Novel cyclic peptide, epichlicin, from the endophytic fungus, Epichloe typhina

Article abstract of DOI:10.1271/bbb.60700

The novel cyclic peptide, epichlicin, was isolated from Epichloe typhina, an endophytic fungus of the timothy plant (Phleum pretense L.). Its structure was determined by NMR studies and by mass spectrometry. Enantiomers of 3-amino tetradecanoic acid, a constituent amino acid of epichlicin, were synthesized as authentic standards. The stereochemistry of each amino acid was elucidated through a combination of the advanced Marfey method and chemical manipulation. Epichlicin showed inhibitory activity toward the spore germination of Cladosporium phlei, a pathogenic fungus of the timothy plant at an IC ₅₀ value of 22 nM.

Full text of DOI:10.1271/bbb.60700

Biosci. Biotechnol. Biochem., 71 (6), 1470–1475, 2007
Novel Cyclic Peptide, Epichlicin, from the Endophytic Fungus,
Epichloe typhina
Yoshiya SETO,1 Kosaku TAKAHASHI,1 Hideyuki MATSUURA,1 Yasunori KOGAMI,1
y
1;
Hiroshi YADA,2 Teruhiko YOSHIHARA,1 and Kensuke NABETA
1Laboratory of Bioorganic Chemistry, Division of Applied Bioscience, Graduate School of Agriculture,
Hokkaido University, Kita-9 Nishi-9 Kitaku, Sapporo 060-8589, Japan
2National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan
Received December 15, 2006; Accepted February 27, 2007; Online Publication, June 7, 2007
[doi:10.1271/bbb.60700]
The novel cyclic peptide, epichlicin, was isolated from
Epichloe typhina, an endophytic fungus of the timothy
plant (Phleum pretense L.). Its structure was determined
by NMR studies and by mass spectrometry. Enantiom-
ers of 3-amino tetradecanoic acid, a constituent amino
acid of epichlicin, were synthesized as authentic stand-
ards. The stereochemistry of each amino acid was
elucidated through a combination of the advanced
Marfey method and chemical manipulation. Epichlicin
showed inhibitory activity toward the spore germination
of Cladosporium phlei, a pathogenic fungus of the
timothy plant at an IC50 value of 22 nM.
choke, although these compounds have not been found
in the aerial parts of timothy plants.4–6)
In order to explain this beneficial mutual relationship
between the fungus and the host plant, we examined
the metabolites of E. typhina, since a culture broth of
E. typhina has been found to inhibit the spore germina-
tion in C. phlei.3) We therefore attempted to isolate the
germination inhibitor from a culture broth of E. typhina
to clarify the mechanism for the disease resistance in-
duced by infection by the endophyte at the level of
antifungal substances produced by the endophyte.
We report here the isolation and structural elucidation
of the novel cyclic peptide, epichlicin (1), from a culture
broth of E. typhina, and its biological activity as a spore
germination inhibitor.
Key words: endophyte; Epichloe typhina; Cladosporium
phlei; germination inhibitor; cyclic peptide
Endophytes are microorganisms which infect and live
symbiotically with host plants, and have been shown
with a broad spectrum of host plants to induce strong
resistance to some types of stress during their symbiosis.
For instance, infection of barley (Hordeum vulgare L.)
