Antimicrobial cuparene-type sesquiterpenes, enokipodins C and D, from a mycelial culture of Flammulina velutipes

Article abstract of DOI:10.1021/np000593r

Two new cuparene-type sesquiterpenes, enokipodins C (1) and D (2), were isolated from culture medium of an edible mushroom, Flammulina velutipes, along with enokipodins A (3) and B (4). The structures of 1 and 2 were determined using spectroscopic methods (HRMS, ¹H and ¹³C, and 2D NMR). The absolute configuration of enokipodin C was determined from the observed ¹H NMR chemical shifts and NOEs in NOESY experiments after conversion into the corresponding esters with the chiral reagent 2-(2′-methoxy-1′-naphthyl)-3,4-dichlorobenzoic acid. All the metabolites showed antimicrobial activity against a fungus, Cladosporium herbarum, and Gram-positive bacteria, Bacillus subtilis and Staphylococcus aureus.

Full text of DOI:10.1021/np000593r

932
J . Nat. Prod. 2001, 64, 932-934
An tim icr obia l Cu p a r en e-Typ e Sesqu iter p en es, En ok ip od in s C a n d D, fr om a
Mycelia l Cu ltu r e of Fla m m u lin a velu tipes
Noemia Kazue Ishikawa,† Yukiharu Fukushi,‡ Keiko Yamaji,‡ Satoshi Tahara,‡ and Kunihide Takahashi*,†
Division of Environmental Resources and Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University,
Kita-ku, Sapporo 060-8589, J apan
Received December 8, 2000
Two new cuparene-type sesquiterpenes, enokipodins C (1) and D (2), were isolated from culture medium
of an edible mushroom, Flammulina velutipes, along with enokipodins A (3) and B (4). The structures of
1
1 and 2 were determined using spectroscopic methods (HRMS, H and 13C, and 2D NMR). The absolute
1
configuration of enokipodin C was determined from the observed H NMR chemical shifts and NOEs in
NOESY experiments after conversion into the corresponding esters with the chiral reagent 2-(2′-methoxy-
1′-naphthyl)-3,4-dichlorobenzoic acid. All the metabolites showed antimicrobial activity against a fungus,
Cladosporium herbarum, and Gram-positive bacteria, Bacillus subtilis and Staphylococcus aureus.
Sch em e 1
Global antibacterial resistance is an increasing public
health problem, because bacterial resistance has developed
to almost all available antibiotics.1 Although fungi are well
known for the production of important antibiotic com-
pounds (e.g., penicillins, streptomycins, rifamycins, and
others), the occurrence of antibiotics in mushrooms is less
well documented.2 Flammulina velutipes (Curt.:Fr.) Sing.
(enokitake in J apanese), which belongs to the Tricolomata-
ceae (Hymenomycetes, Basidiomycota), is an edible mush-
room frequently consumed in J apan. Compounds with
medicinal properties have been isolated from this mush-
room,3-8 including proteins, glycoproteins, and polysaccha-
rides with antitumor and immunomodulatory activities,
lectins, sterols, and monoterpenetriols. We report here the
isolation, structure elucidation including the absolute
configuration, and antimicrobial activities of two new
cuparene-type sesquiterpenes, named enokipodins C (1)
and D (2), from culture medium of F. velutipes. In our
search for antimicrobial compounds in edible mushrooms,
we had reported enokipodins A (3) and B (4) from F.
velutipes.9 Compounds 1 and 2 are cuparene-type sesquit-
erpenes that are more highly oxygenated than 3 and 4,
respectively. The carbon atoms in 1 and 2 are numbered
as indicated in Scheme 1 on the basis of biosynthetic
considerations.
Sch em e 2
Three protons were attributed: one (δ 4.35) to phenolic
OH and two (δ 2.79 and 1.89) to alcoholic hydroxyl protons.
