Endoplasmic reticulum stress suppressive compounds from the edible mushroom Mycoleptodonoides aitchisonii

Article abstract of DOI:10.1021/np500075m

Two novel compounds, 1 and 7, along with six known compounds (2-6 and 8), were isolated from the edible mushroom Mycoleptodonoides aitchisonii (bunaharitake in Japanese). The structures of the new compounds were determined by the interpretation of spectro

Full text of DOI:10.1021/np500075m

Note
pubs.acs.org/jnp
Endoplasmic Reticulum Stress Suppressive Compounds from the
Edible Mushroom Mycoleptodonoides aitchisonii
†
,#
†,#
‡
§
†
Jae-Hoon Choi, Tomohiro Suzuki, Hiroshi Okumura, Keiichi Noguchi, Mitsuru Kondo,
⊥
†,‡
,†,‡,∥
Kaoru Nagai, Hirofumi Hirai, and Hirokazu Kawagishi*
†
Research Institute of Green Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan
Graduate School of Agriculture, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan
‡
§
Instrumentation Analysis Center, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan
⊥
Department of Epigenetic Medicine, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi,
Yamanashi 409-3898, Japan
∥
Graduate School of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan
*
S Supporting Information
ABSTRACT: Two novel compounds, 1 and 7, along with six
known compounds (2−6 and 8), were isolated from the edible
mushroom Mycoleptodonoides aitchisonii (bunaharitake in
Japanese). The structures of the new compounds were
determined by the interpretation of spectroscopic data.
Compounds 1−4 and 6−8 showed protective activity against
endoplasmic reticulum stress-dependent cell death.
ndoplasmic reticulum (ER) stress is caused by disturban-
E
ces in the structure and function of the ER with the
accumulation of misfolded proteins and alterations in calcium
homeostasis. 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
1
−3
diseases.
ER stress has been reported to cause not only
neurodegenerative diseases but also some other diseases, such4
as diabetes, atherosclerosis, and heart and liver disease.
Therefore, protective activity against ER stress is possibly an
important target for addressing these diseases, and the demand
for new lead compounds prompted us to screen the protective
activity of mushroom extracts. We have reported new ER stress
protective compounds from the mushrooms Hericium erina-
8
5
,6
7
ceum, Termitomyces titanicus, and Leccinum extremiorientale.
Three furanones and a phenylpentanone from the edible
mushroom Mycoleptodonoides aitchisonii have also been
reported as protective compounds. In the course of our
continuing search for ER stress protecting compounds from the
mushroom M. aitchisonii, we found the active compounds
described below.
9
soluble part. By comparison of the NMR data, mass spectra,
and specific rotation of compounds 2−6 and 8 with those
10−16
reported previously,
the six known compounds were
identified as shown. Although 5 and 8 have been
14−16
synthesized,
this is the first report of the isolation of
Fresh fruiting bodies of M. aitchisonii were extracted with
EtOH and acetone, successively. After the solutions were
combined and concentrated under reduced pressure, the
concentrate was divided into a CH Cl -soluble fraction, an
these compounds from nature.
Compound 1 was purified as a colorless oil. Its molecular
formula was determined as C H O by HRESIMS (m/z
7
10
4
2
2
EtOAc-soluble fraction, and a water-soluble fraction. Com-
pounds 1, 2, and 5 were purified from the EtOAc-soluble part,
and compounds 3, 4, and 6−8 were obtained from the CH Cl -
Received: January 27, 2014
2
2
©
XXXX American Chemical Society and
American Society of Pharmacognosy
A
dx.doi.org/10.1021/np500075m | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Note
+
1
59.0687 [M + H] ; calcd for C H O , 159.0657), indicating
2, H-4/H-5), the HMBC correlations (H-1/C-3, H-2/C-1, H-
2/C-3, H-3/C-2, H-4/C-3, H-4/C-5, H-5/C-3, H-5/C-4), and
the molecular formula. The connection between the benzene
and pentane moieties was determined by the HMBC
correlations (H-5/C-1′, H-5/C-2′, H-2′/C-5). As a result, the
planar structure of 7 was determined to be 5-phenylpentane-
7
11
4
the presence of three degrees of unsaturation in the molecule.
