Gymnoascolides A-C: Aromatic butenolides from an Australian isolate of the soil ascomycete Gymnoascus reessii

Article abstract of DOI:10.1021/np050145p

Three new aromatic butenolides, gymnoascolides A-C (1-3), have been isolated from the Australian soil ascomycete Gymnoascus reessii and assigned structures on the basis of detailed spectroscopic analysis. The absolute configurations of gymnoascolides B (2

Full text of DOI:10.1021/np050145p

1226
J. Nat. Prod. 2005, 68, 1226-1230
Gymnoascolides A-C: Aromatic Butenolides from an Australian Isolate of the
Soil Ascomycete Gymnoascus reessii
Ben Clark,† Robert J. Capon,*,† Ernest Lacey,‡ Shaun Tennant,‡ Jennifer H. Gill,‡ Benjamin Bulheller,§ and
Gerhard Bringmann*,§
Centre for Molecular Biodiversity, Institute for Molecular Biosciences, University of Queensland,
St. Lucia, Queensland 4072, Australia, Microbial Screening Technologies Pty. Ltd.,
Smithfield, New South Wales 2164, Australia, and Institute of Organic Chemistry, University of Wu¨rzburg,
Am Hubland, D-97074 Wu¨rzburg, Germany
Received April 28, 2005
Three new aromatic butenolides, gymnoascolides A-C (1-3), have been isolated from the Australian
soil ascomycete Gymnoascus reessii and assigned structures on the basis of detailed spectroscopic analysis.
The absolute configurations of gymnoascolides B (2) and C (3) at C-5 were solved using a combination of
chemical derivatization and quantum chemical simulations.
Microbes have long been recognized for their capacity
to yield a wide range of structurally novel and biologically
potent metabolites. Many of these substances have either
found direct application as medicines or provided the
inspiration for successful drugs in the fields of human and
animal health and crop protection. The pre-eminent role
that microbes have played in drug discovery encourages
the view that Australia’s largely unexplored microbial
biodiversity remains an attractive resource worthy of study.
This microbial resource encompasses an extraordinary
array of varied ecosystemssfrom the tropics through
temperate climes to sub-Antarctic and Antarctic regions,
from deserts and tropical rainforests through tropical reefs,
estuaries, and all manner of intertidal and marine habitatss
all of which can be expected to host diverse microbial
communities.
In recent years, we have sought to draw Australian
microbial biodiversity into the arena of drug discovery
through a concerted and systematic exploration of an
Australian microbial isolate library (>250 000 cultures).
One dimension of this research targets isolates whose
extracts display potent antibiotic properties not readily
attributed to known microbial metabolites. Of the hundreds
of microbial isolates encountered in our study that meet
these criteria, this report focuses on a single strain of
Gymnoascus reessii (MST-F9977) which in our hands
yielded a series of novel aromatic butenolides, the gym-
noascolides (1-3). This discovery is perhaps all the more
noteworthy given that literature accounts of chemical
investigations into Gymnoascus are rare and appear to be
limited to reports of such common metabolites as penicillic
acid,1 G-type penicillins,2 and patulin.3
extract was concentrated in vacuo and the resulting residue
fractionated by C18 solid-phase extraction and HPLC, then
subjected to chemical (HPLC), spectroscopic (Photodiode
array, ESI(()MS, 1H NMR), and biological profiling to
disclose the presence of interesting metabolites. During this
fractionation process, a weak but selective antifungal
activity was noted in a number of the otherwise inactive
fractions. This antifungal selectivity was demonstrated by
growth inhibitory activity against the plant pathogen
Septoria nodorum and a corresponding absence of growth
inhibitory activity against the human pathogen Candida
albicans. Further purification of two of these antifungal
fractions yielded the novel aromatic butenolides, gymnoas-
colides A-C (1-3).
