’s culture filtrate extract into the diet of spruce budworm larvae significantly reduced their weight and head capsule size.4 The similarity of these metabolite profiles suggests the undescribed Rhytismataceae endophyte studied here synthesizes metabolites toxic to spruce budworm, warranting further investigation. Metabolites of Lachnum cf. pygmaeum DAOMC 250335. Lachnum cf. pygmaeum DAOMC 250335 was obtained from ascospores originating from a collection of apothecia occurring on a dead P. rubens twig but was included in this study because of its close relationship with plant root fungal associates and previous reports of biologically active metabolites from Lachnum spp.32−34 Compound 8 was isolated from the fermentation broth as a yellow oil with the molecular formula C14H14O4Cl2 established by HRMS, indicative of seven units of unsaturation. The presence of two chlorine atoms

absolute stereochemistry of podoblastin A at C-6 was determined to be R by stereoselective synthesis, which had a specific rotation of [α]D −27.08 (c 1.12, CHCl3).14,15 Dictyopyrone A shares the same core structure and has an S configuration at C-6. The specific rotation of dictyopyrone A is reported as [α]25D 33.8 (c 0.07, CHCl3) when isolated as a natural product and [α]25D 33.9 (c 1.23, CHCl3) when prepared synthetically using chiral reagents.16 Since all these structurally similar dihydropyrones possess a single chiral center at the same position, compound 1 is reported here as a new structure, (R)-4-hydroxy-5-octanoyl-6-oxo-3,6-dihydropyran-2-carboxylic acid (rhytismatone A). Compound 2 was also isolated as a light yellow solid with the molecular formula C15H22O6 determined by HRMS. The increase in mass for compound 2 compared to 1 corresponds to the addition of a methyl group. The MS/MS spectrum of compound 2CF revealed the same dominant m/z 127.1117 [M + H]+ fragment ion, suggesting it also possessed a 1-octanal moiety. The 1H and 13C NMR data (Table 1) of compounds 1 and 2 were very similar. However, the 1H and 13C spectra of 2 displayed additional resonances at δ 3.67 (s) and 52.5, respectively, indicative of a methoxy functionality. An HMBC correlation from the singlet methyl H-8 (δ 3.67) to C-7 (δ 171.4) supported the presence of a methyl ester in compound 2, compared to the carboxylic acid in compound 1 (Figure 1). The specific rotation of compound 2, [α]D −68 (c 0.4 CHCl3), was also negative, indicating an R configuration at C-6. Compound 2 is reported here as a new structure, (R)-methyl 4-hydroxy-5-octanoyl-6-oxo-3,6-dihydropyran-2-carboxylate (rhytismatone B). Rhytismatones A (1) and B (2) are reported here as new structures that share the same 3-acyl-4-hydroxy-5,6-dihydro-2pyrone core structure as alternaric acid, originally isolated from Alternaria solani,17,18 and podoblastins A−C obtained from Podophyllum (may-apple), a medicinal plant.14,19 The methylated dihydropyrone (2) exhibited moderate antifungal activity, inhibiting the growth of Saccharomyces cerevisiae at 25 μg mL−1 (Table 4). Alternaric acid is antifungal at low μg mL−1 concentrations toward specific fungi.18 Podoblastins were identified as the primary antifungal constituents of a Podophyllum extract that inhibited the growth of Pyricularia Table 2. 1H (400 MHz) and 13C (100 MHz) NMR Data in CD3OD for Chloromycorrhizinone A (8) from Lachnum cf. papyraceum DAOMC 250335 position

δC, type

1 2 3 4 5 6 8 9 10

117.0, C 179.9, C 140.8, C 122.8, C 178.3, C 153.2, C 82.6, C 64.4, CH 26.5, CH2

11 12 1′ 2′ 3′

21.1, CH3 25.0, CH3 143.5, C 131.6, CH 14.3, CH3

δH (J in Hz)

3.78, 2.70, 2.48, 1.38, 1.33,

t (5.1) dd (18.4, 5.1) dd (18.4, 5.1) s s

5.95, q (6.7) 1.91, d (6.7) 1477



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Table 3. 1H (400 MHz) and 13C (100 MHz) NMR Data in CD3OD for Isocoumarins (12−14) from Mycosphaerellaceae sp. DAOMC 250863 12 position

δC, type

1 3 4 4a 5

172.3, C 81.0, CH 73.8, CH 34.9, CH 33.3, CH2

6 7

64.1, CH 38.0, CH2

8 8a 9

175.0, C 95.7, C 18.9, CH3

13 δH (J in Hz)

δC, type

4.20 dq (9.3, 6.3) 3.11 dd (10.5, 9.3) 2.71, m 2.29, m 1.25, m 4.28, m 2.64, m 2.33, m

173.3, C 78.6, CH 36.7, CH2 27.7, CH 37.9, CH2 64.3, CH 38, CH2 173.8, C 97.6, C 22, CH3

1.40, d (6.3)