The proton (δ 2.79) assigned to a hemiketal OH, 10-C-OH,
similarly resonated in the H NMR spectrum of 3 (δ 2.74).9
1
The proton at δ 1.89 was located on carbon C-8 (δ 77.2)
because HMBC experiments revealed a clear correlation
of C-8 (δ 77.2) with a methyl group C-14 (δ 1.29), via three
atomic bonds. The presence of a hydroxyl group at C-8 was
further supported by the fact that C-10-OH was correlated
with the only methylene carbon attributable to the cyclo-
pentane ring C-9 (δ 46.5). Full assignments of H and 13C
data for 1 were done on the basis of 2D NMR experiments,
and NOE correlations revealed the relative stereostructure
of 1 depicted in Figure 1, Supporting Information.
The molecular formula of enokipodin D (2) was estab-
lished as C15H18O4 by HREIMS. IR spectroscopy revealed
the presence of a hydroxyl group (3446 cm-1) and carbonyl
groups (1733 and 1690 cm-1) that were attributed to
cyclopentanone and benzoquinone moieties in 2. The 13C
NMR spectrum revealed the presence of 15 carbons com-
prised of four methyls, one methylene, three methines, and
seven quaternary carbons. The quaternary carbons in-
cluded an isolated ketonic carbonyl (δ 216.0), two benzo-
quinone carbonyls (δ 188.7 and 187.5), four sp2 carbons,
and a methine carbon bearing a hydroxyl group (δ 69.2).
Enokipodin D (2) appeared to be an oxocyclopentylbenzo-
Resu lts a n d Discu ssion
The molecular formula of enokipodin C (1) was estab-
lished as C15H20O4 by HREIMS. The absorption at 3384
cm-1 in the IR spectrum and a dehydration ion at m/z 246
as a base peak in the MS of 1 indicated the presence of a
hydroxyl group. A methine carbon corresponding to the
carbinyl carbon appeared at δ 77.2, indicating that 1 was
a secondary alcohol. UV absorption (λmax at 206 and 299
nm) and signals in the 13C NMR for six sp2 carbons
(4×C+2×CH) suggested the presence of a tetrasubstituted
benzene ring. The remaining nine carbons were classified
as four methyls, one methylene, one methine, and three
quaternary carbons. Comparison of 13C NMR data for 1 and
enokipodin A (3) strongly suggested that they had identical
carbon skeletons.
1
* To whom correspondence should be addressed. Tel: +81-11-706-2517.
Fax: +81-11-706-4176. E-mail: kun23@for.agr.hokudai.ac.jp.
† Division of Environmental Resources.
‡ Division of Applied Bioscience.
10.1021/np000593r CCC: $20.00
© 2001 American Chemical Society and American Society of Pharmacognosy
Published on Web 07/07/2001

Antimicrobial Cuparene-Type Sesquiterpenes
J ournal of Natural Products, 2001, Vol. 64, No. 7 933
Ta ble 1. Antibacterial Activities of Enokipodins A-D (1-4)
C (1)
D (2)
A (3)
B (4)
PCP
12.5 µg
species/strain
25 µga
50 µg
25 µg
50 µg
25 µg
50 µg
25 µg
50 µg
25 µg
Gram positive
Bacillus subtilis LMA0011
Staphylococcus aureus AHU1142
Gram negative
20b
18
26
21.2
11
0
16
11.6
28
19.2
32.6
25.2
17
0
21
14
26.6
ntc
31
nt
Escherichia coli AHU1714
Pseudomonas fluorescens AHU1719
0
0
0
0
0
nt
0
nt
0
0
0
0
0
nt
0
nt
nt
nt
nt
nt
a
b
Loaded on 8 mm φ paper disk. Diameter of inhibitory zone in mm (average from three replicates). c nt ) Not tested.
quinone-type derivative, like enokipodin B (4),9 with an
additional hydroxyl group on C-8. Enokipodin A (3) and C
(1) were autoxidized to enokipodin B (4) and D (2) on thin-
layer plates, via the keto-type tautomer illustrated as 69
and 5, respectively, which has not yet been detected.