by the endophytic fungus, Piriformospora indica, in-
duces resistance to fungal diseases caused by the necro-
trophic fungus, Fusarium culmorum, and the biotrophic
fungus, Blumeria graminis, and tolerance to salt stress,
resulting in an overall increase in grain yield.1)
In the case of timothy (Phleum pretense L.), infec-
tion by the choke disease endophytic fungus, Epichloe
thyphina (Pers. ex Fr.) Tul., whose immature form is
Acremonium typhinum,2) induces resistance to leaf spot
disease caused by the pathogen, Cladosporium phlei
(Gregory) de Vries.3) A solution containing spores of
C. phlei sprayed on to being timothy plants infected
with E. typhina resulted in an aberration, a shortened
germ tube length being identified in spore germination.3)
Many antifungal compounds have been isolated from the
Material and Methods
General. FD-MS, FAB-MS and ESI-MS data were
recorded with a JMS-SX102-A mass spectrometer
(Jeol), and FAB-MS-MS data were recorded with a
JMS-700TZ mass spectrometer (Jeol). NMR spectra
were recorded with a JNM-EX 270 FT-NMR system (1H
at 270 MHz, Jeol) and an AM-500 NMR system (1H at
500 MHz and 13C at 125 MHz, Bruker). IR spectra were
recorded with a 270-30 infrared spectrophotometer
(Hitachi), and specific rotation values were determined
with a DIP360 polarimeter (Jasco). Column chromatog-
raphy was conducted with silica gel (Kanto Chemical),
DIAION HP-20 (Mitsubishi Chemical), Sephadex LH-
20 (Amersham Pharmacia Biotech), Develosil C30 (250
mm ꢀ 4.6 mm i.d., Nomura Chemical) and TSK gel
ODS-80 Ts (150 mm ꢀ 4.6 mm i.d., Tosoh).
Incubation of Epichloe typhina. E. typhina was grown
in 500-ml Erlenmeyer flasks which contained 200 ml of
y
To whom correspondence should be addressed. Tel/Fax: +81-11-706-2505; E-mail: knabeta@chem.agr.hokudai.ac.jp
Abbreviations: TFA, trifluoro acetic acid; HPLC, high-performance liquid chromatography; FDLA, 1-fluoro-2,4-dinitorophenyl-5-L-leucinamide;
RT, room temperature

Novel Cyclic Peptide, Epichlicin
1471
Table 1. 1H- and 13C-NMR Spectra for Epichlicin (1) in CD3OD
a potato dextrose medium (200 g/l of potato, 10 g/l of
peptone, 10 g/l of malt extract and 10 g/l of yeast ex-
tract). The fungus was grown in the dark for 50 days at
25 ꢁC.
a
b
ꢂH
ꢂC
Pro
ꢀ
ꢃ
4.25 (1H, t, 7.2 Hz)
2.26 (1H, m)
1.90 (1H, m)
2.15 (1H, m)
2.00 (1H, m)
3.94 (1H, m)
3.8 (1H, m)
63.2
30.2
ꢃ
Germination inhibitory assay. C. phlei was incubated
on a V-8 agar medium (V-8 juice, 200 ml/l; CaCO3, 3
g/l; agar, 20 g/l) for 2–4 weeks at 25 ꢁC. About 1 cm2 of
fungul mycelium was scraped off from the slant and
suspended in 2 ml of water. The suspension was filtered
through gauze, and the filtrate was used for a germina-
tion inhibitory assay. Five ml of the spore solution and
20 ml of each fraction (control; water) were mixed and
pipetted on to a microscope slide. Each slide was put
into a Petri dish and incubated at 25 ꢁC for 12 h in the
dark. After incubation, the percentage of germinated
spores was calculated by comparing with the control.