S. cerevisiae

B. subtilis

E. coli

1 2 3 4 5 6 8 9 10 11 12 13 14 15 16 17 18 19

− − 100 100 − 50 25 100 50 50 − − − − − − − −

50 25 100 − − 100 − − 50 50 − − 100 − − 100 − −

− 100 − 100 − − 25 100 25 25 − − − − − 100 − −

− − − 100 − 25 25 − 25 25 100 100 − − − 50 − −

b

m m m m

4.25, m 2.65, dq (19.4, 2.6) 2.33, dq (19.4, 1.4)

δC, type 170.2, C 81.0, CH 69.5, CH 144.1, C 117.4, CH

δH (J in Hz) 4.56, o 4.55, o 7.08, dd (7.5, 0.8)

137.6, CH 117.5, CH

7.57, dd (8.4, 7.5) 6.94, dd (8.4, 0.8)

162.9, C 108.0, C 17.8, CH3

1.47, d (6.0)

notably different compared to 9−11. The chemical shifts of C-1 and C-6 and COSY cross-peaks from H-9 to both H-10 signals indicated the dimethyloxabicyclohexane moiety of compounds 9−11 was replaced by a hydroxylated dimethyldihydropyran ring in 8. This structural alteration was observed for the related metabolite chloromycorrhizinol previously characterized together with mycorrhizin A (10) and chloromycorrhizin A (11) from an unidentified mycorrhizal fungus of Monotropa hypopitys35 and L. papyraceum.34 The relative stereochemistry of 8 was determined to be R based on an NOE between H-9 and H-12. The chiral configurations of related metabolites of L. papyraceum including mycorrhizins and papyracons possess identical stereochemistry.36 The specific rotations of compound 8, [α]25D 11 (c 0.3, MeOH), and chloromycorrhizinol, [α]25D 30.9 (c 1.7, EtOH), are both positive, which suggests they possess the same stereochemistry at position 9. Here, we report 8 as a new compound, chloromycorrhizinone A. Lachnum papyraceum is closely related to DAOMC 250335 and is a known producer of diverse biologically active metabolites. A single strain originating from woody plant material in Germany yielded more than 30 natural products. Most of these displayed anti-insectan and nematicidal activity.37 That this species produces metabolites toxic to nematodes is not surprising since they have coevolved while occupying similar environments including decaying wood and plant materials. (1′Z)-Dechloromycorrhizin A (9), mycorrhizin A (10), chloromycorrhizin A (11), lachnumon, and lachnumol A were previously isolated from this wood-inhabiting L. papyraceum strain.33,38 Interestingly, one of the phthalides characterized from the German L. papyraceum strain was compound 3, also isolated from the Rhytismataceae endophyte DAOMC 251461 studied here. Using compound 3 as an analytical standard, it was identified from L. cf. pygmaeum DAOMC 250335 by LC-HRMS. Chloromycorrhizinone A (8), mycorrhizin A (10), and chloromycorrhizin A (11) all displayed antibiotic activity against E. coli and B. subtilis. (1′Z)-Dechloromycorrhizin A (9) was less potent in our bioassays. Besides cytotoxic and nematicidal activities, mycorrhizin A (10) and chloromycorrhizin A (11) were previously shown to be antibiotic in the low μg mL−1 range.38 Chloromycorrhizinone A (8) inhibited the growth of M. violaceum, whereas compounds 10 and 11 were active against both S. cerevisiae and M. violaceum at a higher concentration (Table 4).

MIC (μg mL‑1) M. violaceum

4.56, 1.32, 2.88, 1.96,

1.37, d (6.3)

Table 4. Antimicrobial Activity of Natural Products Isolated from Conifer Endophytesa compound

14 δH (J in Hz)

a

Positive controls inhibited the growth of the test organisms at the lowest concentrations tested; there was no effect of DMSO (see text for details). b− no inhibition observed under conditions tested.

within the structure was supported by an increase in the [M + H]+:[M + H + 2]+ isotopic peak ratio. The 1H and 13C NMR spectra (Table 2) showed 11 and 14 resonances, respectively. Proton signals were attributed to an olefinic methine at δ 5.95 (q, J = 6.7, H-2′), an oxygenated methine at δ 3.78 (t, J = 5.1, H-9), an inequivalent methylene at δ 2.48 (dd, J = 18.5, 5.1, H10) and 2.70 (dd, J = 18.5, 5.1, H-10), and three methyl groups at δ 1.33 (s, H-12), 1.38 (s, H-11), and 1.91 (d, J = 6.7, H-3′). Interpretation of the 13C and HSQC spectra indicated eight of the 14 carbon signals were nonprotonated. These signals were resultant of two carbonyls at δ 178.3 (C-5) and 179.9 (C-3), five sp2 carbons at δ 117.0 (C-1), 122.8 (C-4), 140.8 (C-3), 143.5 (C-1′), and 153.2 (C-6), and one sp3 carbon at δ 82.6 (C-8). The NMR spectroscopic data of compound 8 were similar to the para-quinones (1′Z)-dechloromycorrhizin A (9) mycorrhizin A (10) and chloromycorrhizin A (11) also isolated from L. cf. pygmaeum DAOMC 250335. However, the 1H and 13 C NMR chemical shifts of positions 1, 6, 9, and 10 for 8 were 1478