High-resolution ESI(+)MS analysis of gymnoascolide A
(1) returned a pseudo molecular ion ([M + Na]+) consistent
with a molecular formula (C17H14O2) requiring 11 double
1
bond equivalents (DBE). The H and 13C NMR data for 1
(Tables 1 and 2) indicated the presence of two monosub-
stituted aromatic rings, an ester/lactone carbonyl (δC
173.3), two isolated methylene units (δC 71.1, δH 4.69, s
and δC 33.9, δH 3.96, s), and two sp2 carbons (δC 159.7 and
129.6) consistent with a tetrasubstituted double bond
conjugated to the carbonyl. These structural moieties
account for all elements in the molecular formula and all
but one DBE, confirming gymnoascolide A (1) as a lactone
(IR 1759 cm-1).
Two-dimensional NMR analysis of 1 confirmed the
presence of an R,â-unsaturated butyrolactone moiety,
substituted by both phenyl and benzyl functionalities, and
permitted complete proton and carbon NMR assignments
(Tables 1 and 2). The positions of the phenyl and benzyl
substituents were confirmed by NOE difference experi-
ments which revealed enhancements from H-5 to H-2′′/6′′
(2%), and from H-6 to both H-2′/H-6′ (4%) and H-2′′/H-6′′
(4%). Thus, the structure for gymnoascolide A (1) was
assigned as shown.
Results and Discussion
The MeOH extract of a liquid culture of G. reessii
displayed significant growth inhibitory activity against the
bacterium, Bacillus subtilis, the nematode, Haemonchus
contortus, and a tumor cell line (murine NS-1). The MeOH
High-resolution ESI(+)MS analysis of gymnoascolide B
(2) gave a pseudo molecular ion ([M + Na]+) consistent with
a molecular formula (C18H16O4) requiring 11 DBE. Com-
parison of the NMR data for 2 with those of 1 (Tables 1
and 2) confirmed a common phenyl- and benzyl-substituted
R,â-unsaturated butyrolactone moiety. NMR differences
* To whom correspondence should be addressed. Tel: +61 7 3346 2979.
Fax: +61 7 3346 2101. E-mail: r.capon@imb.uq.edu.au (R.J.C.). Tel: +49-
931-888-5323. Fax: +49-931-888-4755. E-mail: bringman@chemie.uni-
wuerzburg.de (G.B.).
† University of Queensland.
‡ Microbial Screening Technologies.
§ University of Wu¨rzburg.
10.1021/np050145p CCC: $30.25
© 2005 American Chemical Society and American Society of Pharmacognosy
Published on Web 07/22/2005

Gymnoascolides A-C from Gymnoascus reessii
Journal of Natural Products, 2005, Vol. 68, No. 8 1227
Table 1. 1H NMR (CDCl3, 400 MHz)a Data for Gymnoascolides A-C (1-3)
1
2
3
H
δH (m, J (Hz))
COSY
HMBCa
δH (m, J (Hz))
COSY
HMBCa
δH (m, J (Hz)) COSY
HMBCa
5
6
4.69 (s)
3.96 (s)
2, 3, 4, 6, 1′, 1′′ 5.48 (s)
2, 4, 5, 1′′, 2′′/6′′ 5.88 (d, 8.8)
3.40 (d, 8.8)
2, 3, 4, 5-OMe, 1′ 5.90 (s)
3, 4, 5, 1′′, 2′′/6′′ 5.85 (d, 6.2) 6-OH 3, 4, 5, 1′′, 2′′/6′′
2, 3, 4, 5-OMe
6-OH
6
6-OH
5-OMe
2′/6′
3′/5′
4′
4, 6
5
3
2.25 (d, 6.2)
3.61 (s)
7.4 (m)
7.4 (m)
7.4 (m)
7.4 (m)
7.4 (m)
7.4 (m)
6
4, 6, 1"
5
3.61 (s)
7.53 (d, 7.0)
7.47 (dd, 7.0, 7.3) 2′/6′, 4′ 2′/6′, 1′
7.42 (t, 7.3)
3′
3′/5′, 3
7.62 (d, 7.6)
7.5 (m)
3′/5′
2′/6′
2′/6′
3′′/5′′
3′/5′
3′′/5′′
2′/6′
7.5 (m)
2′′/6′′ 7.15 (d, 6.9)
3′′/5′′ 7.34 (dd, 6.9, 7.1) 2′′/6′′,4′′ 1′′, 3′′/5′′
4′′ 7.29 (t, 7.1) 3′′/5′′ 2′′/6′′, 3′′/5′′
6, 2′′/6′′, 4′′
7.30 (d, 8.0)
6, 2′′/6′′, 4′′
7.39 (dd, 7.6, 8.0) 2′′/6′′,4′′ 1′′, 3′′/5′′
7.33 (t, 7.6) 3′′/5′′ 2′′/6′′
a Assignments have been made as accurately as possible; however, due to overlapping signals, there is some uncertainty with correlations
to carbons in the range of 127-129 ppm.