Therefore, the structure of enokipodin D (2) was concluded
to be that illustrated in Scheme 1. Full assignment of the
1H and 13C spectroscopic data for 2 was accomplished in a
manner similar to that with 1.
The absolute configuration of enokipodin C (1) was
determined by the 2-(2′-methoxy-1′-naphthyl)-3,5-dichlo-
robenzoic acid (MNCB) method.10 First, enokipodin C (1)
was methylated with dimethyl sulfate, in the presence of
K2CO3, 18-crown-6, and CH3CN, to give 1,4-di-O-methyl-
enokipodin C (7). Then, the secondary alcohol (C-8) in 7
was esterified with (aS)- and (aR)-MNBC to give 8 and 9,
respectively. In the NOESY spectra for 9, NOE was
observed between the methyl protons (H-14) of the alcohol
moiety and methoxy protons of the reagent moiety (Figure
2, Supporting Information). The chemical shift differences
of the proton signals, ∆δ ) 8(aS) - 9(aR), on the right side
of the CB plane have positive values and those on the left
side of the plane have negative values. The value of proton
signals under the border plane were not considered.10
Therefore, C-8 of 7 was confirmed to be R and C-7 and C-10
of 1 were R.
Total synthesis of R-cuparenones has been accomplished
in a variety of ways,11-15 despite the challenge due to steric
congestion created by two contiguous quaternary centers
around the cyclopentane ring.16 However, synthesis or
isolation of R-cuparenone-type sesquiterpenes with high
oxidation levels, such as enokipodins A-D (1-4), has not
yet been reported.
Compounds 1-4 exhibited antibacterial activity against
the Gram-positive bacteria Bacillus subtilis and Staphy-
lococcus aureus, but were inactive against the Gram-
negative bacteria Escherichia coli and Pseudomonas fluo-
rescens. For B. subtilis, the inhibition zones observed with
50 µg of enokipodins C (1) and A (3) were equivalent to
those produced by 12.5 and 25 µg of pentachlorophenol
(Table 1).
F u n gu s a n d Cu ltiva tion . The strain of Flammulina
velutipes Fv-4 used in this work is kept in the culture collection
of the Laboratory of Forest Resource Biology, Graduate School
of Agriculture, Hokkaido University, and maintained on potato
dextrose agar. The mycelia were cultured in 300 mL Erlen-
meyer flasks containing 100 mL of malt peptone broth (3%
Difco malt extract and 0.3% Merck peptone in distilled water,
pH 4.5). Each flask was inoculated with five disks (7 mm in
diameter) of mycelia freshly grown on malt agar plates and
cultured for 45 days at 25 °C under stationary conditions.
An tim icr obia l Assa y. An antifungal assay was carried out
against Cladosporium herbarum AHU9262 (Hyphomycetes).
The culture medium was filtered, and the filtrate was parti-
tioned between EtOAc and H2O. EtOAc extract equivalent to
0.25 mL of the culture medium was charged on thin layer
plates and developed in CHCl3-MeOH ) 25:1. A spore
suspension of C. herbarum was sprayed over the developed
TLC plates, which were incubated at 25 °C under humid
conditions for 3 days.17 The observed inhibitory zones were
correlated with the spots seen on the TLC plates under UV
254 nm light. The Gram-positive bacteria Bacillus subtilis
LMA0011 and Staphylococcus aureus AHU1142 and the Gram-
negative bacteria Escherichia coli AHU1714 and Pseudomonas
fluorescens AHU1719 were used in the antibacterial assays.
A 25 or 50 µg portion of 1, 2, 3, or 4 in acetone (20 µL) was
applied onto a paper disk of φ 8 mm, and the paper disks were
air-dried. Then, the disks were placed on agar plates seeded
with respective organisms. The Petri dishes were allowed to
stand overnight at 4 °C, so that the metabolites could diffuse
into the medium. The plates were then incubated at 37 °C for
18 h. The antibacterial activity was determined by measuring
the diameter of the clear inhibition zone around each paper
disk. Pentachlorophenol (PCP) (12.5 and 25 µg) was used as
positive control antimicrobial compound. All experiments were
done in triplicate.