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Chart 2

Metabolites of Mycosphaerellaceae sp. DAOMC 250863. Based on ITS sequences, the P. mariana endophyte DAOMC 250863 is an undescribed Mycosphaerellaceae species, a putative sister species of Phaeocryptopus gaeumannii (Supporting Information Figure S2). Compound 12 was isolated as a clear solid with the molecular formula C10H14O5 determined by HRMS, indicative of four units of unsaturation. The 1H and 13C NMR spectra (Table 3) displayed 11 and 10 resonances, respectively. Interpretation of the 1H NMR spectrum revealed three oxygenated methines at δ 4.28 (m, H-6), 4.20 (dq, J = 9.3, 6.3, H-3), and 3.11 (dd, J = 10.5, 9.3, H4), a methine at δ 2.71 (m, H-4a), two inequivalent methylenes at δ 2.29 (m, H-5), 1.25 (m, H-5), 2.64 (m, H-7), and 2.33 (dd, J = 19.0, 1.3, H-7), and a methyl at δ 1.40 (d, J = 6.3, H-9). Examination of the 13C and HSQC NMR spectra indicated three of the 10 carbon signals were nonprotonated. These signals were attributed to two sp2 carbons at δ 175.0 (C-8) and 95.7 (C-8a) and a lactone at δ 172.3 (C-1). COSY cross-peaks were observed from H-3 to H-9 and sequentially from H-3 to H-7. HMBC correlations from H-4a to C-8, C-8a, and C-1 also support the presence of an isocoumarin structure (Figure 1). The NMR spectroscopic data for compound 12 were very similar to several isocoumarins isolated here and previously from an undescribed fungus.39 The absolute stereochemistry of these biosynthetically related iscoumarins was determined by spectroscopic methods and comparisons to the literature. For 12, NOE correlations were observed from H-3 to H-4a and from H-4 to H-9, and there was a lack of NOE between H-4a and H-6. These data indicted the configurations of C-3, C-4a, and C-6 were R, whereas C-4 was S. These configurations are in accordance with Findlay et al. (1995).39 Compound 12 is reported here as a new isocoumarin structure, (3R,4S,4aR,6R)4,6,8-trihydroxy-3-methyl-3,4,4a,5,6,7-hexahydroisochromen-1one. The culture filtrate extract of DAOMC 250863 afforded four structurally similar isocoumarins (12−15), including the new metabolite 12. The ramulosin related isocoumarins 13−15 were previously characterized from an undescribed endophyte of P. mariana, designated Conoplea elegantula (Cooke), together with several related structures.39 This common P. mariana

endophyte is most likely closely related to or the same species as DAOMC 250863 studied here. However, it was not deposited in a culture collection, thus preventing direct comparison. On the basis of ITS sequences, DAOMC 250863 is 100% identical to the P. rubens endophytes CBS 121943 and DAOMC 239830.40 Compound 14 and four other biosynthetically related metabolites previously characterized39 were also produced by these P. rubens endophytes, and their culture filtrate extracts were toxic to spruce budworm in feeding assays.40 The culture filtrate of the unidentified P. mariana endophyte and several isocoumarins, including 14, were toxic to spruce budworm larvae and cells.39 Here, compounds 12 and 13 showed modest antibiotic activity to E. coli, whereas 14 modestly inhibited the growth of S. cerevisiae (Table 4). Metabolites of Pezicula sporulosa DAOMC 250862. P. sporulosa DAOMC 250862 (Dermateaceae, Helotiales) was isolated as a P. rubens endophyte and shares a 100% similar ITS sequence to the P. sporulosa ex-type (NR_137161; CBS 224.96). Biologically active secondary metabolites have been reported from endophytic Pezicula strains,41,42 which prompted the investigation of P. sporulosa DAOMC 250862 metabolites in this study. From the fermentation broth of DAOMC 250862, three chlorinated metabolites (17−19) were characterized, and (R)-mellein (20) was detected using an in-house standard. (+)-Crytosporiopsin (17) and related chlorinated cyclopentenone homologues were originally isolated from a coprophilous Sporormia sp.,27 a Periconia sp.,43 and a Cryptosporiopsis sp.44 Several biosynthetically related chlorinated dihydroisocoumarins not isolated here or previously from Cryptosporiopsis were characterized from the other cryptosporiopsin-producing fungi. The structure of (+)-crytosporiopsin (17) was confirmed by Xray crystallography to be of the S configuration at positions 1 and 5.27 It is not surprising that (+)-crytosporiopsin (17) was isolated from a P. sporulosa endophyte since one of the original reports of this metabolite was from its anamorphic state, Cryptosporiopsis, isolated from decaying Betula alleghaniensis (yellow birch).45,46 Interestingly, the P. sporulosa endophyte studied here was collected as an endophyte of red spruce in a yellow birch and red maple stand. In a study of Pezicula endophytes isolated from asymptomatic deciduous and 1479



