Journal of Agricultural and Food Chemistry
ARTICLE
Nuclear Magnetic Resonance Spectroscopy (NMR). 1H
NMR and 13C NMR spectra were acquired on an AMX-400 spectro-
meter (Bruker, Rheinstetten, Germany). For NMR analysis, all com-
pounds were dissolved in pyridine-d5, except for compounds 7 and 10,
which were dissolved methanol-d4 and chloroform-d, respectively.
Chemical shifts were generated as δ values with reference to tetra-
methylsilane (TMS).
% inhibition = [(average control diameter mm ꢀ treatment diameter
mm)/average control diameter mm] ꢁ 100.
In Vitro Fungal Metabolism of Compounds 1 and 2.
Solutions of compound 1 and 2 were separately prepared in ethanol
and water (7:3, v/v), filter sterilized with a 0.22 μm syringe filter, and
incorporated into autoclaved PDA that was allowed to cool to 50 °C.
The media (each plate; 5 mL) were then transferred to polystyrene Petri
dishes (50 mm ꢁ 12 mm) and allowed to solidify. A total of 4 plates were
prepared for each compound. The final concentration for each com-
pound was 100 μM. Plates were inoculated in the center with a 5 mm
plug taken from the periphery of an actively growing stock culture. Plates
were incubated in the dark at 25 °C and harvested at 48 and 72 h,
respectively. At harvest, the total contents of 2 plates (total; 10 mL) were
transferred to a centrifuge tube (50 mL) (Thermo Fisher Scientific Inc.,
Fairlawn, NJ) containing ethanol and water (7:3, v/v; 35 mL). Each
sample was then extracted on a wrist-action autoshaker (15 min)
(Burrell Scientific, Pittsburgh, PA), sonicated in an ultrasonic water
bath (15 min) (B3500A-DTH ultrasonic bath, VWR International Inc.,
West Chester, PA), and centrifuged (5000 rpm for 10 min) (Sorvall RC-
3C Plus, Thermo Fisher Scientific Inc.). The supernatant was then
filtered through a 0.45 μm PTFE syringe filter prior to LC-MS analysis.
Scale-Up Fungal Metabolism of Compound 1. Potato dex-
trose broth (PDB, 39 g Lꢀ1 deionized water) (each; 100 mL) was
prepared and transferred to an Erlenmeyer flask (250 mL) and auto-
claved. Once the broth reached room temperature, it was inoculated
with B. cinerea and incubated on an orbital platform shaker at 200 rpm
for 48 h at 25 °C. After 48 h, compound 1 (35 mg) was dissolved in
ethanol and water (7:3, v/v; 1 mL), filter sterilized with a 0.22 μm sterile
syringe filter, and introduced to the flask. The reaction was monitored by
sampling aliquots (each; 0.5 mL) every 24 h for 96 h. Each aliquot was
diluted (1:3, v/v) with ethanol and water (7:3, v/v; 1.5 mL), and filtered
through a 0.45 μm PTFE syringe filter prior to LC-MS analysis. At the
completion of the reaction, the content of the flask was immediately
frozen under liquid nitrogen, lyophilized, and stored at ꢀ80 °C until
extraction.
Liquid ChromatographyꢀMass Spectrometry (LC-MS).
LC-MS analysis was performed using a HP 1100 series HPLC system
(Agilent Technologies Inc., Santa Clara, CA) equipped with an auto-
injector, quaternary pump, column heater, and diode array detector,
interfaced to a Bruker 6300 series ion-trap mass spectrometer equipped
with an electrospray ionization chamber. Chromatographic separations
were performed using a Prodigy C18 column (250 mm ꢁ 4.6 mm i.d.;
5.0 μm particle size) (Phenomenex, Torrance, CA). The flow rate was
set to 1.0 mL minꢀ1, and the column temperature was set to 25 °C. The
binary mobile phase composition consisted of (A) 0.1% formic acid in
deionized water and (B) 0.1% formic acid in acetonitrile. Separations
were performed using a linear gradient of 15ꢀ43% B over 40 min and
then to 95% B over 5 min; thereafter, elution with 95% B was
performed for 10 min. The re-equilibration time was 10 min. For
instrumentation control and data acquisition, HP ChemStation and
BrukerData Analysis software were used. All mass spectra were
acquired in positive ion mode over a scan range of m/z 100ꢀ2000.
Ionization parameters included capillary voltage, 3.5 kV; end
plate offset, ꢀ500 V; nebulizer pressure, 50 PSI; drying gas flow,
10 mL minꢀ1; and drying gas temperature, 360 °C. Trap parameters
included ion current control, 30000; maximum accumulation time,
200 ms; trap drive, 61.2; and averages, 12 spectra.