Extr a ction a n d Isola tion . After incubation, 1400 mL of
culture medium was separated from the mycelia by filtration.
The culture filtrate was extracted with EtOAc (750 mL × 3).
The combined extracts were washed with a saturated solution
of NaCl (1000 mL × 2), dried (MgSO4), and evaporated to give
937 mg of an oily residue. Part of the crude extract (200 mg)
was charged on PTLC (Silica Gel 60 F254 plates, 0.25 mm thick,
Merck) and developed in CHCl3-MeOH ) 25:1. The bands at
Rf 0.68 (1.4 mg), 0.35 (38.8 mg), and 0.07 (56.2 mg) were
collected. The constituents were rechromatographed on TLC
plates in toluene-acetone ) 4:1, along with authentic 3 and
4. The eluate from the top band (Rf 0.68) yielded a single
product (1.4 mg), indistinguishable from 4. The central band
yielded 35.6 mg of 3 and a small amount of 2. The bottom band
gave 45.7 mg of 1 and 2.4 mg of 2.
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. 1H and 13C NMR
spectra were recorded in CDCl3 on J EOL EX270 and Bruker
AMX500 spectrometers, respectively. 2D NMR (1H-1H COSY,
HMQC, HMBC, and NOESY) was performed on a Bruker
AMX500. The chemical shifts are relative to TMS (1H) and
the solvent peak (δ ) 77.0 ppm; 13C). EIMS and HREIMS
spectra were recorded on a J EOL DX 500 mass spectrometer
and the FDMS on a J EOL J MS-SX102A. The UV and IR
spectra were recorded on Hitachi model U-3210 and Perkin-
Elmer System 2000 FT-IR spectrophotometers, respectively.
Melting points were determined on a Yanako MP-30 micro-
melting point apparatus and are uncorrected. Optical rotations
were recorded on a J ASCO DIP-370 digital polarimeter.
En ok ip od in C (1): colorless oil; [R]24D - 9.4° (c 1.0, MeOH);
UV (MeOH) λmax (log ꢀ) 209 (4.24) and 299 (3.63) nm; IR νmax
(NaCl cell) 3384, 2947, 2362, 1507, 1457, 1417, 1308, 1174,
and 1066 cm-1; 1H NMR (CDCl3, 270 MHz) δ 6.55 (1H, s, H-5),
6.51 (1H, s, H-2), 4.35 (1H, s, 4-OH), 3.89 (1H, d, J ) 8.1 Hz,
H-8), 2.79 (1H, s, 10-OH), 2.26 (1H, dd, J ) 8.1, 15.3 Hz, H-9b),
2.22 (1H, dd, J ) 2.9, 15.3 Hz, H-9a), 2.17 (3H, s, H-15), 1.89
(1H, br, J ) 2.9 Hz, 8-OH), 1.29 (3H, s, H-14), 1.25 (3H, s,
H-12), 0.74 (3H, s, H-13); 13C NMR (CDCl3, 125 MHz) δ 147.8
(s, C-4), 145.8 (s, C-1), 128.5 (s, C-6), 123.4 (s, C-3), 117.2 (s,
C-2), 111.1 (s, C-5), 108.7 (s, C-10), 77.2 (s, C-8), 51.7 (s, C-7),
46.5 (s, C-9), 42.6 (s, C-11), 19.7 (s, C-12), 16.7 (s, C-13), 15.5

934 J ournal of Natural Products, 2001, Vol. 64, No. 7
Ishikawa et al.