Table 2. 13C NMR (CDCl3, 100 MHz)a Data (δ) for
Gymnoascolides A-C (1-3)
were C-6 epimers, and opposite optical rotations if 2 and 3
were C-5 epimers. In the event, gymnoascolide B (2) yielded
(+)-4, while gymnoascolide C (3) gave (-)-4, confirming
that 2 and 3 were indeed epimers at C-5.
C
1
2
3
2
3
4
5
6
173.3
127.6b
159.7
71.1
169.9
131.4b
154.2
102.0
68.7
170.0
130.5b
157.3
102.3
71.5
The absence of structurally related natural or synthetic
compounds of known absolute stereostructure in the sci-
entific literature precluded an elucidation of the absolute
configuration of 4 by comparison of circular dichroism (CD)
spectra. Confronted by this challenge, we turned to quan-
tum chemical CD calculations.4-6 Arbitrarily starting with
the R-enantiomer of 4, the conformational analysis resulted
in 112 minimum structures within the range of 3 kcal/mol
above the global minimum. For each geometry optimized
by means of the PM37 Hamiltonian, the respective CD
curve was computed using the semiempirical CNDO/S
method.8 The single CD spectra thus received were summed
up and weighted following the Boltzmann statistics, that
is, according to the heats of formation of the conformers.
The resulting overall theoretical CD spectrum was submit-
ted to a UV correction9 and compared with experimental
data (Figure 1, top), indicating that the oxidation product
(+)-4 of gymnoascolide B (2) is R-configured, and conse-
quently, (-)-4 has the S-configuration. Although the two
main peaks of the experimental CD curve are, in principle,
reproduced in the calculated spectrum, the agreement is
not very good and the comparison can thus not be used as
a sole proof. For this reason, a second theoretical approach
to simulate the CD curve of 4 was chosen, this time based
on a molecular dynamics (MD) simulation.10 The MD run
was carried out using the TRIPOS force field11 at a virtual
temperature of 500 K for a total time period of 500 ps,
recording the structure every 0.5 ps. For the 1000 struc-
tures thus obtained, the single CD spectra were calculated
again by means of the CNDO/S8 method and averaged
arithmetically to give the overall theoretical spectrum of
4. As shown in Figure 1 (bottom), the CD spectrum
calculated for (5R)-4 matches now almost perfectly with
the experimental data for (+)-4, whereas the curve simu-
lated for (5S)-4 is virtually opposite. Thus, the absolute
configurations at C-5 for (+)-4 and 2 were assigned as R,
and those of (-)-4, and hence 3, as S.
33.9
5-OMe
1′′
2′′/6′′
3′′/5′′
4′′
58.1
57.8
129.6b
128.9c
128.7c
128.8
136.1
128.4
129.1
127.4
128.1b
129.3
128.9
129.8
140.1
125.3
128.8
128.2
128.7b
129.2c
128.9c
129.3d
140.3
127.3
128.4c
128.7d
1′′
2′′/6′′
3′′/5′′
4′′
a Assignments assisted by HMBC and HMQC correlations.
b-d Values within a column may be interchanged.
between the two were consistent with addition of oxygen
substituents to the sp3 centers at C-5 and C-6 (δC 102.0,
δH 5.48, s, and δC 68.7, δH 5.88, d) and the appearance of
OMe (δC 58.1, δH 3.61) and OH (δH 3.40, d) functionalities.