ꢄ
26.3
49.8
ꢄ
ꢂ
ꢂ
C=O
175.2
52.5
Asn-1
ꢀ
ꢃ
4.62 (1H, m)
2.75 (1H, dd, 7.9. 15.0 Hz)
2.55 (1H, m)
36.7
ꢃ
C=O
C=Od
173.1
174.6
57.6
Ser
ꢀ
4.39 (1H, dd, 3.8, 4.5 Hz)
4.02 (1H, dd, 4.5, 12.0 Hz)
3.78 (1H, dd, 3.8, 12.0 Hz)
ꢃ
62.8
ꢃ
C=O
172.7
51.7
Gln
ꢀ
4.68 (1H, dd, 4.5, 10.0 Hz)
2.15 (1H, m)
2.00 (1H, m)
ꢃ
Extraction and isolation. The isolation of the active
compound was monitored with a germination inhibitory
assay. The culture broth of E. typhina (1.7-liter) was
partitioned with ethyl acetate (1.7-liter ꢀ 3). The water
layer was subjected to DIAION HP-20 column chroma-
tography (2-liter), the column being successively eluted
with H2O, MeOH:H2O = 1:1 and MeOH. The MeOH
eluent was concentrated in vacuo (560 mg), and then
dissolved in MeOH. The soluble portion (200 mg) was
subjected to Sephadex LH-20 column chromatography
(70 g), using MeOH as the eluent. The active fraction
(33 mg) was purified by HPLC (Develosil C30 column;
CH3CN:H2O = 1:1; flow rate, 0.5 ml/min; A220) to give
27.7
ꢃ
ꢄ
C=O
C=Od
2.32 (2H, t, 6.2 Hz)
31.8
172.8
177.7
51.7
Asn-2
Tyr
ꢀ
4.53 (1H, dd, 4.5, 7.0 Hz)
2.93 (2H, m)
ꢃ
C=O
C=Od
36.4
173.2
175.2
57.6
ꢀ
ꢃ
ꢃ
4.32 (1H, dd, 4.0, 9.0 Hz)
3.10 (1H, dd, 4.0, 14 Hz)
2.91 (1H, m)
36.7
Bz-o 7.06 (2H, d, 8.4 Hz)
Bz-m 6.72 (2H, d, 8.4 Hz)
131.2
116.2
128.9
157.3
174.2
52.5
Bz-I
Bz-p
C=O
25
3 mg of compound 1 (tR ¼ 9:3 min, ½ꢀꢂD +5.6ꢁ, c 0.33,
Asn-3
ꢀ
4.59 (1H, m)
2.68 (1H, m)
2.50 (1H, m)
MeOH). Epichlicin (1): IR ꢁmax (nujol): 3600–3300
(br.), 3300–3000 (br.), 1680 cmꢃ1. FAB-HR-MS m=z
(½M ꢃ Hꢂꢃ): calcd. for C48H73N12O14, 1041.5369;
ꢃ
37.8
ꢃ
C=O
C=Od
174.6
174.7
43.5
1
found, 1041.5338. H- and 13C-NMR spectral data are
ꢃ-AAc
ꢀ
ꢀ
ꢃ
ꢄ
ꢄ
2.40 (1H, d, 15.5 Hz)
2.55 (1H, dd, 10.8, 15.5 Hz)
4.18 (1H, m)
shown in Table 1.
48.1
36.3
Synthesis of (R) and (S)-3-amino tetradecanoic acids.
Preparation of (E)-2-tetradecanoate (3) from dodecyl-
aldehyde (2). Dodecylaldehyde (2, 1.4 g, 6.5 mmol) and
(tert-butoxycarbonylmethylene)triphenylphosphorane (2.8
g, 6.5 mmol) in CH2Cl2 (5 ml) were stirred in an argon
atmosphere at room temperature for 12 h. After puriy-
ing by silica gel column chromatography (n-hexane:
Et2O = 19:1), tert-butyl (E)-2-tetradecenoate (3, 1.3 g,
4.6 mmol) was obtained in a 64% yield (E:Z = 96:4).
FD-HR-MS m=z (Mþ): calcd. for C18H34O2, 282.2558;
found, 282.2538. 1H-NMR (CDCl3) ꢂ: 6.83 (1H, dt,
J ¼ 7:0, 15.4 Hz), 5.71 (1H, d, J ¼ 15:4 Hz), 2.14 (2H,
dt, J ¼ 6:5, 7.0 Hz), 1.46 (9H, s), 1.40 (2H, m), 1.24
(16H, s), 0.86 (3H, t, J ¼ 6:5 Hz). IR ꢁmax (film): 2928,
1.59 (1H, m)
1.47 (1H, m)
9CH2 1.23–1.35 (18H, m)
0.89 (3H, t, 6.9 Hz)
33.1, 30.6, 27.0, 23.7e
14.4
174.5
CH3
C=O
a 1H-NMR at 500 MHz referenced to CD3OD (ꢂ 3.30)
b 13C-NMR at 125 MHz referenced to CD3OD (ꢂ 49.0)
cꢃ-AA, ꢃ-amino acid
dC=O, ꢄC=O of Asn residue and ꢂC=O of Gln residue
eSome signals overlapped.
