Absolute configurations of ()-hirsutanol A and ()-hirsutanol C produced by Gloeostereum incarnatum

Full text of DOI:10.1038/ja.2011.73

The Journal of Antibiotics (2011) 64, 693–696
2011 Japan Antibiotics Research Association All rights reserved 0021-8820/11 $32.00
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NOTE
Absolute configurations of (ꢀ)-hirsutanol A and
(ꢀ)-hirsutanol C produced by Gloeostereum incarnatum
Ryo Asai1, Shinya Mitsuhashi1, Kengo Shigetomi1, Toshizumi Miyamoto2 and Makoto Ubukata1
The Journal of Antibiotics (2011) 64, 693–696; doi:10.1038/ja.2011.73; published online 31 August 2011
Keywords: antitumor substance; Gloeostereum incarnatum; hirsutanol; incarnal; mushroom; sesquiterpene
rated from an Indo-Pacific sponge Haliclona species.4 However,
Gloeostereum incarnatum is an edible mushroom that appears on
the specific optical rotations of 1 ([a]2D5¼ꢀ41.9 (c 0.97, MeOH))
broad-leaved wood in eastern Asia, northern Japan, northern China
and eastern Siberia.1,2 We have focused our attention on library
construction of fungal strains of mushrooms as a screening source
for the biologically active small molecules useful for forward chemical
genetics and development of medicinal remedy. During the course of
the screening program for potential chemopreventive agents against
murine B16 melanoma cells, we found potent antiproliferative activity
and 2 ([a]2D5¼ꢀ8.8 (c 0.31, MeOH)) were not identical to those of
hirsutanols A ([a]2D5¼ꢀ23.5 (c 0.97, MeOH)) and C ([a]2D5¼+20.6
(c 0.31, MeOH)), respectively.
Significant differences between the data of 2 and those of hirsutanol C
(13C NMR, see Supplementary Table S1) made us to pursue unambig-
uous structure determination of both 1 and 2 (Figure 1). First, we
in a culture broth of Gloeostereum incarnatum HUWCB-0029 picked in determined the relative configuration of 1 and 2 through differential
NOE experiments and molecular modeling5 (Figure 1b). The NOE data
the Sapporo campus of Hokkaido University, Japan. The active
are tabulated with assignments of 1H NMR of 1 and 2 in Supplementary
compound, (ꢀ)-hirsutanol A (1), was purified with an inactive related
compound, (ꢀ)-hirsutanol C (2), through bioassay-guided isolation. Table S2. Observed NOEs of 1 between H-9 and 1-OH, between Me-12
and H-11b, and between Me-12 and Me-14, and NOEs of 2 between H-9
and 1-OH, between Me-12 and H-11b, between H-11b and Me-14, and
between H-3 and 1-OH are critical, and the data assisted molecular
modeling of 1 and 2. We next determined each absolute configuration at
C-9 positions of 1 and 2 to be S configuration by the advanced Mosher
method6 in conjunction with conformational analysis.7 (ꢀ)-Hirsutanol
After derivatizing 1 to (+)-incarnal (3), we confirmed that the strain
produced trace amount of 3 too.
In this article, we describe full details of isolation, unambiguous
determination of the absolute configurations of (ꢀ)-hirsutanol A (1)
and (ꢀ)-hirsutanol C (2) and their biological activities.
Bioassay-guided separation of the active compound (1) was achieved A (1) was converted into the corresponding (S)-MTPA ester (1a) and
(R)-MTPA ester (1b), and (ꢀ)-hirsutanol C (2) was converted into the
by the following procedures: acetone extraction of a culture of
Gloeostereum incarnatum, EtOAc extraction, silica gel column chro-
corresponding (S)-MTPA ester (2a) and (R)-MTPA ester (2b). The
matography, HPLC and recrystallization. The most potent fraction chemical-shift differences (Dd¼d1aꢀd1b and Dd¼d2aꢀd2b) by the
advanced Mosher method are shown in Supplementary Figure S1,
thus 1 and 2 have the absolute configurations depicted in Figure 1a.