Analysis of the 2D NMR data (Table 1) for 2 unambigu-
ously positioned the OH at C-6 and the OMe at C-5. The
phenyl/benzyl positions for 2 were confirmed by comparable
NOE enhancements to those observed for 1 (H-5 to H-2′′/
6′′ (3%), and H-6 to both H-2′/H-6′ (10%), and H-2′′/
H-6′′ (5%)), with additional strong enhancements be-
tween H-5 and the 5-OMe (12%). The observations outlined
above provide evidence for the structure for gymno-
ascolide B (2) as shown, except for its configuration at C-5
and C-6.
Gymnoascolide C (3) was found to be closely related to
and isomeric with gymnoascolide B (2). Unfortunately, the
1H NMR data for 3 (Table 1) failed to disperse the aromatic
proton resonances, preventing interpretations comparable
1
to those for 1 and 2. Significant H NMR shift differences
between 2 and 3 were observed for H-5 (∆δ 0.42) and 6-OH
(∆δ -1.15), with additional small differences observed for
C-4, C-6, and C-2′′/6′′ in the 13C NMR spectrum (Table 2).
As both gymnoascolides B (2) and C (3) were resolved by
achiral HPLC, possessed unique 1H NMR spectra, and
displayed significant specific rotations, they were deemed
to be diastereomers.
Assignment of the absolute configuration at C-6 in 2 and
3 proved more elusive. While the secondary alcohol at C-6
was an obvious target for a Mosher analysis, the poorly
1
dispersed aromatic region in the H NMR spectra of both
To establish whether gymnoascolides B (2) and C (3)
were C-5 or C-6 epimers, a sample of each was oxidized
with pyridinium dichromate to yield the keto derivative 4.
The ketones derived individually from 2 and 3 would be
expected to possess the same optical rotation if 2 and 3
2 and 3 precluded such an approach. We did, however,
attempt a modified Horeau analysis, relying on monitoring
kinetic resolution during the formation of diastereomeric
R-phenylbutyrate esters by chiral HPLC. Unfortunately,

1228 Journal of Natural Products, 2005, Vol. 68, No. 8
Clark et al.
Table 3. Antifungal Properties for Gymnoascolide A (1) versus
Commercially Available Standardsa
C. albicans
LD99 mg/mL
S. nodorum
LD99 mg/mL
compound name
gymnoascolide A
cycloheximide
antimycin A3
monorden
amphotericin B
blasticidin S HCl
na
13
na
6.3
0.20
12
50
12
3.1
0.39
3.1
1.6
a na ) not active at 50 mg/mL.
are limited to two patents (1996 and 1997) which document
insecticidal properties.16,17
Figure 1. Attribution of the absolute configuration of the oxidized
product of gymnoascolide B, (+)-4 by (a) the PM3-Boltzmann approach;
(b) the TRIPOS-MD method. The ellipticities in the calculated CD
spectra have been calibrated to fit with the experimental spectra.
Given that methanol was used in the isolation of the
gymnoascolides, it is possible that gymnoascolides B (2) and
C (3) are derived from the hypothetical precursor 10.
