Termitomycamides A to E, fatty acid amides isolated from the mushroom termitomyces titanicus, suppress endoplasmic reticulum stress

Article abstract of DOI:10.1021/ol102186p

Five fatty acid amides, termitomycamides A to E (1 to 5), were isolated from the giant edible mushroom Termitomyces titanicus. The structures of 1-5 were determined by the interpretation of spectral data and/or synthesis. Compounds 2 and 5 showed protective activity against endoplasmic reticulum stress-dependent cell death.

Full text of DOI:10.1021/ol102186p

ORGANIC
LETTERS
2010
Vol. 12, No. 21
5012-5015
Termitomycamides A to E, Fatty Acid
Amides Isolated from the Mushroom
Termitomyces titanicus, Suppress
Endoplasmic Reticulum Stress
Jae-Hoon Choi,† Kohei Maeda,† Kaoru Nagai,‡ Etsuko Harada,§ Mitsuo Kawade,§
Hirofumi Hirai,† and Hirokazu Kawagishi*,†,¶
Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka
UniVersity, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan, Department of
Epigenetic Medicine, Interdisciplinary Graduate School of Medicine and Engineering,
UniVersity of Yamanashi, Yamanashi 409-3898, Japan, and Iwadekingaku Laboratory,
Tsu 514-0012, Japan, Graduate School of Science and Technology, Shizuoka
UniVersity, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan
achkawa@ipc.shizuoka.ac.jp
Received September 13, 2010
ABSTRACT
Five fatty acid amides, termitomycamides A to E (1 to 5), were isolated from the giant edible mushroom Termitomyces titanicus. The structures
of 1-5 were determined by the interpretation of spectral data and/or synthesis. Compounds 2 and 5 showed protective activity against
endoplasmic reticulum stress-dependent cell death.
Endoplasmic reticulum (ER) stress is caused by disturbances
in the structure and function of the ER with the accumulation
of misfolded proteins and alterations in the calcium homeo-
stasis. The ER response is characterized by changes in
specific proteins, causing translational attenuation, induction
of ER chaperones, and degradation of misfolded proteins.
In the case of prolonged or aggravated ER stress, cellular
signals leading to cell death are activated. ER stress has been
suggested to be involved in some human neuronal diseases,
such as Parkinson’s, Alzheimer’s, and prion disease, as well
as other disorders.1-4 Therefore, the protective activity
against ER stress is an important target for the cure or
† Department of Applied Biological Chemistry, Shizuoka University.
‡ University of Yamanashi.
§ Iwadekingaku Laboratory.
¶ Graduate School of Science and Technology, Shizuoka University.
10.1021/ol102186p  2010 American Chemical Society
Published on Web 10/11/2010

