Bioactivity profiling using HPLC/microtiter-plate analysis: Application to a New Zealand marine alga-derived fungus, gliocladium sp.

Article abstract of DOI:10.1021/np0504917

Using HPLC/microtiter-plate-based generation of activity profiles the extract of a marine alga-derived fungus, identified as Gliocladium sp., was shown to contain the known strongly cytotoxic metabolite 4-keto-clonostachydiol (1) and also clonostachydiol (2) as well as gliotide (3), a new cyclodepsipeptide containing several D-amino acids. The absolute configuration of 1 was elucidated by reduction to 2, and two further oxidized derivatives of clonostachydiol (5, 6) were prepared and evaluated for biological activity.

Full text of DOI:10.1021/np0504917

J. Nat. Prod. 2006, 69, 621-624
621
Bioactivity Profiling Using HPLC/Microtiter-Plate Analysis: Application to a New Zealand
Marine Alga-Derived Fungus, Gliocladium sp.
Gerhard Lang,† Maya I. Mitova,† Gill Ellis,† Sonia van der Sar,† Richard K. Phipps,† John W. Blunt,† Nicholas J. Cummings,‡
Anthony L. J. Cole,‡ and Murray H. G. Munro*,†
Department of Chemistry and School of Biological Sciences, UniVersity of Canterbury, PriVate Bag 4800, Christchurch, New Zealand
ReceiVed NoVember 27, 2005
Using HPLC/microtiter-plate-based generation of activity profiles the extract of a marine alga-derived fungus, identified
as Gliocladium sp., was shown to contain the known strongly cytotoxic metabolite 4-keto-clonostachydiol (1) and also
clonostachydiol (2) as well as gliotide (3), a new cyclodepsipeptide containing several D-amino acids. The absolute
configuration of 1 was elucidated by reduction to 2, and two further oxidized derivatives of clonostachydiol (5, 6) were
prepared and evaluated for biological activity.
Fungi from the marine environment are well-known producers
of novel and pharmacologically active secondary metabolites.1
Chemically productive strains have been isolated from a wide
variety of marine substrates, e.g., sponges,2 algae,3 driftwood,4 and
sediment.5 During the investigation of fungi derived from marine
algae and driftwood collected around the South Island of New
Zealand, the highly cytotoxic and antibacterial extract of a
Gliocladium sp., cultured from a thallus sample of the macroalga
DurVillaea antarctica, attracted our attention. Fungi of this genus
have been described before as producers of structurally diverse
metabolites, e.g., glycosylated polyketides,6 cytotoxic diketopip-
erazines,7 and cyclopeptides.8
Results and Discussion
To identify which of the components (HPLC analysis) of the
Gliocladium extract was responsible for the biological activities
observed, a “bioactivity chromatogram” was generated. To achieve
this, the eluent of an analytical HPLC separation of the extract (100
µg) was collected over 22 min into a 96-well microtiter plate (88
× 0.25 mL fractions), and a daughter plate was then prepared (5
The cytotoxic compound (1) and the other two major compounds
µL transferred from each well of the master plate; equivalent to a
(2 and 3) were then isolated by semipreparative HPLC. Evaluation
total of 2 µg of extract). This daughter microtiter plate, after drying,
of NMR and MS data showed the active compound to be the
was inoculated with P388 cells and incubated for 3 days. The
macrodiolide 4-keto-clonostachydiol (1), a structure recently pat-
distribution of the bioactivity across the plate was revealed using
ented for its cytotoxic and antibacterial activities.10 Compound 2
the yellow MTT dye, which is reduced by living cells to the purple
was, by analysis of the NMR and MS data, unequivocally identified
MTT formazan.9 Correlating the plate-reader output against well
as clonostachydiol,11 a related fungal macrodiolide. Comparison
position (each well is equivalent to a 15 s fragment of the HPLC)
of NMR data and the optical rotation of 2 with values published
allows generation of a HPLC-bioactivity profile. Typically, under
for the natural product and two diastereomers12,13 confirmed the
the HPLC conditions used, peak widths are around 30-60 s, which
stereochemistry to be that of the previously described natural
corresponds to two to four consecutive wells on the microtiter plate,
product. The relative and absolute configuration of 1 was established
allowing ready identification of the bioactive peak(s) in an HPLC
by reduction of the compound with NaBH4/CeCl3. This reaction
chromatogram (Figure 1). In our experience it has been useful to
resulted in two products, 4a and 4b (Scheme 1). Compound 4a
compare the bioactivity profile to both the ELSD data and the UV-
was, by HPLC retention time, 1H NMR, and optical rotation,
DAD output, as the ELSD output reflects the actual mass of
identical to clonostachydiol 2, thus indicating for 1 a stereochemistry
compound in a peak and is not biased toward compounds with
analogous to that of 2, i.e., (5R,10R,13R). Compound 4b was the
strong UV chromophores.
previously undescribed 4-epi-clonostachydiol.