(s, C-15), 11.4 (s, C-14); EIMS m/z 264 [M]+ (70), 246 [M -
H2O]+ (100), 231 (67), 205 (63), 203 (24), 177 (20), 176 (38),
175 (50), 161 (20), 151 (43); FDMS m/z 265 [M + 1]+ (17), 264
[M]+ (100); HREIMS m/z 264.1403 (calcd for C15H20O4,
264.1356).
encouragement during the course of this work. Thanks are also
due to Mr. Kenji Watanabe and Dr. Eri Fukushi (GC-MS &
NMR Laboratory) and Mr. Hiroshi Ishimoto and Mr. Hiroki
Fukui (Laboratory of Ecological Chemistry), in our Graduate
School, for recording the EI- and FDMS and 2D NMR spectra.
Financial support (to S.T. and Y.F.) from CREST, J apan
Science and Technology Corporation, and a Scholarship to
N.K.I. from the Ministry of Education, Science, Sports and
Culture of J apan, are gratefully acknowledged.
En ok ip od in D (2): yellow powder; mp 116.0-117.0 °C;
[R]24 +130° (c 0.1, MeOH); UV (MeOH) λmax (log ꢀ) 254 (3.83)
D
nm; IR νmax (NaCl cell) 3446, 2970, 2340, 1733, 1690, 1653,
1457, 1375, and 1250 cm-1; 1H NMR (CDCl3, 270 MHz) δ 6.86
(1H, s, H-5), 6.59 (1H, d, J ) 1.6 Hz, H-2), 5.04 (1H, dd, J )
8.6, 9.6 Hz, H-8), 2.83 (1H, dd, J ) 8.6, 19.0 Hz, H-9a), 2.43
(1H, dd, J ) 9.6, 19.0 Hz, H-9b), 2.05 (3H, d, J ) 1.6 Hz, H-15),
1.28 (3H, s, H-14), 1.24 (3H, s, H-12), 0.83 (3H, s, H-13); 13C
NMR (CDCl3, 125 MHz) δ 216.0 (s, C-10), 188.7 (s, C-1), 187.5
(s, C-4), 150.6 (s, C-6), 144.5 (s, C-3), 135.8 (s, C-5), 135.2 (s,
C-2), 69.2 (s, C-8), 55.4 (s, C-11), 53.3 (s, C-7), 41.7 (s, C-9),
22.8 (s, C-13), 19.8 (s, C-12), 17.0 (s, C-14), 15.0 (s, C-15); EIMS
m/z: 262 [M]+ (6.6), 244 [M - H2O]+ (20), 202 (20), 192 (78),
191(92), 190 (36), 178 (33), 177 (60), 176 (22), 175 (100); FDMS
m/z 264 [M + 2]+ (63), 263 [M + 1]+ (60), 262 [M]+ (100);
HREIMS m/z 262.1223 (calcd for C15H18O4, 262.1200).
Su p p or tin g In for m a tion Ava ila ble: This material is available
free of charge via the Internet at http://pubs.acs.org.
Refer en ces a n d Notes
(1) Bax, R.; Mullan, N.; Verhoef, J . Int. J . Antimicrob. Ag. 2000, 16, 51-
59.
(2) Miles, P. G.; Chang, S. T. Mushroom Biology: Concise Basics and
Current Developments; World Scientific: Singapore, 1997; Part III,
Chapter 6, pp 103-117.
(3) Zhang, H.; Gong, F.; Feng, Y.; Zhang, C. Inter. J . Med. Mushrooms
1999, 1, 89-92.
(4) Breene, W. M. J . Food Prot. 1990, 53, 883-894.
(5) Wasser, S. P.; Weis A. L. Crit. Rev. Immunol. 1999, 19, 65-96.
(6) Wang, H.; Ng, T. B.; Ooi, V. E. C. Mycol. Res. 1998, 102, 897-906.
(7) Yaoita, Y.; Amemiya, K.; Ohnuma, H.; Furumura, K.; Masaki, A.;
Matsuki, T.; Kikuchi, M. Chem. Pharm. Bull. 1998, 46, 944-950.