However, it is worth noting that methanol was also used
extensively in the isolation of microperfuranone (6), and
no methoxylated products were reported in that instance.14
Gymnoascolide A (1) possessed moderate, selective activ-
ity against the pathogenic plant fungus Septoria nodorum
(MIC ) 13 µg/mL), while none of gymnoascolides A-C (1-
3) displayed activity against the human fungal pathogen
C. albicans, the gram-positive bacteria B. subtilis, the
livestock nematode H. contortus, or a mammalian tumor
cell line (murine NS-1). The potency and selectivity of the
antifungal properties of gymnoascolide A (1) were com-
pared against a range of known antifungal metabolites (as
detailed in Table 3), which revealed a selectivity between
C. albicans and S. nodorum comparable with that of
cycloheximide, and a potency against S. nodorum compa-
rable with that of monorden. Further biological studies on
the gymnoascolides are ongoing, as are efforts at total
synthesis.
despite demonstrating the sensitivity and reliability of this
approach on suitable standards, this method proved in-
conclusive when applied to gymnoascolides, possibly due
to a lack of adequate steric discrimination between C-6
substituents. A lack of available material prevented further
degradative/chemical approaches to assign the absolute
configuration at C-6 in either 2 or 3.
The gymnoascolides possess a rare structure motif. While
butenolides and substituted aromatics are common among
microbial metabolites,12 very few natural products, micro-
bial, plant, or other, possess the constellation of phenyl and
benzyl substituents about a γ-butenolide as presented in
the gymnoascolides A-C (1-3). The only comparable
published compounds are eutypoid A (5), isolated in 2002
from a South China Sea marine fungus of the genus
Eutypa,13 and microperfuranone (6)14 and the anhydride
7,15 isolated in 1998 and 1983 from the terrestrial fungi
Anixiella micropertusa and Aspergillus nidulans, respec-
tively. Similarly, the carbonarins are a more highly sub-
stituted suite of related metabolites isolated from the
terrestrial fungus Aspergillus carbonarius and first re-
corded in the patent literature in 1996.16 These include the
acetal, carbonarin A (8), and the butenolide, carbonarin H
(9), both of which possess a clear structural overlap with
the gymnoascolides. Literature accounts of the carbonarins
Experimental Section
General Experimental Procedures. As for previous
work,18 except for the following: Initial HPLC work was
carried out on a system consisting of two Shimadzu LC-8A
preparative liquid chromatographs with a static mixer, Shi-
madzu SPD-M10AVP diode array detector, and Shimadzu
SCL-10AVP system controller. UV-vis absorption spectra
were obtained using a Shimadzu UV-1650PC spectrophotom-

Gymnoascolides A-C from Gymnoascus reessii
Journal of Natural Products, 2005, Vol. 68, No. 8 1229
eter, while infrared (IR) spectra were acquired using a Shi-
madzu FTIR-8400 spectrometer.
(0.01% TFA) over 20 min, through a 5 µm Phenomenex LUNA
C18(2) 150 × 21.2 mm column) to yield gymnoascolide A (1) (9.1
mg, 1.8%).
In addition, for the oxidation products (+)-4 and (-)-4,
optical rotations were obtained using a Jasco P-1010 intelligent
remote module-type polarimeter, and circular dichroism (CD)
spectra were acquired using a JASCO J-810 spectropolarim-
Subsequently, a more polar fraction (44 mg) that appeared
to contain similar metabolites was fractionated using C18 SPE
(as for the previous fraction), followed by C8 HPLC (10
injections, 2.5 mL/min gradient elution from 60% H2O/MeCN
(0.01% TFA) to 20% H2O/MeCN (0.01% TFA) over 20 min,
through a 5 µm Phenomenex LUNA C8(2) 10 × 250 mm column)
then phenyl-hexyl HPLC (20 injections, 1 mL/min isocratic
elution with 50% H2O/MeCN, through a 5 µm Phenomenex
phenyl-hexyl 4.6 × 150 mm column), yielding gymnoascolide
B (2) (2.2 mg, 0.43%) and gymnoascolide C (3) (1.5 mg, 0.29%).
All percent yields for purified metabolites were calculated
against the combined residue (0.5 g) recovered after elution
of the extract from two parallel C18 SPE preparative cartridges.