In the same way a microtiter plate using 500 µg of extract was
In comparison with 4-keto-clonostachydiol (1), which is strongly
prepared and tested against Bacillus subtilis. For an appropriate
cytotoxic against P388 cells (IC50 0.55 µM) and active against
response from this organism it is necessary to use a considerably
Bacillus subtilis and the fungi Trichophyton mentagrophytes and
higher concentration of sample per well. Both the resulting
Cladosporium resinae, clonostachydiol (2) is significantly less
“cytotoxicity chromatogram” and the “anti-Bacillus chromatogram”
cytotoxic and does not possess antimicrobial activity (Table 1). To
(Figure 1) showed the bioactivity to be located around a retention
explore the biological activity potential of related structures, 2 was
time of 12.5 min, correlating to one of the major HPLC peaks.
oxidized with Dess-Martin periodinane (Scheme 2). Under these
conditions the alcohol at C-10 was readily oxidized to the ketone,
* To whom correspondence should be addressed. Tel: +64-3-3642434.
while oxidation at C-4 proceeded only slowly. This made it possible
Fax: +64-3-3642429. E-mail: murray.munro@canterbury.ac.nz.
† Department of Chemistry.
to isolate compound 5, an isomer of 4-keto-clonostachydiol (1) with
the oxidation states of C-4 and C-10 interchanged. Full oxidation
‡ School of Biological Sciences.
10.1021/np0504917 CCC: $33.50
© 2006 American Chemical Society and American Society of Pharmacognosy
Published on Web 03/17/2006

622 Journal of Natural Products, 2006, Vol. 69, No. 4
Lang et al.
Scheme 2. Oxidation of Clonostachydiol (2): (i) Dess-Martin,
(ii) MnO2
1
organism. The H NMR spectrum was characteristic of a peptide,
with six partially overlapping signals for the R-protons of the
R-amino acid and the R-hydroxy acid residues between 3.8 and
5.2 ppm. A TOCSY experiment revealed the presence of five amino
acid units, namely, glycine, two alanine, and two tyrosine residues
and an R-hydroxy acid, 3-phenyllactic acid. Long-range H,C-
correlations of the R-protons to the carbonyl carbons of the adjacent
amino acid provided the information necessary for assembling the
sequence of the amino acids and the 3-phenyllactic acid (Figure
2). The carbonyl resonances were narrowly spaced in the range of
171 to 175 ppm, so the IMPRESS technique14 was applied, as this
approach provides better resolution in the F1 dimension than a
conventional HMBC experiment. This established that the peptide
had the sequence phenyllactoyl-Ala-Gly-Tyr-Ala-Tyr with the ring-
closing ester bond between the hydroxyl group of the phenyllactic
1
acid and the second tyrosine residue. The H NMR resonances of
two singlet methyl groups, one double-bond proton, and an
oxymethylene group were also identified and assigned to an
O-prenyl residue. This O-prenyl group was attached to the
C-terminal tyrosine unit, as the methylene protons (H-10) showed
an HMBC correlation to C-7 as well as an ROE interaction with
one of the aromatic proton signals (H-6/H-8) of this amino acid
(Figure 2). A further IMPRESS experiment was required to establish
that these aromatic protons did not belong to one of the two other
aromatic groups.
Figure 1. HPLC of the Gliocladium extract: UV absorption at
235 nm, ELSD signal, cytotoxicity against P388 cells, and anti-B.
subtilis activity.
Scheme 1. Reduction of 4-Keto-clonostachydiol (1)
Acid hydrolysis of 3 and Marfey’s analysis15 of the amino acid
mixture showed that both tyrosine units were of D-configuration,
while one of the alanines was D- and the other L-configured. The
3-phenyllactic acid was found to have an S-configuration by reacting
the acid hydrolyzate of 3 with (S)-MTPA-Cl and HPLC comparison
of the resulting (R)-MTPA-phenyllactic acid ester with standards.