1
226.1932; found, 282.1922, H-NMR (CDCl3) ꢂ: 7.07
(1H, dt, J ¼ 6:8, 15.7 Hz), 5.80 (1H, d, J ¼ 15:7 Hz),
2.21 (2H, dt, J ¼ 6:8, 7.0 Hz), 1.45 (2H, t, 6.5 Hz), 1.25
(16H, s), 0.86 (3H, t, 6.5 Hz). IR ꢁmax (film): 3300–2500
1872, 1720, 1040 cmꢃ1
.
Deprotection of compound 3. Compound 3 (1.3 g,
4.6 mmol) was dissolved in 3 ml of TFA and the solution
stirred for 12 h at room temperature. The reaction mix-
ture was evaporated to give tetradecenoate (4, 1.1 g) in a
99% yield. FD-HR-MS m=z (Mþ): calcd. for C14H26O2,
(br.), 2956, 1696, 986 cmꢃ1
.
Synthesis of compounds 5a and 5b. (S)-ꢀ-methyl-
benzylamine (270 mg, 2.2 mmol) was added to the so-
lution of 4 (100 mg, 0.44 mmol) in 10 ml of pyridine,
and the mixture was refluxed for 60 h. The reaction

1472
Y. SETO et al.
mixture was successively washed with 1 M HCl and
water, and the organic layer was then concentrated in
vacuo and purified by silica gel column chromatography
(MeOH:CHCl3 = 3:17). Two diastereomers, 5a (20 mg,
0.057 mmol, 13%, =99% d.e.) and 5b (12 mg, 0.036
mmol, 8%, =99% d.e.) were obtained. (3S, 10S)-3-(1-
Phenyl ethylamino)tetradecanoic acid (5a) was charac-
terized as follows. EI-HR-MS m=z (Mþ): calcd. for
aqueous TFA for 45 min at a flow rate of 1.0 ml/min.
The UV absorbance was recorded at a wavelength of
340 nm. The retention times (min) of the FDLA de-
rivatives of the hydrolysate of compound 1 were as fol-
lows (standard figure in brackets): L-Ser; 19.4 (19.3); L-
Asp, 20.4 (20.3); L-Glu, 22.8 (22.8); L-Pro, 27.7 (27.8);
D-Tyr, 37.2 (37.1). The ꢃ-amino acid was analyzed by
using another solvent system (MeOH:0.1% aqueous
TFA = 9:1, 1.0 ml/min). The retention times of FDLA
derivatives of the standards of the (S)- and (R)-ꢃ-amino
acids were 5.5 and 9.7 min, respectively, and that of the
hydrolysate of compound 1 was 9.5 min.
25
C22H37NO2, 347.2824; found, 347.2811. ½ꢀꢂD +16.3ꢁ (c
1
0.16, MeOH), H-NMR (CDCl3) ꢂ: 7.50–7.25 (5H, m),
4.18 (H-ꢀ, 1H, q, J ¼ 6:8 Hz), 2.87 (1H, br.s), 2.58 (H-
ꢃ, 1H, dd, J ¼ 4:1, 16.2 Hz) 2.30, (H-ꢃ, 1H, dd, J ¼ 4:1,
16.2 Hz), 1.66 (3H, d, J ¼ 6:8 Hz), 1.40–1.10 (20H, m),
0.85 (3H, t, J ¼ 6:8 Hz). IR (film): 3300–2500, 2928,
1720 cmꢃ1. (3R, 10S)-3-(1-phenyl ethylamino)tetradeca-
noic acid (5b) was characterized as follows. EI-HR-MS
m=z (Mþ): calcd. for C22H37NO2, 347.2824; found,
Results and Discussion
Isolation and planar structure of compound 1
Isolation of the germination inhibitory compound was
monitored by a germination inhibitor assay. The culture
broth of E. typhina was purified by DIAION HP-20,
Sephadex LH-20 and reverse-phase HPLC with a C30
column to give compound 1.