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insectan activities.28,42 As previously noted, this metabolite has been characterized from a white spruce endophyte extract that displayed anti-insectan activity toward spruce budworm larvae.4 The P. sporulosa endophyte of P. rubens examined here and the several Pezicula species investigated previously were all isolated from needles of healthy tree branches.42 The synthesis of antifungal metabolites such as cryptosporiopsin (17), which are antagonistic toward plant pathogens, suggests defensive mutualism.42,45 Since these Pezicula endophytes were reported to be recovered from a high percentage of healthy foliage, it appears these strains are normally associated with decaying wood and have low virulence toward their host. The life cycles of foliar endophytes of conifers are currently poorly understood despite their important ecological roles. Continuing to investigate the in planta production of metabolites of taxonomically distant endophytes would contribute to a better understanding of plant−endophyte relationships and their biodiversity, cryptic life cycles, and ecological roles.

coniferous tree branches in Northern Germany, (+)-crytosporiopsin (17) was characterized from six of the 85 strains studied.42 This chlorinated natural product possesses antifungal activity against several other wood rot fungi and is antibacterial. These authors suggested this biologically active metabolite could be applied to prevent the deterioration of forest products or control diseases of agriculture and forests.45 This agrees with the findings in this study, where cryptosporiopsin (17) was antibacterial toward E. coli and B. subtilis and inhibited the growth of S. cerevisiae. The 5-hydroxy cryptosporiopsin analogue (18) was originally isolated from the fermentation broth of a wood-derived Cryptosporiopsis sp. with cryptosporiopsin (17). Chemical derivatization and comparison of the spectroscopic data of compounds 17 and 18 determined that both metabolites were 5S. However, 18 was 1R as opposed to 17, which was 1S.46 Compound 18 did not display any considerable in vitro antifungal activity when assayed previously nor in the present investigation.46 This suggests the C-5 chlorine is necessary for appreciable antifungal activity. Compound 19 was isolated as a clear solid with the molecular formula C10H12Cl2O4 determined by HRMS. The isotopic peak distribution indicative of two chlorine atoms was also observed for 19. The NMR spectroscopic data for compound 19 and crytosporiopsin (17) were similar; however, subtle differences were observed (Supporting Information Table S2). A COSY cross-peak between δ 4.44 (d, J = 6.7, H-4) and 4.17 (d, J = 6.7, H-5) not observed for compounds 17 and 18 and the chemical shift of C-4 (δ 75.9) were notable differences compared to the previously elucidated structures. These spectroscopic data and the HRMS data indicated compound 19 possessed a hydroxy group at C-4 instead of a ketone functionality. The 1H and 13C data for compound 19 were in accord with those for cryptosporiopsinol.43,47 However, comparison of the experimentally determined specific rotation of 19, [α]D 23.6 (c 0.16, MeOH), had the opposite sign, [α]D −90 (c 0.56, MeOH), compared to cryptosporiopsinol isolated from a Periconia species.43 The absolute stereochemistry of (−)-cryptosporiopsinol was determined to be 1S, 4S, and 5R by X-ray crystallography.43,47 For compound 19, an NOE between H-4 and H-5 indicated these protons are in the same plane. This observation was consistent with (−)-cryptosporiopsinol.43 The absolute configuration of positions 1, 4, and 5 could not be determined for compound 19; however, it is reported here as (+)-cryptosporiopsinol. An in-house standard of (R)-mellein (20) isolated from an undescribed Rhytismataceae foliar endophyte of white spruce was utilized to confirm its production by P. sporulosa DAOMC 250862.4 At the appropriate retention index, a [M + H]+ ion at m/z 179.0702 was observed, identifying the simple isocoumarin within the culture filtrate extract. In a survey of the secondary metabolites produced by endophytic Pezicula species, (R)mellein (20) was detected from all 85 extracts studied.42 While (R)-mellein (19) could be characteristic of the genus, the antifungal metabolites cryptosporiopsin (17), (−)-mycorrhizin A, and 4-epi-ethiosolide appear to be more species specific, although metabolite profiles were influenced by culture conditions.42 Several other structurally related nonchlorinated isocoumarins have been characterized from endophytic Pezicula species (reported as Cryptosporiopsis).48 (R)-Mellein (20) was originally reported from Aspergillus melleus49 and subsequently from several fungal genera, plants, and insects.4,50 This metabolite possesses antifungal, herbicidal, algicidal, and anti-