Gymnoascolide A (1): colorless oil; IR (CHCl3) νmax 3064-
3011, 2928, 1759, 1495, 1467, 1126, 1045, 957 cm-1; UV
(EtOH) λmax (ꢀ) 248 (10500) nm; 1H NMR data (CDCl3, 400
MHz) see Table 1; 13C NMR data (CDCl3, 100 MHz) see Table
2; ESI(+)MS (30 kV) m/z 523 [2M + Na]+, 273 [M + Na]+, 251
[M + H]+; HRESI(+)MS m/z 273.0880 ([M + Na]+, C17H14O2-
Na requires 273.0891).
Gymnoascolide B (2): white solid; [R]D -105° (c 0.044,
CHCl3); IR (CHCl3) νmax 3543, 2930, 1774, 1495, 1448, 1369,
1331, 1234, 1109 cm-1; UV (EtOH) λmax (ꢀ) 259 (4900) nm; 1H
NMR data (CDCl3, 400 MHz) see Table 1; 13C NMR data
(CDCl3, 100 MHz) see Table 2; ESI(+)MS (30 kV) m/z 319 [M
+ Na]+; HRESI(+)MS m/z 319.0942 ([M + Na]+, C18H16O4Na
requires 319.0946).
Gymnoascolide C (3): white solid; [R]D -110° (c 0.022,
CHCl3); IR (CHCl3) νmax 3570, 2928, 2855, 1767, 1494, 1446,
1371, 1334, 1117 cm-1; UV (EtOH) λmax (ꢀ) 255 (5500) nm; 1H
NMR data (CDCl3, 400 MHz) see Table 1; 13C NMR data
(CDCl3, 100 MHz) see Table 2; ESI(+)MS (30 kV) m/z 319 [M
+ Na]+; HRESI(+)MS m/z 319.0941 ([M + Na]+, C18H16O4Na
requires 319.0946).
1
eter. H NMR spectra were acquired using a Bruker Avance
600 spectrometer, and ESI(()MS data were obtained using a
Agilent 1100 Series Separations module equipped with a
Agilent 1100 Series LC/MSD mass detector. High-resolution
(HR) ESI-MS measurements were obtained on a Finnigan
MAT 900 XL-Trap instrument with a Finnigan API III source.
Computational Methods. All calculations were carried out
with Linux AMD MP 2400+ workstations. The conformational
analysis was performed by means of the semiempirical PM37
method as implemented in the program package, Gaussian
98,19 starting from preoptimized geometries generated by the
TRIPOS11 force field as part of the molecular modeling
package, SYBYL 7.0.11 The molecular dynamics simulations
of 4 were run at a virtual temperature of 500 K using the
TRIPOS11 force field with a time step of 2 fs. Bond lengths
were constrained using the SHAKE algorithm.20 The overall
simulation time was 500 ps, and every 0.5 ps a single geometry
was extracted.
The wave functions required for the computation of the
rotational strengths for the electronic transitions from the
ground state to excited states were obtained by CNDO/S8
calculations followed by single configuration interaction (SCI)
computations, including 784 singly occupied configurations and
the ground state determinant. These computations were
carried out using the BDZDO/MCDSPD21 program package.
The single CD spectra were summed up and weighted follow-
ing the Boltzmann statistics, that is, according to the respec-
tive heats of formation. The rotational strengths were trans-
formed into ∆ꢀ values and, for a better visualization, super-
imposed with a Gaussian band shape function.
Biological Material. The fungal strain MST-F9977 was
isolated from a roadside soil sample collected near Sussex Inlet
on the southern coast of New South Wales, Australia, in an
area regenerating from a recent bushfire. The isolate was
identified as an ascomycete, Gymnoascus reessii, on morpho-
logical grounds. On malt extract agar, it is characterized by
orange hyphae and a pinkish reverse.
Assay Details. Procedures for antibacterial,22 cytotoxicity,22
and nematocidal23 assays have been described previously.