25
347.2808. ½ꢀꢂD ꢃ25:4ꢁ (c 0.13, MeOH). 1H-NMR
(CDCl3) ꢂ: 7.42–7.29 (5H, m), 4.31 (1H, q, J ¼ 6:8 Hz),
2.86 (1H, br. s), 2.47 (2H, br. s), 1.71 (3H, d, J ¼ 6:8
Hz), 1.20–1.10 (20H, m), 0.86 (3H, t, J ¼ 6:8 Hz). IR
(film): 3300–2500 (br.), 2928, 1720 cmꢃ1
.
Compound 1 was isolated as a colorless powder. The
molecular formula of compound 1 was established as
C48H74N12O14 by the FAB-HR-MS data. The 13C-NMR
data together with the molecular formula showed that
compound 1 had 12 amide-type carbonyls. The 1H-
NMR data showed many peaks around ꢂ2.00–4.00. The
1H- and 13C-NMR spectra of compound 1 showed close
similarity to those of mixirin A, a cyclic peptide, which
has been isolated from marine Bacillus sp.7) The COSY
data showed the presence of one Ser, one Pro, one Gln,
one Tyr, three Asn residues and a residue having a long
alkyl chain. Further analyses of COSY, HMQC and
HMBC data revealed this residue to be 3-amino tetra-
decanoic acid (ꢃ-amino acid). The presence of 17 ex-
changeable protons was shown by ESI-MS data meas-
ured after dissolution in CD3OD, showing an ½M þ
Na þ 17ꢂþ ion peak at m=z 1082, supporting the pre-
dicted amino acid composition. As a result, the con-
stituent amino acids of compound 1 were determined to
be the same as those of mixirin A.7)
The HMBC spectrum showed correlation between ꢀ-
hydrogen of each amino acid and the carbonyl carbon
of the adjacent amino acid (Fig. 1). An analysis of the
fragmentation of compound 1 obtained from the FAB-
MS-MS data supported the amino acid sequence de-
termined from the HMBC spectrum (Fig. 1). Accord-
ingly, the planar structure of compound 1 was deter-
mined to be identical to that of mixirin A (Fig. 1),
although the specific rotation value differed from that of
Hydrogeneration of compounds 5a and 5b. Com-
pounds 5a (20 mg, 0.057 mmol) and 5b (12 mg, 0.036
mmol) were separately dissolved in 3 ml of acetic acid
and hydrogenated for 3 h under H2 catalyzed by Pd/C.
Each reaction mixture was filtered through Celite and
purified by silica gel column chromatography (MeOH:
CHCl3 = 2:3) to give the desired ꢃ-amino acids, 6a
(12 mg, 0.047 mmol, 83%, =99% e.e.) from 5a and 6b
(5.2 mg, 0.021 mmol, 60.1%, =99% e.e.) from 5b. (3S)-
Amino tetradecanoic acid (6a) was characterized as fol-
lows. FAB-HR-MS m=z ðM ꢃ HÞꢃ: calcd. for C14H28-
25
NO2, 242.2120; found, 242.2126. ½ꢀꢂD +16.0ꢁ (c 0.90,
H2O). 1H-NMR (CD3OD) ꢂ: 2.48 (1H, dd, J ¼ 4:1, 16.5
Hz), 2.27 (1H, dd, J ¼ 8:9, 16.5 Hz), 1.61 (2H, m), 1.29
(20H, s), 0.89 (3H, t, J ¼ 6:8 Hz). IR (film): 3300–2500
(br.), 2924, 1720 cmꢃ1. (3R)-Amino tetradecanoic acid
(6b) was characterized as follows. FAB-HR-MS m=z
ðM ꢃ HÞꢃ: Calcd. for C14H28NO2, 242.2120; found,
25
242.2126. ½ꢀꢂD ꢃ15:3ꢁ (c 0.90, H2O). 1H-NMR
(CD3OD) ꢂ: 2.48 (1H, dd, J ¼ 4:1, 16.5 Hz), 2.27 (1H,
dd, J ¼ 8:9, 16.5 Hz), 1.61 (2H, m), 1.29 (20H, s), 0.89
(3H, t, J ¼ 6:8 Hz). IR (film) 3300–2500 (br), 2924,
1720 cmꢃ1
.
Determination of the stereochemistry of compound 1
by the advanced Marfey method. Compound 1 (500 mg)
was hydrolyzed in 1 ml of 6 M HCl at 120 ꢁC for 12 h.