The only stereochemical detail remaining unclear was which alanine
was of L- and which was of D-configuration. The conformational
flexibility of the molecule limits the value of the observed ROE
correlations when applied to potential molecular modeling studies
of the two stereochemical options, while attempts to crystallize the
compound have been as yet unsuccessful. Gliotide (3) was inactive
in the cytotoxicity and antimicrobial assays.
Table 1. Cytotoxic and Antimicrobiala Activities of Compounds
1, 2, 5, and 6
IC50(P388) [µM]
B. subtilis
T. ment.
C. resinae
1
2
5
6
0.55
25
1.9
3.9
6
2
1
The value of the microtiter-plate-based system lies in the simple
generation of bioactivity profiles of the HPLC chromatogram of
not active
3
1
not active
3
1
not active
5
not active
a Widths of inhibition zones (in mm) around disks loaded with 40
µg of compound.
of 2 to the diketone 6 was achieved with MnO2. Both oxidation
products of clonostachydiol are antimicrobial and markedly cyto-
toxic (Table 1), but less so than the natural product 1. This suggests
that the presence of at least one R,â-unsaturated ketone moiety is
essential for the biological activity. The reduced activity of 6, in
comparison to 1 and 5, may be due to the nonpolar nature of this
compound, which is barely soluble in water.
HRESIMS indicated a m/z of 742.3424, corresponding to a
molecular formula of C40H48N5O9, for the [M + H]+ ion of
compound 3, which was named gliotide after the producing
Figure 2. Long-range H,C-couplings (arrows) and one ROE
interaction (dashed arrow) define the amino acid sequence of 3
and the position of the prenyl group.

HPLC/Microtiter-Plate Analysis of Gliocladium sp.
Journal of Natural Products, 2006, Vol. 69, No. 4 623
Table 2. NMR Data of Gliotide (3; 500 MHz, MeOH-d4)
position
δC
δH
position
δC
δH
Ala1
pTyra
1
2
3
174.7
50.3
18.2
1
2
3
173.8
57.6
37.3
4.39 dd 7.0, 7.0
1.09 d 7.2
4.48 dd 9.1, 5.3
3.05 m
a
Gly
1
2
b
2.92 dd 13.5, 9.2
172.5
43.9
4
129.4
131.8
116.3
159.9
66.0
121.5
138.9
18.4
a
b
3.88 d 16.2
3.47 d 16.2
5/9
6/8
7
10
11
12
13
14
PLAb
1
7.00 d
6.58 d 8.6
Tyr
1
2
173.6
58.2
37.2
4.43 d 6.5
5.34 bt 6.1
4.31 m
3.01 m
2.82 dd 14.1, 9.7
3
a
b
1.64 s
1.67 s
4
129.1
131.5
116.6
157.7
26.1
5/9
6/8
7
Ala2
1
7.00 d m
6.68 d 8.6
171.2
76.1
38.8
2
3
5.19 dd 5.7, 4.2
3.03 m
2.74 dd 14.3, 3.9
a
b
174.5
50.8
18.9
2
3
4.31 m
1.35 d 7.2
4
136.9
131.5
129.5
128.4
5/9
6/8
7
6.88 bd 7.7
7.19 m
7.18 m
a O-Prenyltyrosine. b 3-Phenyllactic acid.
subsequently partitioned between petroleum ether and methanol/water
(9:1). The resulting methanol/water phase was concentrated to dryness
(184 mg).
crude extracts. This then allows strategic decisions to be made for
the subsequent purification steps. The bioactivity profile can also
be used to assist in the dereplication of extracts containing known
bioactive compounds.
Generation of Bioactivity Profiles. An aliquot of the extract (100
µg) was analyzed by HPLC (solvents: (A) H2O + 0.05% TFA, (B)
MeCN; gradient: 0 min 10% B, 2 min 10% B, 14 min 75% B, 24 min
75% B, 26 min 100% B; flow: 1 mL/min; 40 °C). The eluent from
the DAD was split in a 1:10 ratio between the ELSD and the fraction
collector configured to collect into a 96-well microtiter plate (15 s/well).