prevention of these diseases and the demand for new
protective substances prompted us to screen the protective
activity of the mushroom extracts. We previously reported
the protective function of dilinoleoylphosphatidylethanol-
amine that was isolated from the mushroom Hericium
erinaceum.5 Moreover, we have found new ER stress
protective compounds from the mushroom H. erinaceum6,7
and Mycoleptodonoides aitchisonii.8 During the screening
for ER stress protecting effects of the extracts of various
mushrooms, we found relatively strong activity in the extract
of the mushroom Termitomyces titanicus and tried to isolate
the active principles from the mushroom. T. titanicus with a
cap diameter of up to 1 m is the largest edible mushroom in
the world according to Guinness Book of Records. It is
typified by symbiotic life together with termites. Termites
cultivate the mycelia in their nest and fruiting bodies can be
seen arising on or near the mounds.9 Here, we describe the
isolation, structural elucidation, and biological activity of
termitomycamides A to E (1 to 5).
column chromatography, followed by HPLC to afford
compounds 1, 2, and 5 (0.5, 3.4, and 1.0 mg, respectively).
On the other hand, the EtOH soluble fraction was partitioned
between 1-BuOH and H2O. The BuOH soluble part was
fractionated by gel permeation chromatography, silica gel
flash column chromatography, and HPLC to give compounds
3 (3.3 mg) and 4 (1.7 mg).
Termitomycamide A (1) was isolated as a colorless oil.
Its molecular formula was determined as C26H39NO3
by HRESIMS [m/z 436.2824 [M + Na]+ (calcd for
C26H39NaNO3, 436.2828)]. The complete assignment of all
the protons and carbons was accomplished by DEPT,
HMQC, COSY, and HMBC experiments as shown in Table
1. The DEPT experiment indicated the presence of a methyl,
Table 1. NMR Spectroscopic Data for Termitomycamide A (1)a
position δH (mult, J in Hz)
δC
HMBC
1
2
3
174.2
36.6
25.6
2.32 (t, 7.8)
1.67 (m)
C-1
C-2
4-7, 15 1.23-1.37 (m)
29.1, 29.2, 29.2
29.3, 29.6
8, 14
9, 13
10, 12
11
2.03 (m), 2.03 (m) 27.2, 27.2
C-9, 10, 12, 13, 16
C-8, 11, 14
C-8, 11, 14
5.36 (m), 5.36 (m) 130.2, 130.5
5.33 (m), 5.33 (m) 127.9, 128.0
2.75 (dd, 6.8, 6.4) 25.6
C-9, 10, 12, 13
16
17
18
1′
1.28 (m)
1.28 (m)
0.87 (t, 6.9)
4.64 (s)
31.5
22.6
14.1
46.0
C-16, 17
C-1, 2′
2′
1′′
2′′, 6′′
3′′, 5′′
4′′
192.2
126.9
130.5
116.0
161.9
7.77 (d, 8.5)
6.91 (d, 8.5)
C-2′, 1′′, 4′′
C-1′′, 4′′
a CDCl3, 500 MHz.
Powders of the dried fruiting bodies of T. titanicus (3.3
kg) were extracted with hexane, EtOAc, and EtOH, succes-
sively. Since the EtOAc and EtOH soluble fractions showed
the protective activity against ER stress-dependent cell death,
the EtOAc soluble fraction was separated using silica gel
13 methylenes, 8 methines, and 4 quaternary carbons. The
COSY and HMBC correlations are illustrated in Figure 1.
The NMR spectra suggested the presence of a linoleyl moiety
including two disubstituted double bonds [δH 5.36 (H-9, H-13,
2H), δH 5.33 (H-10, H-12, 2H); δC 127.9 (C-9), 128.0 (C-10),
130.2 (C-12), 130.5 (C-13), 174.2 (C-1)]. The 2-amino-1-(4-
hydroxyphenyl)ethanone moiety was confirmed by the COSY
correlations (H-2′′/H-3′′, H-5′′/H-6′′), HMBC correlations (H-
1′/C-2′, H-2′′/C-2′, H-2′′/C-1′′, H-2′′/C-4′′, H-3′′/C-1′′, H-3′′/
C-4′′), and chemical shifts of H-1′ (δH 4.64) and C-2′ (δC 192.2).
The connection between the amine and linoleyl moiety was
indicated by the HMBC correlation from H-1′ to the amide
carbonyl at C-1 (δC 174.2), the lower chemical shift of H-1′
(δH 4.64), and the molecular formula of 1.
Termitomycamide B (2) was purified as a colorless oil. Its
molecular formula was determined as C28H40N2O2 by HRES-
IMS [m/z 459.2990 [M + Na]+ (calcd for C28H40NaN2O2,
459.2988)]. The 1H and 13C NMR data of the position from 1
to 18, C-1′, and C-2′ of 2 were very similar to those of 1. The
indole moiety was constructed by the COSY correlations
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Org. Lett., Vol. 12, No. 21, 2010
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was determined by the COSY correlation (H-1′′/H-2′′),
HMBC correlations (H-1′′/C-2′′, H-1′′/C-3′′, H-2′′/C-1′′,
H-2′′/C-3′′), and the downfield chemical shift C-3′′ (δC
177.7). The three-methylene chain was constructed by COSY
correlations (H-1′/H-2′, H-2′/H-3′) and HMBC correlations
(H-1′/C-2′, H-1′/C-3′, H-2′/C-1′, H-2′/C-3′, H-3′/C-1′, H-3′/
C-2′). The connection between linoleyl moiety and the other
parts (the acid and the three-methylene chain) was deter-
mined by HMBC correlations (H-1′/C-1, H-3′/C-1′′, H-1′′/
C-3′) and the molecular formula of 4. As a result, the
structure of 4 was determined.
Termitomycamide E (5) was isolated as a colorless oil. Its
molecular formula was determined as C26H41NO2 by HRESIMS
[m/z 422.3034 [M + Na]+ (calcd for C26H41NaNO2, 422.3035)].
The NMR data of 5 were very similar to those of 1. Comparison
of the molecular formula and NMR data of 5 with those of 1
indicates that 5 possesses a methylene instead of a carbonyl of 1.
Although compound 5 already has been reported as an intermediate
of organic synthesis,12 this is the first reported isolation of it from
a natural source. The structure was confirmed by synthesis.
Figure 1. COSY and HMBC correlations of 1-5.
In addition, there is no report of the isolation of the
corresponding amines in 1 to 4 from nature.
(H-1′′/H-2′′, H-4′′/H-5′′, H-6′′/H-7′′), HMBC correlations
(H-1′′/C-3′′, H-1′′/C-3a′′, H-2′′/C-3′′, H-2′′/C-3a′′, H-4′′/C-3′′,
H-4′′/C-6′′, H-4′′/C-7a′′, H-5′′/C-3a′′, H-5′′/C-7′′, H-6′′/C-4′′,
H-6′′/C-7a′′, H-7′′/C-3a′′, H-7′′/C-5′′), and the molecular for-
mula of 2. The structure for 2 was confirmed by its synthesis
from linoleic acid and the corresponding amine.
The compounds were subjected to the protective activity assay
against ER stress-dependent cell death caused by tunicamycin
(TM). ER stress was induced by addition of TM into the culture
medium of Neuro2a cells in the presence or absence of each
compound. TM is an inhibitor of N-linked glycosylation and the
formation of N-glycosidic protein-carbohydrate linkages.11 It
specifically inhibits dolichol pyrophosphate-mediated glycosylation
of asparaginyl residues of glycoproteins12 and induces “ER stress”.
Compounds 2 and 5 showed significant protective activity against
TM-toxicity dose-dependently (Figure 2). On the other hand, 3
Termitomycamide C (3) was isolated as a colorless oil. Its
molecular formula was determined as C28H44N2O3 by HRES-
IMS [m/z 479.3262 [M + Na]+ (calcd for C28H44NaN2O3,
479.3250)]. The 1H and 13C NMR data of position 1 to 18 of
3 were similar to those of 1. The moiety of a 3,4-disubstituted
pyridine ring in 3 was suggested by coupling constants and
chemical shift: δH 8.72 (1H, d, J ) 6.1 Hz), δH 7.84 (1H, d, J
) 6.1 Hz), and δH 8.79 (br s) and the COSY correlations (H-
2′′/H-6′′, H-5′′/H-6′′). The 13C NMR, DEPT spectra and HMBC
correlations (H-2′′/C-3′′, H-2′′/C-4′′, H-2′′/C-6′′, H-5′′/C-6′′,
H-6′′/C-2′′, H-6′′/C-4′′, H-6′′/C-5′′) also indicated the presence
of a 3,4-disubstituted pyridine ring. The position of the methyl
group at the pyridine was elucidated by HMBC correlations
(H-2′′/C-3′′-Me, H-3′′-Me/C2′′, H-3′′-Me/C-3′′, H-3′′-Me/C-4′′).
The position of the carboxyl group in the ring was assigned by
the HMBC correlation from H5′′ to 4′′-COO. The propyl group
was determined by the COSY correlations (H-1′/H-2′, H-2′/H-
3′) and HMBC correlations (H-1′/C-2′, H-1′/C-3′, H-2′/C-
1′, H-2′/C-3′, H-3′/C-1′, H-3′/C-2′). The connection between
the propyl and the ring was determined by HMBC correlations
(H-3′/C-2′′, H-3′/C-6′′, H-2′′/C-3′, H-6′′/C-3′). The junction be-
tween the linoleyl moiety and propylpyridinium group was
determined by HMBC correlation from H1′ to C-1 (δC 176.7). As
a result, the structure of 3 was determined as shown.
Termitomycamide D (4) was purified as a colorless oil.
Its molecular formula was determined as C24H44N2O3
by HRESIMS [m/z 431.3252 [M + Na]+ (calcd for
Figure 2. Protective activity of 2 and 5 against ER stress-dependent
cell death. The cell viabilities were analyzed by MTT assay, and
the values were represented as the mean ( SEM of the relative
percentage of surviving cells compared with the untreated cells (n
) 16). (/) p < 0.05, (//) p < 0.01, Tukey-Kramer multiple
comparisons tests.
1
C24H44NaN2O3, 431.3250)]. The H and 13C NMR data of
position 1 to 18 of 4 were very similar to those of 3. The
structure elucidation using NMR was accomplished in the
same manner as for 1. The moiety of the propanoic acid
5014
Org. Lett., Vol. 12, No. 21, 2010