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EXPERIMENTAL SECTION

General Experimental Procedures. NMR spectra of isolated metabolites were recorded with a Bruker Avance 400 spectrometer (Milton, On) at 400.1 (1H) and 100 MHz (13C) using a 5 mm autotuning broadband probe with a Z-gradient. Secondary metabolites were dissolved in CD3OD (CDN Isotopes, Point Claire, Quebec) and referenced to the solvent peak (δH 3.31and δC 49.1). LC-HRMS and LC-MS/MS data were acquired with a Thermo Q-Exactive Orbitrap mass spectrometer (Thermo Scientific, Waltham, MA, USA), coupled to an Agilent 1290 HPLC system. Crude extracts and pure metabolites were separated by an Agilent Zorbax Eclipse Plus RRHD C18 (2.1 × 50 mm, 1.8 μm) at a flow rate of 0.3 mL min−1 using mobile phases consisting of acetonitrile (ACN)−doubly distilled water (ddH2O) with 0.1% formic acid (FA) (v/v). Column fractions were screened by LCUV-MS using a Waters 2795 separation module, Waters 996 diode array detector, and Micromass Quatro LC mass spectrometer. Fractions were separated by a Phenomenex Kinetix C18 (100 × 4.60 mm, 2.6 μm) column (Torrance, CA, USA) and a mobile phase consisting of ACN−ddH2O with 0.1% FA (v/v). Metabolites were isolated by semipreparative HPLC utilizing an Agilent 1100 HPLC system equipped with a diode array detector and a mobile phase consisting of ACN−ddH2O. Linear gradients were programmed accordingly for each metabolite with a flow rate of 4 mL min−1. Silica gel (Silicycle; 40−63 μm) was utilized for flash chromatography. Optical rotations were determined using an Autopol IV polarimeter (Rudolph Analytical, Hackettown, NJ, USA). UV spectra were recorded on a Varian Cary 3 UV−vis spectrophotometer scanning from 190 to 800 nm. Fungal Material. Branches of P. mariana and P. rubens trees of various age classes were collected in New Brunswick, Canada, and stored in plastic bags at 4 °C. Needles were removed using forceps and surface sterilized by serial passage in 70% ethanol (1 min), 3% NaClO solution (7.5 min), and 70% ethanol (30 s). Needles were rinsed in sterile ddH2O, blotted dry using sterile Kimwipes, and cut into ca. 2 mm sections. Needle sections, excluding the petioles, were placed on 2% malt extract agar (MEA; 20 g of Bacto malt extract, Difco Laboratories, Sparks, MD; 15 g of agar, EMD Chemicals Inc., NJ; 1000 mL of ddH2O) in 9 cm Petri dishes and incubated in the dark at 16 °C until sufficient mycelial growth was observed from the sections. Mycelia emerging from needle tissue were excised axenically and subcultured on 6 cm Petri dishes containing MEA. Strains selected for investigation of natural products were maintained on MEA and incubated at 8 °C. Fungal Identifications. Total genomic DNA was extracted from 4−12-week-old cultures on MEA using the Ultraclean Microbial DNA isolation kit (Mo Bio Laboratories, Carlsbad, CA, USA) following the manufacturer’s protocol. The ITS region was amplified and sequenced 1480



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using the primers ITS4 and ITS551 and partial 28S nuclear ribosomal LSU rRNA gene region was amplified using the primer pairs LR0R and LR5 and sequenced using LR0R, LR3, LR3R, and LR5.52 DNA amplification used a PCR master mix consisting of 0.5 μm of 2 mM dNTPs, 0.04 μm of 20 μM forward primer, 0.04 μm of 20 μM reverse primer, 1 μL of 10× Titanium Taq buffer (Clontech, Mountain View, CA, USA), 0.1 μL of 50× Titanium Taq enzyme (Clontech), 1 μL of DNA template, and 7.32 μL of sterile ddH2O per reaction. ITS and LSU were amplified using the following PCR profile: 95 °C for 3 min, then 35 cycles at 95 °C for 1 min, 56 °C for 45 s, and 72 °C for 1.5 min, followed by a final extension at 72 °C for 10 min. PCR products were verified by agarose gel electrophoresis and sequenced with Big Dye Terminator (Applied Biosystems, Foster City, CA, USA). Sequence contigs were assembled, trimmed, and manually checked using Geneious R6 6.1.8 (Biomatters, Auckland, New Zealand). Strain identification involved comparing sequence similarity with identified sequences in NCBI GenBank using the BLASTN function with default parameters. ITS and LSU sequences were also compared with unpublished sequences from personal collections of J. B. Tanney (refer to the Supporting Information for ITS and LSU GenBank accessions for studied endophytes, Table S1). Attempts to induce sporulation in vitro included prolonged incubation (over 2 years) on MEA, corn meal agar (Acumedia Manufacturers Inc., Lansing, MI, USA), oatmeal agar,53 spruce needle potato agar (modified from Su et al. 2012),54 1.5% water agar (within 1 mL trace metal solution)53 with or without the addition of sterile P. rubens needles on the agar surface, and floating mycelial plugs in sterile tap water under ambient light conditions. Cultures were incubated under several light treatments including a 24 h dark, 12 h fluorescent light cycle and ambient light, and temperature treatments included incubation at 5 °C intervals from 5 to 40 °C. Fermentation, Extraction, and Isolation. For inoculation, a portion of a 2% MEA plate for each endophyte studied was macerated in sterile ddH2O under aseptic conditions. The resulting suspensions were used to inoculate (5% v/v) 15 250 mL Erlenmeyer flasks each containing 50 mL of 2% malt extract broth. Starter cultures were incubated for 1 week on a rotary shaker (100 rpm) in the dark at 25 °C. Resulting endophyte cultures were macerated, individually transferred to Glaxo bottles containing 1 L of the same liquid medium, and incubated without agitation as described above for 7 weeks. After the incubation period, mycelia were separated from the culture filtrate by suction through Whatman #4 (Whatman GE Healthcare, UK) filter papers. The resulting volume and pH of the culture filtrates were recorded. Mycelia were lyophilized and stored at −20 °C. Culture filtrates were saturated with NaCl and exhaustively extracted with ACS grade EtOAc. The aqueous culture filtrate was discarded, and the organic layer was filtered through a Whatman #1 with anhydrous Na2SO4 prior to drying under reduced pressure. The resulting crude extracts were suspended in HPLC grade MeOH, passed through 0.2 μm PTFE (25 mm) syringe filters (Tisch Scientific, USA), dried under a gentle stream of nitrogen gas, and weighed. Crude endophyte culture filtrate extracts were stored dry in amber vials at −20 °C prior to further study. Crude culture filtrate extracts of selected endophytes generated to obtain adequate metabolite amounts were generally separated by flash column chromatography prior to semipreparative HPLC. Short silica gel columns employing a step gradient elution system of hexanes− EtOAc (0−100% v/v) in 10% increments followed by 5, 10, 20, and 50% EtOAc−MeOH (v/v) were typically implemented. Rhytismataceae sp. DAOMC 251461 Metabolites. Fractionation of the crude EtOAc culture filtrate extract (3.0 g) of DAOMC 251461 by a short silica gel column yielded nine fractions. LC-UV-MS screening identified three chemically distinct fractions that were selected for subsequent metabolite purifications by semipreparative HPLC. Fraction 1 (12.3 mg) eluted with 10% EtOAc in hexanes (v/v) and was further separated by HPLC using a linear gradient programmed from 55% to 85% ACN−ddH2O over 23 min to afford compounds 5 (2.3 mg) and 6 (3.0 mg). Fraction 4 (117.0 mg) eluted with 40% EtOAc in hexanes (v/v) and was chromatographed with a