A total of 88 wells were collected (2.5-24.5 min). A daughter plate
was prepared by transferring an aliquot (5 µL) from each well of the
master plate. After complete evaporation of the solvent the wells in
the plate were analyzed for activity against P388 murine leukemia cells
as follows: aliquots of P388 cell suspension (150 µL; 1.26 × 104 cells)
were added to each well except wells H1 and H2, which as a positive
control were loaded with medium (150 µL) only. After incubation (36
°C, 3 days) an MTT solution was added to each well (20 µL, 3.8 mg/
mL in PBS) and incubated (4 h at 36 °C). To dissolve the formazan
product, HCl in isopropanol (170 µL, 0.08 M) was added to each well.9
Cell viability was assessed by measuring the absorption of each well
at 540 nm, subtracting the absorption at 690 nm and using the negative
control (wells H1 and H2) and the analyte free cell control (wells H3-
H5) as 0% and 100% growth reference, respectively.
Experimental Section
General Experimental Procedures. Optical rotations were mea-
sured with a Perkin-Elmer 341 polarimeter and UV spectra on a GBC
UV/vis 920 spectrometer. NMR spectra were recorded on a Varian
(UNITY INOVA) AS-500 spectrometer (500 and 125 MHz for 1H and
13C NMR, respectively), using the signals of the residual solvent protons
(δH 3.30 and 2.60 for MeOH-d4 and DMSO-d6, respectively) and the
solvent carbons (δC 49.3 and 39.6 for MeOH-d4 and DMSO-d6,
respectively) as internal references. HRESIMS were acquired using a
Micromass LCT TOF mass spectrometer, which was also used for LC-
MS. HPLC was performed on a Dionex system equipped with an ISCO
Foxy Jr. sample collector using a reversed-phase analytical column
(Phenomenex Prodigy C18, 4.6 × 250 mm, 5 µm) with photodiode
array (DAD) and evaporative light scattering detection (ELSD; Alltech).
Solvents used for extraction and isolation were distilled prior to use.
Biological Assays. For the P388 cytotoxicity assays the following
medium was used: MEM, fetal calf serum (10%), penicillin (266 u/mL),
streptomycin (132 µg/mL), L-glutamine (2 mM), NaHCO3 (2.2 g/L),
HEPES (7.4 mM). For the Bacillus subtilis assay a RPMI-1640 medium
was used, supplemented with L-glutamine (2 mM) and MOPS (34.53
g/L) and adjusted to pH 7 with NaOH (1 M). Cytotoxicity against P388
cells and antimicrobial activities were measured using standard
protocols.16
Fungus. A small cube from the interior of the alga DurVillaea
antarctica, collected at the coast of Tauranga Bay, New Zealand, was
placed on filtered seawater agar (fresh filtered seawater, agar 15 g/L,
chloramphenicol 100 µg/L, ampicillin 100 µg/L, and streptomycin
sulfate 50 µg/L). After cultivation at 15 °C for 7 days, hyphae growing
on the substrate were transferred onto seawater potato dextrose agar
(fresh filtered seawater, Gibco PDA 39 g/L, chloramphenicol 100 µg/
L, ampicillin 100 µg/L, and streptomycin sulfate 50 µg/L). One of the
isolates was identified as a species of Gliocladium due to the densely
penicillate conidiophores bearing one-celled, ellipsoidal, smooth-walled
conidia in slimy heads.17
For the antibacterial profile a second microtiter plate was prepared
using identical conditions, but injecting 500 µg of extract. After
evaporation of the solvent this plate was tested directly for activity
against B. subtilis: 200 µL of a B. subtilis suspension (OD(610 nm) )
0.04) was added to each well except the negative control wells H1 and
H2, which were loaded with 200 µL of medium each. After incubation
(24 h; 30 °C) a resazurin solution (0.2 mg/mL PBS; 30 µL) was added
to all wells except H4 and the plate further incubated (15 min; 30 °C).
Cell viability was assessed the same way as described above for the
cytotoxicity profile, but measuring the absorption at 600 nm and
subtracting the absorption at 690 nm.
Isolation. The extract was subjected to semipreparative HPLC
(Phenomenex Luna C18, 10 × 250 mm, 5 µm; H2O + 0.05% TFA
(A), MeCN (B); 0 min 20% B, 13 min 75% B; 5 mL/min; 40 °C).