(8) Hirai, Y.; Ikeda, M.; Murayama, T.; Ohata, T. Biosci. Biotechnol.
Biochem. 1998, 62, 1364-1368.
1,4-Di-O-m eth ylen ok ip od in C (7). The alcohol 1 (12.3 mg,
47.7 µmol), anhydrous K2CO3 (40.8 mg, 286.2 µmol), 18-
crown-6 (1.3 mg, 4.9 µmol), and dimethyl sulfate (18.1 µL,
190.8 µmol) in CH3CN (0.5 mL) were stirred at room temper-
ature for 4 h.18 The reaction mixture was directly applied to
PTLC (CHCl3-MeOH ) 20:1) to give 1,4-di-O-methylenoki-
podin C (7) (Rf 0.53, 5.2 mg, 42% yield); EIMS m/z 292 [M]+
(35), 206 (30), 179 (100); HREIMS m/z 292.1667 (calcd for
(9) Ishikawa, N. K.; Yamaji, K.; Tahara, S.; Fukushi, Y.; Takahashi, K.
Phytochemistry 2000, 54, 777-782.
C
17H24O4, 292.1668).
(10) Fukushi, Y.; Yajima, C.; Mizutani, J . Tetrahedron Lett. 1994, 35, 599-
602.
(a S)- a n d (a R)-MNCB Ester of 1,4-Di-O-m eth ylen ok i-
(11) Honda, T.; Kimura, N.; Tsubuki, M. Tetrahedron: Asymmetry 1993,
4, 21-24.
(12) Chavan, S. P.; Ravindranathan, T.; Patil, S. S.; Dhondge, V. D.;
Dantale, S. W. Tetrahedron Lett. 1996, 37, 2628-2630.
(13) Cossy, J .; Gille, B.; BouzBouz, S.; Bellosta, V. Tetrahedron Lett. 1997,
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(14) Kulkarni, M. G.; Pendharkar, D. S. Tetrahedron 1997, 53, 3167-
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(15) Pal, A.; Gupta, P. D.; Roy, A.; Mukherjee, D. Tetrahedron Lett. 1999,
40, 4733-4734.
p od in C (8 a n d 9). To a solution of 7 (2.6 mg, 13.5 µmol),
(aS)-MNCB (4.9 mg, 14.1 µmol), and 4-pyrrolidinopyridine
(0.13 mg, 0.8 µmol) in CHCl3 (0.5 mL) was added dicyclohexy-
lcarbodiimide (DCC) (2.4 mg, 11.6 µmol), and the solution was
stirred at room temperature for 12 h.19 The reaction mixture
was directly applied to PTLC (hexanes-EtOAc ) 3:1) to give
8 (Rf 0.29, 1.7 mg, 65% yield). Under essentially the same
conditions, compound 7 was esterified with (aR)-MNCB to give
9 (Rf 0.40, 2.0 mg, 91% yield). EIMS m/z: 8, 622 [M+2]+ (8),
620 [M]+ (11), 348 (46), 346 [M - C17H22O3]+ (70), 274 (85),
259 (100); 9. 622 [M + 2]+ (24), 620 [M]+ (33), 348 (12), 346
[M - C17H22O3]+ (19), 329 (20), 275 (22), 274 (100), 259 (50).
(16) Parker, W.; Ramage, R.; Raphael, R. A. J . Chem. Soc. 1962, 1558-
1563.
(17) Homans, A. L.; Fuchs, A. J . Chromatogr. 1970, 51, 327-329.
(18) Hatsuno, S.; Ito, N.; Sugasawa, S. Proc. J apan Acad. 1979, 55 Ser.
B, 165-167.
(19) Hassner, A.; Alexanian, V. Tetrahedron Lett. 1978, 4475-4478.
Ack n ow led gm en t. The authors are grateful to Dr. Kiyoshi
Miura, Dr. Takashi Yajima, and Dr. Masato Shibuya for
NP000593R