Journal of Natural Products
Article
stem was placed into ethanol for 3 s followed by brief flame
sterilization, stripped of its outer bark, and divided into three sections,
which were plated onto water agar (WA; 15 g of bacteriological agar
and 1 L of deionized water) and a 1:10 dilution of potato dextrose agar
deionized H O, and analyzed by LC-ELSD. Polyol analysis was
performed on a 4.0 × 250 mm Hi-Plex Ca USP L19 (Agilent) column
2
eluted at 0.3 mL/min with an isocratic solution of H O−CH CN
2
3
(7:3). Injection of 5 μL of the reconstituted H O layer from the
2
(
PDA; 2.4 g of Difco potato dextrose broth, 15 g of bacteriological
hydrolysis revealed a single peak (t = 11.46 min). Injection of 5 μL of
R
agar, and 1 L of deionized water). The fungal culture E11018A was
isolated from a 1:10 PDA plate 8 days after the initial plating. The
sample was subsequently transferred to 1× PDA plates 19 days later.
Permanent stocks of the organism were made by growing E11018A on
triple-autoclaved barley seeds and storing them at −80 °C in the Yale
Peabody Culture Collection [YU.101029].
a solution containing 0.05 mg/mL of glucose (control) and the linear
sugar standards showed well-resolved peaks for D-glucose (t = 5.94
R
min), D-talitol (tR = 11.08 min), D-mannitol (tR = 11.48 min), D-
sorbitol (t = 14.69 min), and L-iditol (t = 17.20 min). Co-injection of
R
R
the hydrolysis product and D-mannitol provided a single peak (t
=
R
11.60 min).
Culture Conditions. Approximately 5 mm diameter agar plugs of
E11018A were inoculated into 10 mL culture tubes containing 5 mL of
× potato dextrose broth (PDB; 24 g of Difco potato dextrose broth
Antimicrobial Assay. For Petri dish assays of the fungal extracts,
plates were divided into four quadrants: blank (bacteria alone), vehicle
1
control (bacteria + 5 μL of MeOH), CH Cl extract (bacteria + 5 μL
2 2
in 1 L of deionized water) and grown under static conditions for 7
days. Fungal matter was subsequently transferred to 100 mL of 1×
PDB in 500 mL Erlenmeyer flasks and incubated with shaking at 150
rpm and 30 °C for 7 days. Again, the fungal matter was transferred to 1
L of 1× PDB in 2 L Erlenmeyer flasks and incubated under shaking
conditions at 150 rpm and 30 °C for another 14 days.
Isolation of Genomic DNA and Phylogenetic Analysis.
Genomic DNA was extracted from approximately 100 mg of fresh
mycelium scraped from PDA plates, using the Qiagen DNeasy plant
mini kit to process. The ITS rDNA, SSU rDNA, and LSU rDNA and
TEF1 and RPB2 nuclear gene regions were amplified. The PCR
amplicons were cleaned and sequenced by the W. M. Keck Foundation
of 58.6 mg/mL solution of extract), and EtOAc extract (bacteria + 5
μL of 143.2 mg/mL solution of extract). Extracts were resuspended in
500 μL of MeOH prior to spotting on plates. For liquid-based assays, a
culture of S. aureus was grown overnight in 5 mL of tryptic soy broth
(30 g of tryptic soy broth in 1 L of deionized water), with shaking at
37 °C. Cells were diluted to a final OD600 of 0.0325 and treated with
media alone, 1% MeOH (v/v), 0.05 μg/mL ciprofloxacin (positive
control), or dilutions of compounds 1 and 2 (250, 100 μg/mL, 75, 50,
25, 10, 1, 0.1, and 0.01 μg/mL). Treated cells and controls were grown
for 8 h, taking OD600 measurements every 10 min to produce kinetic
growth curves. The final concentration of MeOH for all conditions
never exceeded 1% total volume.
(
Yale School of Medicine). A subset of taxa in the order Pleosporales
was assembled. Sequences were aligned with MUSCLE v3.8.31, and
ASSOCIATED CONTENT
Supporting Information
■
gaps present in more than 20% of the sequences removed with
14,15
*
S
Phyutility.
Jmodeltest v2.1.6 identified the substitution model
SYM+I+G for LSU, K80+I+G for SSU, and GTR+I+G for RPB2 and
16,17
18
TEF1.