In addition, (ꢀ)-hirsutanol A (1) was converted into an oxidized
compound 3 by the Dess–Martin oxidation8 as shown in Figure 1a.
The physicochemical properties of 3 agreed well with the data including
the m.p. of incarnal, whose relative configuration was unambiguously
determined by X-ray crystallography.9 Supplementary Table S3 indicates
13C NMR differences between 3 and incarnal. Although neither specific
optical rotation nor CD data of incarnal were given in the literature, the
separated by silica gel column chromatography showed two distinct
peaks on HPLC (column: Xterra RP18, 19ꢁ300 mm and solvent: 30%
MeOH), and 84.6 mg of (ꢀ)-hirsutanol A (1) as an active compound
(retention time: 65.2min) and 76.6mg of (ꢀ)-hirsutanol C (2) as an
inactive compound (retention time: 83.9min) were isolated by peak-
based fraction collection.
The structures of 1 and 2 deduced from FD-MS and NMR data,
including 1H-1H COSY, HMQC and HMBC, were, respectively,
identical to the proposed structures of hirsutanols A and C, hirsu- authors mentioned that incarnal was isolated from culture fluid of
Gloeostereum incarnatum.9 To confirm whether our strain (Gloeostereum
tane-type3 sesquiterpenes isolated from an unidentified fungus sepa-
1Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo, Japan and 2Division of Environmental Resources, Graduate School of Agriculture,
Hokkaido University, Sapporo, Japan
Correspondence: Professor M Ubukata, Research Faculty of Agriculture, Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University,
Kita-9, Nishi-9, Kita-ku, Sapporo 060-8589, Japan.
E-mail: m-ub@for.agr.hokudai.ac.jp
Received 13 June 2011; revised 6 July 2011; accepted 11 July 2011; published online 31 August 2011

Absolute configurations of (ꢀ)-hirsutanol A and (ꢀ)-hirsutanol C
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7
Table 1 Biological activities of (ꢀ)-hirsutanol A (1), (ꢀ)-hirsutanol C (2)
OH
OH
8
5
6
and (+)-incarnal (3)
9
14
1
4
2
10
Compound
11
3
O
O
OH
OH
15
12
Test organism
1
2
3
Incarnala
13
B16 melanoma cells (IC50 (mM))
Staphylococcus aureus (MIC (mM))
Bacillus subtilis (MIC (mM))
25.6
4200
4100
4100
4100
14.4
NDb
25.6c
51.2c
4400c
(-)-hirsutanol A (1)
(-)-hirsutanol C (2)
4100
4100
4100
4100
4100
4100
O
Escherichia coli (MIC (mM))
aBiological activities reported in ref. 9.
bNot determined.
cMueller–Hinton agar dilution method.
DMP, CH2Cl2, rt
99%
O
OH
(+)-incarnal (3)
respectively, whereas (ꢀ)-hirsutanol C (2) did not show activity at
200 mM as shown in Table 1. Both 1 and 2 did not show antibacterial
activity against Gram-positive bacteria, Staphylococcus aureus and
Bacillus subtilis, and Gram-negative bacteria, Escherichia coli, at
100 mM, which was evaluated by a broth microdilution method
based on National Committee for Clinical Laboratory Standards
(NCCLS) guideline.10
(-)-hirsutanol A (1)
(-)-hirsutanol C (2)
In conclusion, the relative structure of 1 was unambiguously
determined correlating with that of incarnal, which was determined
by X-ray crystallography. Assignments of 1H and 13C NMR of incarnal
were unambiguously determined by HMQC and HMBC analyses.