linear gradient that increased from 20% to 70% ACN−ddH2O over 16 min, yielding compounds 7 (5.4 mg) and 3 (4.0 mg). Compound 4 (4.7 mg) was purified from fraction 5 (373.2 mg) with a program that increased linearly from 20% to 90% ACN−ddH2O over 21 min. Reexamination of the crude extracts’ LC-UV-MS chromatograms indicated two dominant peaks had yet to be isolated from investigated fractions. A portion (200 mg) of fraction 9 (621.5 mg) that eluted with 50% EtOAc−MeOH (v/v) was next separated by PTLC (Whatman Partisil K6, 20 × 20 cm, 250 μm) with 10% MeOH−CHCl3 (v/v). Six bands were scrapped from the silica plate, filtered through a 0.2 μm PTFE syringe filter, and screened by LC-UV-MS. The two dominant metabolites present within the crude extract were detected in two of the six silica scrapings. An HPLC method that linearly increased from 40% to 80% ACN−ddH2O over 17 min afforded compounds 1 (9.9 mg) and 2 (9.3 mg) from respective silica scrapings. Rhytismatone A (1): 9.9 mg; light yellow solid; [α]25D −30 (c 0.4, CHCl3); UV (MeOH) λmax (log ε) 205 (3.72), 230 (3.70), 270 (3.90); for 1H and 13C NMR spectroscopic data, see Table 1; HRMS m/z 285.1335 [M + H]+ (calcd for [C14H21O6]+ 285.1333). Rhytismatone B (2): 9.3 mg; light yellow solid; [α]25D −68 (c 0.4, CHCl3); UV (MeOH) λmax (log ε) 230 (4.02), 270 (4.21); for 1H and 13 C NMR spectroscopic data, see Table 1; HRMS m/z 299.1491 [M + H]+ (calcd for [C15H23O6]+ 299.1489). Lachnum cf. pygmaeum DAOMC 250335 Metabolites. The crude culture filtrate extract (1.9 g) of DAOMC 250335 was separated by normal-phase column chromatography into 11 fractions. Fraction 1 (215.0 mg) eluted with 10% EtOAc in hexanes (v/v). A linear HPLC gradient was programmed from 25% to 100% ACN−ddH2O over 25 min to afford compounds 9 (5.0 mg), 10 (9.6 mg), and 11 (25.9 mg). Fraction 9 (106.2 mg) eluted with 30% EtOAc in hexanes (v/v), and the same HPLC gradient utilized for compounds 9−11 afforded compound 8 (6.0 mg). Chloromycorrhizinone A (8): 6.0 mg; yellow oil; [α]25D 11 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 206 (4.04), 269 (3.85); for 1H and 13 C NMR spectroscopic data, see Table 2; HRMS m/z 317.0346 [M + H]+ (calcd for [C14H15O4Cl2]+ 317.0342). Mycosphaerellaceae sp. DAOMC 250863 Metabolites. The amount of culture filtrate extract (0.6 g) obtained from the 1 L screening fermentation of DAOMC 250863 was unusually high compared to most other foliar endophytes studied. Additionally, LCUV-MS chromatograms of its extract revealed a relatively simple metabolite profile. As such, metabolite purifications were conducted directly on the crude extract obtained from the 1 L fermentation. A linear HPLC gradient was programmed from 20% to 60% ACN− ddH2O over 16 min and provided compounds 12 (3.6 mg), 13 (2.9 mg), 14 (6.3 mg), 15 (3.0 mg), and 16 (14.5 mg). (3R,4S,4aR,6R)-4,6,8-Trihydroxy-3-methyl-3,4,4a,5,6,7hexahydroisochromen-1-one (12): 3.6 mg; clear solid; [α]25D 17.8 (c 0.18, MeOH); UV (MeOH) λmax (log ε) 200 (3.12), 265 (3.50); for 1 H and 13C NMR spectroscopic data, see Table 3; HRMS m/z 215.0912 [M + H]+ (calcd for [C10H15O5]+ 215.0914). Pezicula sporulosa DAOMC 250862 Metabolites. The culture filtrate extract (1.6 g) of DAOMC 250862 was separated into 11 fractions by normal-phase column chromatography. LC-UV-MS analysis indicated major metabolites were predominantly within three fractions. Fraction 2 (117.3 mg) eluted with 20% EtOAc in hexanes (v/v), and a linear gradient increasing from 35% to 60% ACN−ddH2O over 18 min afforded compound 17 (37.4 mg). Fraction 4 (116.1 mg) eluted with 40% EtOAc in hexanes (v/v), and a linear gradient programmed from 20% to 55% ACN−ddH2O over 21 min yielded compound 18 (13.8 mg). Fraction 5 eluted with 50% EtOAc in hexanes (v/v) and was subsequently separated by a linear HPLC gradient increasing from 10% to 70% ACN−ddH2O over 21 min and yielding compound 19 (6.2 mg). Compound 20 was detected in the crude culture filtrate of DAOMC 250862 by LC-HRMS using a previously isolated in-house standard. Antimicrobial Assays. Initial antifungal activity of studied endophytes’ crude extracts (50 mg mL−1) was screened using the Oxford diffusion assay against M. violaceum and S. cerevisiae homogeneously dispersed on 2% MEA.55 Subsequently, purified 1481