The reaction product was dissolved in 50 ml of water
and 20 ml of 1 M NaHCO3, and reacted with 100 ml of
the advanced Marfey reagent, FDLA (1 mg/100 ml in
acetone), at 45 ꢁC for 1 h. The reaction was stopped by
adding 20 ml of 1 M HCl, and the mixture diluted with
810 ml of acetonitrile. To determine the stereochemistry
of ꢀ-amino acid, the sample was analyzed by reverse-
phase HPLC in a C18 column (Tosoh TSK-gel 80Ts),
using a linear gradient of 15–45% CH3CN in 0.1%
25
25
mixirin A (compound 1, ½ꢀꢂD +5.6ꢁ; mixirin A, ½ꢀꢂD
ꢃ18:2ꢁ). We therefore expected that epichlicin was a
diastereoisomer of mixirin A.
Synthesis of (R)- and (S)-3-amino tetradecanoic acid
Compound 1 contained a ꢃ-amino acid as a constit-
uent. To determine the stereochemistry of this residue,
(S)- and (R)-3-amino tetradecanoic acids were synthe-

Novel Cyclic Peptide, Epichlicin
1473
O
O
HN
(CH2)10CH3
O
HO
NH
HN
H2N
O
O
O
O
NH
NH2
N
O
O
O
NH
H2N
NH
OH
O
HN
O
→
key HMBC correlations
NH2
Asn
541
β−AA
Asn
818
Tyr
704
Gln
427
Ser
299
Asn
212
Pro
Fig. 1. Absolute Structure of Epichlicin (1) and the Fragmentation Pattern of Compound 1 Obtained from FAB MS-MS Data (positive ion mode).
ꢃ-AA, ꢃ-amino acid
Ph
Me
NH
(c)
(a) (b)
64%
CH3(CH2)10CHO
COOH
COOH
21%
CH3(CH2)10
CH3(CH2)10
2
4
5
Me
Ph
NH
NH2
(d)
5
COOH
COOH
COOH
83%
CH3(CH2)10
CH3(CH2)10
5a
6a
Me
Ph
NH
(d)
NH2
COOH
61%
CH3(CH2)10
CH3(CH2)10
5b
6b
Scheme 1. Synthesis of (S)- and (R)-3-Amino Tetradecanoic Acid.
Reagents and conditions: (a) (tert-butoxycarbonylmethylene)triphenylphosphorane, CH2Cl2, RT, 12 h; (b) TFA, RT, 12 h; (c) (S)-ꢀ-
methylbenzylamine, pyridine, reflux for 60 h; (d) Pd/C, AcOH, H2, 50–60 ꢁC, 5 h.
sized as authentic standards as shown in Scheme 1.
Starting with dodecyl aldehyde (2), an ꢀ, ꢃ-unsaturated
ester (3) was obtained by the Wittig reaction in a 69%
yield. After deprotecting the tert-butyl group in TFA,
ꢀ, ꢃ-unsaturated carboxylic acid (4) was reacted with
(S)-ꢀ-methyl benzylamine (Aldrich, enantiomeric ratio
=99:5%) to give two diastereoisomers. These were
separated by silica gel column chromatography (5a,
13%; 5b, 8%). The diastereomeric excesses of 5a and 5b
NMR signals for H-ꢀ and H-ꢃ of 5a and 5b. Each of the
diastereoisomers was separately hydrogenated in acetic
acid under an H2 atmosphere catalyzed by Pd/C to
remove the benzyl group, giving the desired compounds
(6a, 83%; 6b, 61%). The enantionmeric excesses of 6a
and 6b were both estimated to be =99% on the basis of
the diastereomeric excess of 5a and 5b (reported data,
e.e. = 93%8)). The specific rotation value of (S)-3-
amino tetradecanoic acid has been reported as +28.8ꢁ (c
0.90, H2O, 25 ꢁC),8) the specific rotations values of 6a
1
were both calculated to be 99% on the basis of the H-

1474
Y. SETO et al.
and 6b being +16.0ꢁ (c 0.90, H2O, 25 ꢁC) and ꢃ15:3ꢁ (c
0.90, H2O, 25 ꢁC), respectively. Thus, the stereochem-
istry of 6a was determined to be (S), and that of 6b to be
(R). The reported specific rotation value is thought to be
in error, and this may be responsible for the difference
between the reported and our data. Although the
enantioselective synthesis of 3-amino tetradecanoic acid
has been reported,8) the synthesis of these compounds
via diastereomers is novel to our knowledge.