Partial Acid Hydrolysis of Compound 1. Compound 1 (1 mg)
was refluxed in a reaction vial (1 mL) (Reacti-Vial, Thermo Fisher
Scientific Inc., Fairlawn, NJ) at 80 °C for 2 h in a solution of 1 N HCl in
methanol (0.5 mL). After hydrolysis and titration to pH 7 with NaOH
(4 N), the sample was evaporated to dryness under reduced pressure
(30 °C; 1.0 ꢁ 10ꢀ3 mbar) using a Labarota 4003 rotary evaporator
(Heidolph Brinkman LLC, Elk Grove Village, IL). The residue was
dissolved in ethanol and water (7:3, v/v; 2 mL), mixed on a vortex mixer
(1 min), and filtered through a 0.45 μm PTFE syringe filter (Thermo
Fisher Scientific Inc., Fairlawn, NJ) prior to LC-MS analysis.
B. cinerea Growth Inhibition Assay. Antifungal activity was
assessed by an in vitro fungal growth inhibition assay modified from
Nicol et al.34 Fungi were maintained on potato dextrose agar (PDA,
39g Lꢀ1 deionizedwater) andincubatedinthedark at25°C. The cultures
were continuously maintained by transferring a 5 mm plug of mycelium
cut with a cork bore from the periphery of actively growing colonies to
freshly prepared media. The fungal growth inhibition of compounds
1ꢀ5 were evaluated at three concentrations (1, 10, and 100 μmol) in the
final media. Solutions of compounds 1ꢀ5 were prepared in ethanol and
water (7:3, v/v), filter sterilized with a 0.22 μm sterile syringe filter
(Thermo Fisher Scientific Inc., Fairlawn, NJ), and incorporated into
autoclaved PDA that was allowed to cool to 50 °C. The media (each
plate; 5 mL) were then transferred to polystyrene Petri dishes
(50 mm ꢁ 12 mm) (VWR International Inc., West Chester, PA)
and allowed to solidify. The final concentration of the carrier solvent
was 1% of the final volume of media for all treatments and the control.
Plates were inoculated with a 5 mm plug taken from the periphery of
an actively growing stock culture. Plates were incubated in the dark at
25 °C, and the radial growth of each colony was measured using an
ABS Solar Digimatic caliper (Mitutoyo America Corporation,
Aurora, IL). Treatment and control colonies were measured when the
control colonies reached approximately 80% of the plate diameter. The
average control colony diameter minus the average treatment colony
diameter was used to calculate the relative growth inhibition.
Semipreparative RP-HPLC Isolation of the Fungal Meta-
bolites of Compound 1. The lyophilized reaction mixture, as
described above, was ground into a fine powder with a laboratory mill
(IKA Labortechnik, Staufen, Germany) and extracted with ethanol and
water (7:3, v/v; 2 ꢁ 50 mL) on an autoshaker at room temperature for
15 min. After centrifugation (5000 rpm for 10 min), the supernatant was
collected and the residue discarded. The supernatant was then evapo-
rated under reduced pressure, dissolved in a mixture of 0.1% formic acid
in deionized water and 0.1% formic acid in acetonitrile (75:25, v/v;
5 mL), and filtered through a 0.45 μm PTFE syringe filtered prior to
purification. Chromatographic separations were achieved by semipre-
parative RP-HPLC performed on a Luna C18 column (250 mm ꢁ 21.2
mm i.d.; 10 μm particle size) (Phenomenex, Torrance, CA). Chroma-
tography was performed on a Shimadzu LC-6AD liquid chromatograph
(Shimadzu Scientific Instruments Inc., Columbia, MD) using a UV/vis
detector and a 2 mL injection loop. Mixtures of (A) 0.1% formic acid in
deionized water and (B) 0.1% formic acid in acetonitrile were used as the
mobile phase. The flow rate was set to 20 mL minꢀ1, the column
temperature was 23 ( 2 °C, and UV detection was recorded at λ =
210 nm. Chromatography was performed using a linear gradient of
5ꢀ30% B over 45 min and then to 90% B over 10 min; thereafter, elution
with 90% B was performed for 10 min. The re-equilibration time was
10 min. The target compounds were collected, freed from solvent under
reduced pressure, and lyophilized, yielding 6 (2 mg), 7 (5 mg), and 10
(1 mg) (Figure 1) as white amorphous powders in high purity >98%, as
determined by LC-MS and NMR.
Compound 6 , (22R,25R)-Spirosol-5-en-3β-yl O-R-L-rhamnopyra-
nosyl-(1f2)-β-D-glucopyranoside. 1H NMR and 13C NMR were
consistent with the literature.32
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dx.doi.org/10.1021/jf200093q |J. Agric. Food Chem. 2011, 59, 5945–5954