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compounds 1−6 and 8−19 were tested for in vitro antimicrobial activity against M. violaceum, S. cerevisiae, B. subtilis (ATCC 23857), and E. coli (ATCC 67878). M. violaceum was grown in 20 g L−1 malt extract (Bacto), 2.5 g L−1 peptone (Bacto), and 2.5 g L−1 yeast extract (Sigma, St. Louis, MO, USA), whereas S. cerevisiae was inoculated and grown in 1 g L−1 yeast extract supplemented with 10 g L−1 glucose. Bacteria were inoculated and grown in 5 g L−1 yeast extract, 10 g L−1 peptone, and 10 g L−1 NaCl. Nystatin was the positive control for antifungal assays, and chloramphenicol was the antibacterial positive control. DMSO was the negative control for all assays. Isolated metabolites and positive controls were individually tested at 100, 50, 25, and 12.5 μg mL−1 in sterile 96-well microplates (Falcon 353072 Microtest-9, Franklin Lakes, NJ, USA). A 10 μL aliquot of each individual metabolite solution dissolved in DMSO was added to 200 μL of fungal or bacterial suspension. Assays were performed in triplicate and incubated at 28 °C with a rotary table shaker providing gentle agitation (500 rpm). Optical density (OD) measurements were made at 600 nm with a Molecular Devices Spectra Max 340PC reader (Sunnyvale, CA, USA). Antimicrobial OD data were also analyzed by ANOVA followed by Tukey’s test (p < 0.05) for significant differences (Systat V13.1; Systat Software Inc., Chicago, IL, USA) compared to the negative control (DMSO). Positive controls inhibited the growth of all test organisms at the lowest concentrations tested. Previous studies had shown the MIC of nystatin in the S. cerevisiae culture used was 4 μM and for M. violaceum, 2 μM. In the antibiotic assays, for B. subtilis chloramphenicol had an MIC of 2.5 μM.9,13 All test organisms grew in the presence of the negative control DMSO.