Physicochemical properties of 1 were similar to those of hirsutanol A,
whose structure was given as the same as that of 1. Although the
absolute value of specific rotation, UV, IR, 1H and 13C NMR data of 1
showed little differences in comparison with the reported data of
hirsutanol A, and m.p. of hirsutanol A was not given in the literature,4
hirsutanol A could have the same structure with 1 in considering
purity of the sample. The absolute configuration of hirsutanol A has
not been determined, whereas the absolute configuration of 1 was
unambiguously determined as shown in Figure 1. The reported data of
Predicted interproton distances (Å)
Me-12 to Me-14: 2.28
Me-12 to H-11β: 2.25
H-9 to 1-OH: 3.29
H-3 to 1-OH: 2.44
Me-12 to H-11β: 2.23
H-9 to 1-OH: 2.99
H-11β to Me-14: 2.41
H-11α to H-9: 3.24
H-9 to Me-15: 2.45
Figure 1 The stereochemistry of (ꢀ)-hirsutanol A (1), (ꢀ)-hirsutanol C (2)
and (+)-incarnal (3). (a) Absolute configurations of 1 and 2, and conversion
of 1 into 3 by the Dess–Martin oxidation. (b) Stable conformers of 1 and 2
based on the MM2 force field calculation. Numbering corresponds to
Supplementary Table S2. The predicted minimum interproton distances
support the interpretation of NOE results (see text). A full color version of
this figure is available at The Journal of Antibiotics journal online.
1
specific rotation, UV, IR, H and 13C NMR of hirsutanol C, whose
structure was proposed to be the same as that of 2 shown in Figure 1,
incarnatum HUWCB-0029) produced incarnal or not, we further were not identical to those of 2. Therefore, we assigned very carefully
analyzed another active fraction on the silica gel column chromato- the data of 1H and 13C NMR of 2 by using HMQC, HMBC and
graphy. We found a small peak, which shows identical UV pattern and differential NOE experiments. In addition to those assignments, we
retention time to those of 3 on analytical HPLC with a photodiode determined the absolute configuration of 2 by the advanced Mosher
array detector (each retention time: 9.5 min, Nova-Pak C18, 3.9ꢁ method, and concluded that the structure of 2 is depicted as shown in
150 mm, 30–100% MeOH linear gradient, 0.8 mlminꢀ1, 401C). Thus Figure 1. (ꢀ)-Hirsutanol A (1) and (+)-incarnal (3) exhibited
absolute configuration of incarnal would be the same as that of moderate antiproliferative activity against B16 melanoma cells,
compound 3. Because both optical rotations of 1 and hirsutanol A, whereas (ꢀ)-hirsutanol C (2) showed no activity even at 200 mM.
a major component from an unidentified fungus,4 are negative and Therefore, 1 and 3 bearing carbonyl group conjugated with exomethy-
the 13C NMR data of 1 are in accord with that of hirsutanol A as lene might be cysteine-targeting inhibitors of a functional protein
shown in Supplementary Table S1, compound 1 is identical to related to cell proliferation.11 Antibacterial activity of hirsutanol A
hirsutanol A. On the contrary, optical rotation of 2 is opposite to against B. subtilis was mentioned without data,4 whereas we could not
that of hirsutanol C, a minor component from unidentified fungus;4 detect antibacterial activities of 1, 2 and 3 at the concentration of
however, the 13C NMR data of 2 are well matched with that of 100 mM. In the literature,9 incarnal exhibited antibacterial activity
hirsutanol C except C-3 position, whose chemical shift is in the region against S. aureus and B. subtilis by using the Mueller–Hinton agar
of the solvent signals of CD3OD. We therefore concluded that dilution method. These differences might come from differences of
compound 2 is identical to hirsutanol C, and the sample of hirsutanol assay method and bacterial strains. Both 1 and 3 having carbonyl
C might contain impurities, which have an influence on physico- moiety conjugated with exomethylene could have weak antibacterial
chemical properties of the compound, or some miscue analysis activities at submillimolar concentrations.12 Recently X-ray crystal
was done in the study of hirsutanol C isolated from the unidentified structure of hirsutanol A isolated from the marine fungus Chondros-
fungus.4
tereum sp.13 and its biological activities14 were reported. The relative
configuration of hirsutanol A and its conformation correspond to the
(ꢀ)-Hirsutanol A (1) and (+)-incarnal (3) exhibited antiproliferative molecular model of 1 (Figure 1b), and specific rotation of this sample
activity against murine B16 melanoma cells with IC50 of 25 and 14mM, is approximately consistent with that of 1.
The Journal of Antibiotics

Absolute configurations of (ꢀ)-hirsutanol A and (ꢀ)-hirsutanol C
R Asai et al
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EXPERIMENTAL PROCEDURE
Materials
Fungal mycelium of Gloeostereum incarnatum (Japanese name: Nikawauroko-
take) was collected in the Sapporo Campus of Hokkaido University, Japan.