Clonostachydiol (2; 13.2 mg), 4-keto-clonostachydiol (1; 8.2 mg), and
gliotide (3; 7.1 mg) were eluted after 5.9, 8.4, and 12.5 min,
respectively.
Extraction. For chemical investigation the Gliocladium sp. was
grown at 26 °C in half-strength potato dextrose broth (PDB; 3 × 500
mL). The cultures were agitated on a shaker (first 14 days of growth)
and then left growing under static conditions (16 more days). The
cultures were filtered, and broth and cells were extracted exhaustively
with ethyl acetate. The combined organic phases were concentrated,
partitioned against water, and dried to give a crude extract, which was
4-Keto-clonostachydiol (1): colorless oil; [R]20 +49 (c 0.33,
D
MeOH); 1H and 13C NMR data were consistent with those reported in
the literature;10 for full 1D and 2D NMR data, see Supporting
Information; ESIMS m/z 283.1 [M + H]+.
Clonostachydiol (2): white solid; [R]20D +90 (c 1.0, MeOH) (lit.10
1
+103, c 1.0 MeOH); H and 13C NMR data and results from CIGAR

624 Journal of Natural Products, 2006, Vol. 69, No. 4
Lang et al.
and COSY were identical with, or consistent with, reported data;11
(CH2, C-11), 33.4 (CH2, C-12), 20.4 (CH3, C-14), 18.3 (CH3, C-6);
HRESIMS m/z 283.1147 [M + H]+ (calcd for C14H19O6, 283.1182).
Preparation of 6. A solution of clonostachydiol (2; 2.8 mg; 0.010
mmol) in CH2Cl2 (3 mL) was stirred with freshly prepared MnO2 (30
mg) at room temperature for 24 h. Filtering and drying the reaction
ESIMS m/z 285.2 [M + H]+.
1
Gliotide (3): white solid; [R]20 +6.6 (c 0.33, MeOH); for H and
D
13C NMR data (in MeOH-d4), see Table 2; for additional NMR data
(HMBC, COSY, ROESY data in MeOH-d4; H, 13C, and ROESY in
1
mixture gave 6 (1.6 mg; 0.006 mmol; 60%) as a colorless oil: [R]20
DMSO-d6), see Supporting Information; HRESIMS m/z 742.3424 [M
D
+ H]+ (calcd for C40H48N5O9, 742.3452).
1
+25 (c 0.1, MeOH); H NMR (CDCl3; 500 MHz) δ 7.06 (1H, d, J )
Preparation and Analysis of Marfey Derivatives. Compound 3
(0.8 mg) was hydrolyzed by heating (110 °C for 24 h) in HCl (6 M; 1
mL). After cooling, the solution was evaporated to dryness and
redissolved in H2O (100 µL). A 1% (w/v) solution (100 µL) of FDAA
(Marfey’s reagent, NR-(2,4-dinitro-5-fluorophenyl)-L-alaninamide)15 in
acetone was added to an aliquot (50 µL) of the acid hydrolysate solution
(or to 50 µL of a 50 mM solution of the respective amino acid). After
addition of NaHCO3 solution (1 M; 20 µL) the mixture was incubated
(1 h at 40 °C). The reaction was stopped by addition of HCl (2 M; 10
µL), the solvents were evaporated to dryness, and the residue was
redissolved in MeOH-H2O (1:1; 1 mL). An aliquot of this solution
(10 µL) was analyzed by HPLC (Phenomenex Luna C18, 250 × 4.6,
5 µm; solvents: A H2O + 0.05% TFA, B MeCN; linear gradient: 0
min 35% B, 30 min 45% B; 25 °C; 1 mL min-1). Retention times
(min) of the amino acid derivatives were as follows: L-Ala (6.4), D-Ala
(7.6), L-Tyr (25.1), and D-Tyr (29.5).