Antifungal activity was determined in an agar-based, micro-
titer plate bioassay. Spores of Candida albicans or Septoria
nodorum (20 000 sp/well) were applied to potato dextrose agar
in the wells of a microtiter plate, containing serial 2-fold
dilutions of the test compound. The plates were incubated at
24 °C before a qualitative assessment of fungal growth was
made at 48 h, with the LD99 determined as the lowest
concentration of the test compound at which no growth of the
fungus was observed.
Oxidation of Gymnoascolide B (2). A sample of 2 (1.1
mg) in CH2Cl2 (2 mL) was treated with pyridinium dichromate
(2.2 mg) and the suspension stirred overnight, after which the
mixture was filtered through a small plug of silica to remove
chromium salts and washed well with ether. The combined
washings were concentrated in vacuo to yield (+)-4 (0.9 mg,
82%) as a colorless oil: [R]D +175° (c 0.018, CHCl3); UV (EtOH)
λmax (ꢀ) 256 (6700) nm, 293 (sh) (3400); CD (MeOH) λmax (ꢀ)
1
233 (-41000), 307 (18500) nm; H NMR (CDCl3, 600 MHz) δ
7.78 (2H, m), 7.50 (1H, m), 7.45 (2H, m), 7.33 (2H, m), 7.28
(1H, m), 7.23 (2H, m), 6.20 (1H, s), 3.64 (3H, s); ESI(+)MS
m/z 317 [M + Na]+; HRESI(+)MS m/z 317.0793 ([M + Na]+,
C18H14O4Na requires 317.0790).
Oxidation of Gymnoascolide C (3). A sample of 3 (0.7
mg) was oxidized in the same manner as described above,
yielding (-)-4 (0.7 mg, 100%) as a colorless oil: [R]D -160° (c
0.014, CHCl3); UV (EtOH) λmax (ꢀ) 254 (6200) nm; 293 (sh)
(3000); CD (MeOH) λmax (ꢀ) 233 (34000), 307 (-15000) nm; 1H
NMR (CDCl3, 600 MHz) δ 7.78 (2H, m), 7.50 (1H, m), 7.45 (2H,
m), 7.33 (2H, m), 7.28 (1H, m), 7.23 (2H, m), 6.20 (1H, s), 3.64
(3H, s); ESI(+)MS m/z 317 [M + Na]+; HRESI(+)MS m/z
317.0784 ([M + Na]+, C18H14O4Na requires 317.0790).
Extraction and Isolation. A solid fermentation (100 g
wheat, 21 days, 28 °C) was extracted with MeOH. This extract
was concentrated in vacuo to an aqueous residue that was
diluted with H2O to a final volume of 1 L. This was passed
through two parallel C18 solid-phase extraction (SPE) car-
tridges (2 × 10 g, Varian HF C18), eluting with MeOH (2 × 40
mL each). On evaporation of the combined MeOH eluants, a
residue (∼0.5 g) was obtained that was subjected to prepara-
tive HPLC (60 mL/min with gradient elution of 70 to 10% H2O/
MeCN over 20 min followed by MeCN for 10 min, through a 5
µm Platinum EPS C18 50 × 100 mm column). One hundred
fractions were collected, concentrated in vacuo, and combined
on the basis of analytical HPLC analysis.
Acknowledgment. The authors would like to thank S.
Duck and G. MacFarlane for acquisition of HRESIMS data,
D. Howse for assistance in data management, and A. Hocking
for taxonomic classification. This research was partially funded
by the Australian Research Council and by the Fonds der
Chemischen Industrie.
One of these combined fractions (38 mg) possessed interest-
ing aromatic 1H NMR resonances and was further fractionated
by C18 SPE (10% stepwise gradient elution from 60% H2O/
MeOH to MeOH, through a 500 mg Alltech Extract-Clean C18
cartridge) and C18 HPLC (3 injections, 10 mL/min gradient
elution from 70% H2O/MeCN (0.01% TFA) to 30% H2O/MeCN
References and Notes
(1) Kitano, K.; Kintaka, K.; Katamoto, K.; Nara, K.; Nakao, Y. J. Ferment.
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