[a]D ¼ꢀ52.4 (c 0.21, CH3OH)13). UV (200–400 nm (AU)): 309 (2.41) and
217 (1.62). 1H NMR (270MHz, CDCl3) 0.99 (3H, s, H-14), 1.27 (3H, s, H-12),
1.34 (3H, s, H-15), 1.79 (1H, d, J¼14.8Hz, H-11a), 2.25 (1H, d, J¼14.8Hz,
11b), 4.71 (1H, d, J¼6.9, 2.3Hz, H-9), 5.24 (1H, s, H-13a), 6.10 (2H, H-5 and
13b, overlapping), 6.51 (1H, d, J¼2.3 Hz, H-7); (270MHz, CD3OD, see
Supplementary Figure S2) 0.95 (3H, H-14), 1.23 (3H, s, H-12), 1.29 (3H, s,
H-15), 1.70 (1H, d, J¼14.9Hz, H-11a), 2.27 (1H, d, J¼14.8Hz, H-11b; the
chemical shift, 2.67p.p.m. in ref. 4 may be typographical error), 4.63 (1H, d,
J¼2.3 Hz, H-9), 5.27 (1H, s, H-13a), 5.96 (1H, s, H-13b; 6.00p.p.m. is assigned
to H-13b in ref. 4), 6.02 (1H, s, H-5), 6.50 (1H, d, J¼2.4 Hz, H-7); (270MHz,
dimethylsulfoxide-d6): see Supplementary Table S2. 13C NMR (67.5Hz,
CD3OD): see Supplementary Table S1 and Supplementary Figure S3.
FD (m/z): 246(100) [M]+, 228(25) [M-H2O]+, 218(75) [M-CO]+. FD-HRMS
m/z [M]+ calculated for C15H18O3, 246.12559; observed, 246.12595.
DNA sequencing of isolate
Mycelia of isolated strain were maintained with a malt extract agar (2% malt
extract and 1.5% agar) plate in a dark place at 22–241C. Total DNA was
extracted from a block of mycelia (3mmꢁ3 mmꢁ3 mm) using Isoplant II
DNA extraction kit (Wako Pure Chemical Industries, Osaka, Japan) according
to the manufacturer’s instructions. The extracted DNA was dissolved in 50ml
TE buffer, and 1 ml of the DNA solution was used as template DNA. The primer
pair of ribosomal DNA, ITS1f: 5¢-CTTGGTCATTTAGAGGAAGTAA-3¢ (sense)
and ITS4: 5¢-TCCTCCGCTTATTGATATGC-3¢ (antisense), was used for PCR
amplification.15,16 The sequence of the ITS1-5.8S ribosomal DNA-ITS2 region
was determined by using an ABI Auto Sequencer 3730 (Applied Biosystems,
Foster City, CA, USA). The aligned sequence well accorded with Gloeostereum
incarnatum 3332 (BLAST Locus No. AF141637) with 98% (350/355) of
identities and 0% (3/335) of gaps in a BLAST search.17
(ꢀ)-Hirsutanol C (2), m.p. 205–2061C. [a]25¼ꢀ8.8 (c 0.31, CH3OH; lit.,
D
hirsutanol C: [a]2D5¼+20.6 (c 0.31, CH3OH)4). UV(200–400nm (AU)): 288
(2.01), 206 (0.44, sh). 1H NMR (270MHz, CDCl3): 0.97 (3H, s, H-14), 1.04
(3H, s, H-12), 1.12 (3H, d, J¼7.2 Hz, H-13), 1.34 (3H, s, H-15), 1.67 (1H, d,
J¼14.9Hz, H-11a), 2.11 (1H, d, J¼14.9Hz, H-11b), 2.86 (1H, q, J¼7.2 Hz, H-
3), 4.73 (1H, dd, J¼7.5, 2.4Hz, H-9), 5.87 (1H, s, H-5), 6.46 (1H, d, J¼2.4 Hz,
H-7); (270Hz, CD3OD, see Supplementary Figure S4) 0.92 (3H, s, H-14), 1.02
(3H, s, H-12), 1.06 (3H, d, J¼7.2 Hz, H-13), 1.28 (3H, s, H-15), 1.60 (1H, d,
J¼14.8Hz, H-11a), 2.10 (1H, d, J¼14.8Hz, H-11b), 2.89 (1H, q, J¼7.2 Hz,
H-3), 4.65 (1H, d, J¼2.3 Hz, H-9), 5.76 (1H, s, H-5), 6.44 (1H, d, J¼2.4 Hz,
H-7); (270Hz, dimethylsulfoxide-d6): see Supplementary Table S2. 13C NMR
(67.5 Hz, CD3OD): see Supplementary Table S1 and Supplementary Figure S5.