16.0 Hz, H-9), 6.97 (1H, d, J ) 16.6 Hz, H-3), 6.75 (1H, d, J ) 16.0
Hz, H-8), 6.45 (1H, d, J ) 16.6 Hz, H-2), 5.32 (1H, q, J ) 7.3 Hz,
H-5), 5.10 (1H, m, H-13), 2.71 (1H, ddd, J ) 13.5, 8.9, 3.3 Hz,
Ha-11), 2.53 (1H, ddd, J ) 13.5, 8.9, 3.3 Hz, Hb-11), 2.03 (2H, m,
H-12), 1.57 (3H, d, J ) 7.0 Hz, H-6), 1.30 (3H, d, J ) 6.6 Hz, H-14);
13C NMR (CDCl3, 125 MHz) δ 198.5 (C, C-10), 197.8 (C, C-4), 165.0
(C, C-7), 163.4 (C, C-1), 138.3 (CH, C-9), 135.0 (CH, C-3), 129.6
(CH, C-8), 129.6 (CH, C-2), 75.7 (CH, C-5), 71.6 (CH, C-13), 37.4
(CH2, C-11), 30.4 (CH2, C-12), 17.9 (CH3, C-14), 15.3 (CH3, C-6);
HRMS (APCI, pos.) m/z 281.1028 [M + H]+ (calcd for C14H17O6,
281.1025).
Acknowledgment. This work was supported by a fellowship within
the Postdoc-Programme of the German Academic Exchange Service
(DAAD). We thank Mr. B. Clark for mass spectrometric analyses.
Preparation and Analysis of (R)-MTPA Esters of 3-Phenyllactic
Acid. To (R)- or (S)-3-phenyllactic acid (0.1 mg), synthesized from D-
or L-phenylalanine, respectively,18 or to dried acid hydrolysate of 3
(from 0.4 mg peptide) were added a solution of (S)-MTPA chloride
(0.5 mg) in CH2Cl2 (0.5 mL), Et3N (3 µL), and a small crystal of
DMAP. After 3 h the solvent was evaporated, the residue dissolved in
MeOH (0.5 mL), and the solution analyzed by HPLC (Phenomenex
Luna C18, 250 × 4.6, 5 µm; solvents: A H2O + 0.05% TFA, B MeCN;
linear gradient: 0 min 10% B, 2 min 10% B, 14 min 75% B, 24 min
75% B; 40 °C; 1 mL min-1). Retention times of the (R)- and (S)-3-
phenyllactic acid esters were 17.86 and 18.00 min, respectively.
Reduction of 1. To a stirred solution of 1 (1.3 mg, 4.6 µmol) and
CeCl3‚7H2O (3 mg, 8.0 µmol) in MeOH (2.5 mL) was added NaBH4
(2 mg, 53 µmol). After 5 min the solvent was evaporated in vacuo,
dissolved in EtOAc (3 mL), and washed with HCl (0.5 M; 3 mL).
Semipreparative HPLC on a C18 column yielded the two reduced
products (0.3 mg of each). 4a: [R]20D +100 (c 0.03, MeOH); 1H NMR
data and HPLC retention time identical to those of clonostachydiol
(2). 4b (4-epi-clonostachydiol): [R]20D +60 (c 0.03, MeOH); 1H NMR
(MeOH-d4; 500 MHz) δ 6.84 (1H, dd, J ) 15.8, 4.3 Hz, H-9), 6.77
(1H, dd, J ) 15.7, 3.0 Hz, H-3), 6.14 (1H, dd, J ) 15.7, 2.0 Hz, H-2),
5.85 (1H, dd, J ) 15.8, 1.9 Hz, H-8), 5.25 (1H, qd, J ) 6.5, 2.1 Hz,
H-5), 5.12 (1H, m, H-13), 4.57 (1H, m, H-10), 4.40 (1H, m, H-4),
1.97 (1H, m, Ha-11), 1.70 (1H, m, Hb-11), 1.66 (1H, m, Ha-12), 1.50
(1H, m, Hb-12), 1.38 (3H, d, J ) 6.5 Hz, H-6), 1.21 (3H, J ) 6.5 Hz,
H-14).
Supporting Information Available: 1H and 13C NMR spectra as
well as tabulated NMR data of compounds 1 and 3 and HPLC of the
3-phenyllactic-acid (R)-MTPA esters are available free of charge via
the Internet at http://pubs.acs.org.
References and Notes
(1) Blunt, J. W.; Copp, B. R.; Munro, M. H. G.; Northcote, P. T.; Prinsep,
M. R. Nat. Prod. Rep. 2005, 22, 15-61.
(2) Bringmann, G.; Lang, G.; Steffens, S.; Schaumann, K. J. Nat. Prod.
2004, 67, 311-315.
(3) Klemke, C.; Kehraus, S.; Wright, A. D.; Ko¨nig, G. M. J. Nat. Prod.
2004, 67, 1058-1063.