100
80
60
40
20
0
Determination of the absolute stereochemistry of com-
pound 1
The stereochemistry of each amino acid in mixirin A
has been determined by Marfey’s method as D-Pro, D-
Tyr, L-Ser, L-Asn and L-Gln,7) although the stereo-
chemistry of the ꢃ-amino acid has not been shown. We
therefore determined the stereochemistry of each amino
acid, including the ꢃ-amino acid, with the advanced
Marfey method.9,10)
32
28
24
20
16
Concentration of compound 1(nM)
Fig. 2. Germination Inhibitory Activity of Epichlicin (1) against
C. phlei.
Each value is expressed as the mean ꢄ standard deviation from
three results.
Compound 1 was hydrolyzed in 6 M HCl, and then
reacted with the advanced Marfey reagent, FDLA.9,10)
After its conversion with FDLA, the sample was
analyzed by reverse-phase HPLC. The stereochemistry
of the ꢀ-amino acids was determined as L-Pro, D-Tyr, L-
Ser, L-Asn and L-Gln, respectively. Although the stereo-
chemistry of the proline of mixirin A has been reported
to be D, that of epichlicin was confirmed as L. An
analysis of the ꢃ-amino acid was made by using a
different solvent system. The retention times of the
FDLA derivatives of the synthesized (S)- and (R)-ꢃ-
amino acids were 5.5 and 9.7 min, respectively. The
peak of the FDLA derivative of the hydrolysate of
compound 1 appeared at 9.5 min, enabling the stereo-
chemistry of the ꢃ-amino acid to be determined as
having (R) configuration (6b in Scheme 1). In conclu-
sion, the total absolute stereochemistry of compound 1
was determined to be that shown in Fig. 1. The
stereochemistry of the proline of epichlicin, differing
from that of mixirin A, and the stereochemistry of the ꢃ-
amino acid, which had not been determined in mixirin
A, was found to be (R). We named compound 1 as
epichlicin.
Table 2. Germ Tube Elongation Inhibitory Activity of Epichlicin
against C. phlei
Five ml of the spore solution was pipetted on to a microspore slide
and incubated for 12 h. Then, 20 ml of a solution of epichlicin was
added (control, water) and incubated for more 12 h. After incubating
the length of the germ tube was measured. Each value is the aver-
age ꢄ SD (n ¼ 10).
Concentration
of epichlicin (nM)
32
20
Control (0)
Length of germ
tube (mm)
0.16 (ꢄ0:031) 0.20 (ꢄ0:026) 0.25 (ꢄ0:037)
obtaining resistance against some types of stress through
infection by the endophyte. Our results indicate the
possibility that some organic compounds produced by
the endophyte play an important role in this induced
resistance. Epichlicin may thus be useful in defending
plants from disease stress.
Acknowledgments
We thank Mr. K. Watanabe and Dr. E. Fukushi in
our faculty for measuring the MS data, and Mrs. S.
Oka in Center for Instrumental Analysis at Hokkaido
University for measuring the MS-MS data. We also
thank Dr. T. Shimanuki for kindly presenting E. typhina
and C. phlei.
Germination inhibitory activity of Epichlicin against
C. phlei
Epichlicin (1) showed germination inhibitory activity
against C. phlei at an IC50 value of 22 nM (Fig. 2). This
test was conducted three times and the reproducibility
was confirmed. Epichlicin also inhibited germ tube
elongation of C. phlei at 20 and 32 nM (Table 2).
Under normal conditions, E. typhina produces epi-
chlicin at around 2 mM level in the rapid-growth stage.
The inhibitory effect of epichlicin against E. typhina is
therefore much less than that against C. phlei.
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