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(3) Findlay, J. A.; Li, G.; Miller, J. D.; Womiloju, T. O. Can. J. Chem. 2003, 81, 284−292. (4) Sumarah, M. W.; Puniani, E.; Blackwell, B. A.; Miller, J. D. J. Nat. Prod. 2008, 71, 1393−1398. (5) Sumarah, M. W.; Puniani, E.; Sørensen, D.; Blackwell, B. A.; Miller, J. D. Phytochemistry 2010, 71, 760−765. (6) Tanney, J. B.; McMullin, D. R.; Green, B. D.; Miller, J. D.; Seifert, K. A. Fungal Biol. 2016, 120, 1448−1457. (7) Frasz, S. L.; Walker, A. K.; Nsiama, T. K.; Adams, G. W.; Miller, J. D. Can. J. For. Res. 2014, 44, 1138−1143. (8) Miller, J. D.; Sumarah, M. W.; Adams, G. W. J. Chem. Ecol. 2008, 34, 362−368. (9) Sumarah, M. W.; Kesting, J. R.; Sorensen, D.; Miller, J. D. Phytochemistry 2011, 72, 1833−1837. (10) McMullin, D. R.; Green, B. D.; Miller, J. D. Phytochem. Lett. 2015, 14, 148−152. (11) Sumarah, M. W.; Walker, A. K.; Seifert, K. A.; Todorov, A.; Miller, J. D. Recent Adv. Phytochem. 2016, 45, 195−206. (12) Richardson, S. N.; Walker, A. K.; Nsiama, T. K.; McFarlane, J.; Sumarah, M. W.; Ibrahim, A.; Miller, J. D. Fungal Ecol. 2014, 11, 107− 113. (13) Richardson, S. N.; Nsiama, T. K.; Walker, A. K.; McMullin, D. R.; Miller, J. D. Phytochemistry 2015, 117, 436−443. (14) Miyakado, M.; Inoue, S.; Tanabe, Y.; Watanabe, K.; Ohno, N.; Yoshioka, H.; Mabry, T. J. Chem. Lett. 1982, 10, 1539−1542. (15) Ichimoto, I.; Machiya, K.; Kirihata, M.; Ueda, H. J. Pestic. Sci. 1988, 13, 605−613. (16) Takaya, Y.; Kikuchi, H.; Terui, Y.; Furukawa, K.-I.; Seya, K.; Motomura, S.; Ito, A.; Oshima, Y. J. Org. Chem. 2000, 65, 985−989. (17) Brian, P. W.; Curtis, P. J.; Hemming, H. G.; Unwin, C. H.; Wright, J. M. Nature 1949, 164, 534. (18) Tabuchi, H.; Hamamoto, T.; Miki, S.; Tejima, T.; Ichihara, A. J. Org. Chem. 1994, 59, 4749−4759. (19) Tanabe, Y.; Miyakado, M.; Ohno, N.; Yoshioka, H. Chem. Lett. 1982, 10, 1543−1546. (20) Achenbach, H.; Mühlenfeld, A.; Brillinger, G. U. Liebigs Annalen der Chemie. 1985, 8, 1596−1628. (21) Sommart, U.; Rukachaisirikul, V.; Tadpetch, K.; Sukpondma, Y.; Phongpaichit, S.; Hutadilok-Towatana, N.; Sakayaroj, J. Tetrahedron 2012, 68, 10005−10010. (22) Shan, R.; Stadler, M.; Anke, H.; Sterner, O. J. Nat. Prod. 1997, 60, 804−805. (23) Stadler, M.; Anke, H. J. Antibiot. 1993, 46, 961−967. (24) Cimmino, A.; Andolfi, A.; Avolio, F.; Ali, A.; Tabanca, N.; Khan, I. A.; Evidente, A. Chem. Biodiversity 2013, 10, 1239−1251. (25) Gallo, M. B. C.; Chagas, F. O.; Almeida, M. O.; Macedo, C. C.; Cavalcanti, B. C.; Barros, F. W. A.; de Moraes, M. O.; Costa-Lotufo, L. V.; Pessoa, C.; Bastos, J. K.; Pupo, M. T. J. Basic Microbiol. 2009, 49, 142−151. (26) Stossel, A. Biochem. Biophys. Res. Commun. 1969, 35, 186−191. (27) McGahren, W. J.; van den Hende, J. H.; Mitscher, L. A. J. Am. Chem. Soc. 1969, 91, 157−162. (28) Claydon, N.; Grove, J. F.; Pople, M. Phytochemistry 1985, 24, 937−943. (29) Koshino, H.; Terada, S.; Yoshihara, T.; Sakamura, S.; Shimanuki, T.; Sato, T.; Tajimi, A. Phytochemistry 1988, 27, 1333−1338. (30) Schneider, G.; Anke, H.; Sterner, O. Z. Naturforsch. C 1996, 51, 802−806. (31) Ganley, R. J.; Newcombe, G. Mycol. Res. 2006, 110, 318−327. (32) Shan, R.; Stadler, M.; Sterner, O.; Anke, H. J. Antibiot. 1996, 49, 447−452. (33) Stadler, M.; Anke, H.; Shan, R.; Sterner, O. J. Antibiot. 1995, 48, 154−157. (34) Ondeyka, J.; Harris, G.; Zink, D.; Basilio, A.; Vicente, F.; Bills, G.; Platas, G.; Collada, J.; Gonzálex, A.; de la Cruz, M.; Martin, J.; Kahn, J.; Galuska, S.; Giacobbe, R.; Abruzzo, G.; Hickey, E.; Liberator, P.; Jiang, B.; Xu, D.; Roemer, T.; Singh, S. B. J. J. Nat. Prod. 2009, 72, 136−141. (35) Trofast, J. Phytochemistry 1978, 17, 1359−1361.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b01157. 1 H and 13C NMR spectra for all isolated metabolites (1− 19), culture filtrate extract HRMS chromatograms, spectroscopic and HRMS data for previously reported metabolites, tabulated NMR spectroscopic data for compounds 17−19, ITS phylogenetic analysis of endophytes DAOMC 251461 and DAOMC 250863, collection information including GenBank accessions (ITS and LSU), and brief discussions of studied endophytes (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +1 613 5260 2600, ext. 1053. Fax: +1 613 520 3749. ORCID

J. David Miller: 0000-0002-6680-6563 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Drs. J. Renaud and M. Sumarah (AAFC, London) for acquisition of HRMS data and helpful comments during the preparation of the manuscript. This work was funded by the Natural Science and Engineering Research Council (NSERC), MITACS, and JD Irving, Limited.

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REFERENCES

(1) Clay, K. Funct. Ecol. 2014, 28, 293−298. (2) Miller, J. D. In Endophytes of Forest Trees; Springer: The Netherlands, 2011; pp 237−249. 1482



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Natural Products of Picea Endophytes from the Acadian Forest

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