(4) Tsuda, M.; Mugishima, T.; Komatsu, K.; Sone, T.; Tanaka, M.;
Mikami, Y.; Shiro, M.; Hirai, M.; Ohizumi, Y.; Kobayashi, J.
Tetrahedron 2003, 59, 3227-3230.
(5) Liu, W.; Gu, Q.; Zhu, W.; Cui, C.; Fan, G.; Zhu, T.; Liu, H.; Fang,
Y. Tetrahedron Lett. 2005, 46, 4993-4996.
(6) Kasai, Y.; Komatsu, K.; Shigemori, H.; Tsuda, M.; Mikami, Y.;
Kobayashi, J. J. Nat. Prod. 2005, 68, 777-79.
(7) Usami, Y.; Yamaguchi, J.; Numata, A. Heterocycles 2004, 63, 1123-
1129.
(8) Arai, N.; Shiomi, K. Iwai, Y.; Omura, S. J. Antibiot. 2000, 53, 609-
614.
(9) Alley, M. C.; Scudiero, D. A.; Monks, A.; Hursey, M. L.; Czerwinski,
M. J.; Fine, D. L.; Abbott, B. J.; Mayo, A.; Shoemaker, R. H.; Boyd,
M. R. Cancer Res. 1988, 48, 589-601.
(10) Kano, C.; Adachi, K.; Shizusato, Y. Patent JP 5247737, 2005, 15
pp.
Preparation of 5. A solution of clonostachydiol (2; 3.7 mg; 0.013
mmol) and Dess-Martin periodinane (20 mg; 0.047 mmol) in CH2Cl2
(3 mL) was stirred at room temperature for 3 h. The mixture was washed
twice with saturated NaHCO3 solution and dried over Na2SO4.
Purification by chromatography on a RP18 cartridge (MeOH/H2O)
(11) Grabley, S.; Hammann, P.; Thiericke, R.; Wink, J.; Philipps, S.;
Zeeck, A. J. Antibiot. 1993, 46, 343-345.
(12) Rao, A. V. R.; Murthy, V. S.; Sharma, G. V. M. Tetrahedron Lett.
1995, 36, 139-142.
(13) Rao, A. V. R.; Murthy V. S.; Sharma, G. V. M. Tetrahedron Lett.
1995, 36, 143-146.
yielded compound 5 (1.9 mg; 0.007 mmol; 54%) as a white solid:
1
[R]20 +35 (c 0.1, MeOH); H NMR (CDCl3; 500 MHz) δ 6.87 (1H,
D
(14) Crouch, R.; Boyer, R. D.; Johnson, R.; Krishnamurthy, K. Magn.
Reson. Chem. 2004, 42, 301-307.
d, J ) 16.0 Hz, H-9), 6.63 (1H, d, J ) 15.7 Hz, H-8), 6.60 (1H, dd,
J ) 15.9, 8.4 Hz, H-3), 5.74 (1H, d, J ) 15.8 Hz, H-2), 5.02 (1H, m,
H-5), 5.02 (1H, m, H-13), 3.99 (1H, t, J ) 8.8 Hz, H-4), 2.64 (1H, m,
Ha-11), 2.55 (1H, m, Hb-11), 2.10 (1H, m, Ha-12), 1.97 (1H, m,
Hb-12), 1.49 (3H, d, J ) 6.2 Hz, H-6), 1.23 (3H, d, J ) 6.2 Hz, H-14);
13C NMR (CDCl3, 125 MHz) δ 201.0 (C, C-10), 167.5 (C, C-7), 166.2
(C, C-1), 147.2 (CH, C-3), 140.0 (CH, C-9), 132.6 (CH, C-8), 126.8
(CH, C-2), 77.7 (CH, C-4), 74.1 (CH, C-5), 72.6 (CH, C-13), 40.2
(15) Marfey, P. Carlsberg Res. Commun. 1984, 49, 591-596.
(16) Lang, G.; Blunt, J. W.; Cummings, N. J.; Cole, A. L. J.; Munro, M.
H. G. J. Nat. Prod. 2005, 68, 1303-1305.
(17) Domsch, K. H.; Gams, W.; Anderson, T. H. Compendium of Soil
Fungi; Academic Press: London, 1980; Vol. 1, pp 368-377.
(18) Bauer, T.; Gajewiak, J. Tetrahedron 2004, 60, 9163-9170.
NP0504917