and 4 did not have any activity even at the concentrations up to 1
µg/mL (see Figure S1 in the Supporting Information).
5a-c) and their precursors (the three acids and the two
amines) were examined, all of them showed no significant
protective activity against TM-toxicity (data not shown). In
addition, linoleic acid also did not exhibit the protective
acitity similar to the previous report (data not shown).5 These
results indicated that the linoleyl moiety in 2 and 5 was
indispensable for the activity of the compounds.
To investigate further the structure-activity relationship
on protective activity against ER stress-dependent cell death,
analogues of 2 and 5 that have different fatty acid parts
(stearyl, oleyl, and arachidonyl) from each other were
synthesized (Scheme 1). Although all the amides (2a-c and
Acknowledgment. We thank V. K. Deo (Shizuoka
University) for valuable discussions. This work was partially
supported by a grant-in-aid for scientific research on priority
areas “Creation of Biologically Functional Molecules” (No.
17035037) from the Ministry of Education, Culture, Sports,
Science and Technology of Japan.
Scheme 1. The Structures of Compounds (2a-c and 5a-c)
Supporting Information Available: Experimental pro-
cedures including bioassay and synthesis, 1H and 13C NMR
spectra of 1-5, and protecting effect of 3 and 4. This material
is available free of charge via the Internet at http://pubs.acs.org.
OL102186P
(11) Mahoney, W. C.; Duksin, D. J. Biol. Chem. 1979, 254, 6572.
(12) Olden, K.; Pratt, R. M.; Jaworski, C.; Yamada, K. M. Proc. Natl.
Acad Sci. U.S.A. 1979, 76, 791.
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