Alignments were interleaved using Mesquite v3.01. Trees
were assembled using MrBayes v3.2.2, using 2 runs of 4 chains and a
1
9,20
The
1H NMR, 13C NMR, COSY, HSQC, HMBC, and
ROESY, together with HRESIMS data for compounds 1
Compound Isolation and Purification. After growth in liquid
culture for 28 days, fungal matter from 4 L of cultures was filtered, and
a 1:1 aqueous-solvent extraction was performed on the broth in 2 L
separatory funnels. The aqueous layer was initially extracted with
CH Cl and then with EtOAc. The solvent layers were collected and
2
2
AUTHOR INFORMATION
Corresponding Authors
■
concentrated in vacuo to dryness. The crude CH Cl (29.3 mg) and
2
2
EtOAc (71.6 mg) extracts were pooled together and dissolved in 1.5
mL of MeOH prior to HPLC fractionation using a 250 × 21.2 mm
Luna 5 μm C18 column (Phenomenex) with a flow rate of 10 mL/min
and a 50 min gradient from 0 to 100% acetonitrile in water. Peaks were
monitored at 215 and 254 nm. Stelliosphaerol A (1) (7.1 mg) eluted
between 30.9 and 31.8 min, absorbing strongly at 215 nm.
Stelliosphaerol B (2) eluted between 35.9 and 37.4 min. Due to the
broadness of the peak, compound 2 underwent a subsequent round of
HPLC separation on an analytical 250 × 4 mm Luna, 5 μm C18
column (Phenomenex) with a flow rate of 2 mL/min and a 20 min
gradient from 40% to 60% acetonitrile in water. Stelliosphaerol B (2)
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the Ministerio del Ambiente of Ecuador for a
collecting and research permit provided to S.A.S. We thank P.
Vargus Nunez for help with host plant identification, D.
Spakowicz for guidance with phylogenetic analysis, the
(
3.0 mg) eluted at 12.8 min, absorbing strongly at 215 nm.
Stelliosphaerol A (1): [α] 2D 5 −9.5 (c 0.08, MeOH); UV (MeOH)
λmax (log ε) 270 (1.31) nm; IR NaCl 3347, 2915, 1452, 1372, 1072,
1
−1
1
13
021 cm ; H NMR (600 MHz, CD OD) and C NMR (150 MHz,
3
́
Endophyte Collection Quito Catolica (CEQCA) for cataloging
CD OD) data, see Table 1; HRESIMS found m/z 425.2507 [M +
3
+
the microbial isolates, and S. Tarasov and M. Dyba (Biophysics
Resource Core, Structural Biophysics Laboratory, CCR) for
assistance with high-resolution mass spectrometry. This
research was funded in part by an HHMI Professorship and
NSF grant OISE 853408 awarded to S.A.S. It was also
supported by the Intramural Research Program of the NIH,
National Cancer Institute, Center for Cancer Research, and
with Federal funds from the National Cancer Institute, National
Institutes of Health, under contract HHSN261200800001E.
The content of this publication does not necessarily reflect the
views or policies of the Department of Health and Human
Services, nor does mention of trade names, commercial
Na] , calcd for C H O Na, 425.2515.
21
38
7
25
Stelliosphaerol B (2): [α]D −6.3 (c 0.08, MeOH); UV (MeOH)
λmax (log ε) 270 (1.31) nm; IR NaCl 3390, 2923, 1675, 1439, 1376,
190, 1134, 1072, 1039 cm ; H (600 MHz, CD OD) and C NMR
3
−1
1
13
1
(
4
150 MHz, CD OD) data, see Table 2; HRESIMS found m/z
09.2565 [M + Na] , calcd for C H O Na, 409.2566.
21 38 6
3
+
Cleavage of Stelliosphaerol A (1) and Sugar Determination.
The linear reduced sugar moiety was hydrolyzed from the
sesquiterpene scaffold by adding 200 μg of 1 to a mixture of 100 μL
of 0.05 M HCl and 300 μL of cyclohexane and refluxing at 90 °C for
11
3
0 h, as previously described. After hydrolysis, 200 μL of H O was
2
added, and the reaction was extracted twice with 200 μL of EtOAc.
The H O layer was evaporated in vacuo, reconstituted with 100 μL of
2
E
J. Nat. Prod. XXXX, XXX, XXX−XXX