FD-MS (m/z): 248(100) [M]+, 230(26) [M-H2O]+. FD-HRMS m/z [M]
calculated for C15H20O3, 248.14124; observed, 248.14032.
Fermentation procedure of the producing strain
Each block (10 mmꢁ10 mmꢁ3 mm) cutoff from mycelial pellet of Gloeostereum
incarnatum spread on a malt extract agar plate was inoculated into 20 of 500 ml
K-1 flasks (K-Techno, Toyama, Japan) each containing 150 ml of production
medium consisting of 1% malt extract, 0.5% yeast extract and 0.5% glucose.
Fermentation was carried out in a dark place at 25 1C for 30 days in stationary
culture.
(+)-Incarnal (3), m.p. 132–133 1C (lit., m.p. 132–1331C).9 [a]D25¼+339.8
(c 0.30, CH3OH). UV (200–400 nm (AU)): 309, 217 (lit., 306, 2189). 1H NMR
(270MHz, CDCl3, see Supplementary Figure S6): 1.22 (3H, s, H-14), 1.28 (3H,
s, H-12), 1.41 (3H, s, H-15), 1.73 (1H, s, OH-1), 2.06 (1H, d, J¼13.9Hz, H-
11a), 2.26 (1H, d, J¼13.9 Hz, H-11b), 5.40 (1H, s, H-13a), 6.21 (1H, s, H-13b),
6.43 (1H, s, H-5), 7.20 (1H, s, H-7). 13C NMR (67.5MHz, CDCl3): see
Supplementary Table S3 and Supplementary Figure S7. FD-HRMS m/z [M]+
calculated for C15H16O3, 244.1099; observed, 244.1077.
General experimental procedures for structural study
Unless otherwise stated, chemicals of the highest commercial purity were used
without further purification. The m.p. was measured by a Mettler Melting-
Point Apparatus FP-5 (Mettler, Greifensee, Switzerland) for (ꢀ)-hirsutanol A
(1) and (ꢀ)-hirsutanol C (2), and a Yanagimoto Micro-Melting Point Appa-
ratus (Yanagimoto, Kyoto, Japan) for (+)-incarnal (3). UV spectra of 1, 2 and 3
were recorded on a Hitachi L7455 photodiode array detector (Hitachi, Tokyo,
Japan) at 301C; IR spectra were recorded on a Digilab FTS-50A (Digilab,
Tokyo, Japan); 1H, 13C, HH-COSY, HMBC, HMQC, DIF-NOE and NOESY
NMR spectra were measured with a Bruker AMX-500 (Bruker, Billeria, MA,
USA) (500MHz) or JEOL JNM-EX270 (JEOL, Tokyo, Japan) (270MHz).
Chemical shifts are reported in d p.p.m. using tetramethylsilane as internal
standard, and coupling constants (J) are given in hertz. Mass spectra were
acquired with FD techniques using a JEOL JMS-SX102A. A part of the NMR and
MS spectra were measured at the GC-MS and NMR Laboratory, Faculty of
Agriculture, Hokkaido University. Optical rotations were determined on a JASCO
(JASCO, Tokyo, Japan) P-2200 polarimeter in 3.4 mmꢁ5.0 cm cells at 25 1C.
Computational procedure
The equilibrium geometries of all compounds were optimized by molecular
mechanics (Allinger’s MM2) calculations by using Spartan Student (Ver. 3.0.2,
Wavefunction, Irvine, CA, USA). Stable conformers were confirmed by CON-
FLEX calculations using CAChe 4.5 for Power Macintosh (Fujitsu, Kawasaki,
Japan) and well corresponded to the three dimensional structures of incarnal9
and hirsutanol A13 determined by X-ray crystallography.
Antiproliferative assay
Murine B16 melanoma cells, which were derived from C57BL/6 mouse, were
Extraction and isolation
A volume of 300 ml acetone was added to each culture, after cutting mycelial maintained in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine
pellet into small pieces with scissors, the combined resultant suspension was serum, 1.9 g lꢀ1 sodium bicarbonate, 100 mg mlꢀ1 streptomycin and 20Umlꢀ1
¨
penicillin G at 37 1C under 5% CO2. Antiproliferative activities were measured
stirred overnight at 4 1C and filtered with Buchner funnel (filter paper No. 3,
ADVANTEC, Tokyo, Japan) under reduced pressure. The filtrate was evapo- by Cell Counting Kit-8 (Dojindo, Tokyo, Japan). Cell Counting Kit-8 allows
rated in vacuo, and the aqueous solution was extracted twice with equal part of colorimetric assays using WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-
ethyl acetate. The combined organic layer was evaporated in vacuo to give a 5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt), which produces a
crude extract. The extract was subjected to silica gel column chromatography water-soluble formazan dye upon reduction in the presence of an electron
by stepwise elution with the following solvent systems; CHCl3:MeOH¼25:1, carrier, 1-methoxy PMS. WST-8 is reduced by cellular dehydrogenase to an
9:1, 4:1 and 0:1 to give 17 fractions. The fraction nine showed most potent orange formazan product that is soluble in cell culture medium. The amount of
antiproliferative activity was purified by HPLC (pump: Waters 600, column: formazan produced is directly proportional to the number of living cells.18
Xterra RP18, 7 mm, 19ꢁ300 mm, solvent: 30% MeOH) to give 84.6 mg of 1 and
The B16 cells (1.0ꢁ104 cells per 95 ml per well) were seeded into 96-well
76.6 mg of 2. The fraction three showed antiproliferative activity and showed microplates, and then 5 ml of test sample was added to each well. After
similar Rf value (Rf 0.57–0.85) to that of synthetic 3 (Rf 0.65) on the silica gel incubating for 24h, 10ml of WST-8 solution (Cell Counting Kit-8) was added
TLC (EtOAc:hexane, 4:1). The fraction showed a small peak having the to each well. After a further 1–3h in culture, the OD of the water-soluble
identical UV pattern and retention time (9.5min) to those of 3 on HPLC formazan produced by the cells was measured with a microplate reader (Sunrise
with a photodiode array detector (Nova-Pak C18 Waters ODS, 3.9ꢁ150 mm, Remote, TECAN, Mannedorf, Switzerland) at 450nm (reference: 595 nm). The
30–100% MeOH linear gradient, 0.8 mlminꢀ1, 401C).
test compound was dissolved in dimethylsulfoxide, and the final concentration
(ꢀ)-Hirsutanol A (1), m.p. 149–1501C (lit., 161–163 1C13). [a]D25¼ꢀ41.9 of dimethylsulfoxide in the medium was 1%. The IC50 values were determined
(c 0.97, CH3OH; lit., hirsutanol A: [a]2D5¼ꢀ23.5 (c 0.97, CH3OH);4 graphically.
The Journal of Antibiotics

Absolute configurations of (ꢀ)-hirsutanol A and (ꢀ)-hirsutanol C
R Asai et al
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Antibacterial assay
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0.3% meat extract, 1.75% Bacto Peptone (BD, Franklin Lakes, NJ, USA) and
0.15% soluble starch. After preculture with Mueller–Hinton liquid medium,
5 ml of test compound solutions and 95ml of bacterial inoculums were mixed to
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ACKNOWLEDGEMENTS
This work was partially supported by a research grant donated by the Institute
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Supplementary Information accompanies the paper on The Journal of Antibiotics website (http://www.nature.com/ja)
The Journal of Antibiotics