The structure of 1 was elucidated by interpretation of 2D NMR
spectra including COSY, HMBC, and HMQC (Figure 1). The
1
3
,3,4-triol. The absolute configuration of 7 was determined as
S, 4R by X-ray crystallography analysis on its p-bromoben-
zoate (Figure 2).
Figure 1. COSY and HMBC correlations of 1 and 7.
DEPT experiment and the molecular formula indicated the
presence of two methyls, two methines, two quaternary
carbons, and a carboxy. The complete assignment of all the
protons and carbons was accomplished as shown in Table 1.
Table 1. H and 13C NMR Data for 1 and 7
1
1
(in CD OD)
7 (in CDCl3)
3
δH, mult
(J in Hz) position
δH, mult (J
in Hz)
position
δC, type
δC, type
Figure 2. ORTEP drawing of p-bromobenzoates of 4 (left) and 7
(
right) with ellipsoids at the 50% probability level. Hydrogen atoms
1
2
3
4
5
1
2
3
4
61.1, CH2
33.1, CH2
73.8, CH
75.3, CH
38.4, CH2
3.86, m
1.81, m
3.83, m
3.83, m
2.86, dd
are shown as small spheres of arbitrary radii.
174.9, C
126.4, C
162.7, C
99.6, CH
The stereochemistry of compound 4 has not been previously
12
6.08, s
5a
reported. In this study, the absolute configuration of 4 was
determined as 3R, 4R, 1′R by X-ray crystallography analysis of
its p-bromobenzoate (Figure 2).
Compounds 1−8 were subjected to the protective-activity
assay against ER stress-dependent cell death caused by
tunicamycin (TM) or thapsigargin (TG). ER stress was
induced by the addition of TM or TG 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
(
13.7,
.8)
2
5
b
2.71, dd
13.7,
.2)
(
9
1
′
′
64.9, CH
4.75, m
1′
2′, 6′
138.2, C
2
21.8, CH3 1.42, d
7.0)
129.3, C H
7.22, d
(7.5)
2
2
2
(
3
-Me
8.8, CH3 1.89, s
3′, 5′
128.6, C H
7.30, dd
2
(
7.0, 7.5)
17
of N-glycosidic protein−carbohydrate linkages. It specifically
inhibits dolichol pyrophosphate-mediated glycosylation of
asparaginyl residues of glycoproteins and induces ER stress.
4
′
126.6, CH
7.23, t
(
7.0)
18
2
+
The furanone moiety was determined by the HMBC
correlations (H-5/C-2, H-5/C-3, H-5/C-4) and the chemical
shifts of the two quaternary carbons (δ 126.4, 162.7) and the
carboxy carbon (δ 174.9). A methyl was determined to be
connected to C-3 by the HMBC correlations (C-3−Me/C2, C-
−Me/C3, C-3−Me/C-4). The linkage between the hydrox-
yethyl group [COSY (H-1′/H-2′), HMBC (H-1′/C-2′, H-2′/
C-1′)] and the furanone ring was determined by the HMBC
correlations (H-2′/C-4, H-1′/C-5). The presence of the
hydroxy group at C-5 was suggested by the molecular formula
and the chemical shift [δ 6.08 (1H, s) and δ 99.6]. All the
data allowed us to conclude that 1 was 5-hydroxy-4-(1-
hydroxyethyl)-3-methylfuran-2(5H)-one. Its stereochemistry
remains undetermined.
Compound 7 was isolated as a colorless oil. Its molecular
formula was determined as C H O by HRESIMS. The
TG, an inhibitor of sarcoplasmic/endoplasmic reticulum Ca -
ATPase, also induces ER stress by disrupting the homeostatic
balance of the Ca2+ concentration in the ER. None of the
compounds showed protective activity against TM toxicity. On
the other hand, in the assay using TG, all the compounds
except for 5 showed protective activity at more than 50 μM.
Compounds 5 and 6 are diastereomers of each other,
suggesting that the stereochemistry played an important role
in the activity. Similar γ-lactones and a phenylpentanol having
protective activity against TM or TG toxicity have been
19
C
C
3
9
reported previously. Sodium valproate, which is known as an
ER stress protector in vivo, showed a similar protective effect to
these compounds (Figure S11, Supporting Information).
H
C
20
EXPERIMENTAL SECTION
■
1
General Experimental Procedures. H NMR spectra (one- and
two-dimensional) were recorded on a Jeol Lambda-500 spectrometer
at 500 MHz, while 13C NMR spectra were recorded on the same
instrument at 125 MHz. The HRESIMS spectra were measured on a
JMS-T100LC mass spectrometer. A Jasco grating infrared spectropho-
tometer was used to record the IR spectra, and the specific rotation
values were measured by a Jasco DIP-1000 polarimeter. HPLC
separations were performed with a Jasco Gulliver system using normal-
phase HPLC columns (Senshu Pak AQ, Senshu Scientific Co., Ltd.,
Japan; Develosil Silica-HILIC, Nomura Chemical, Japan; Cosmosil
1
1
16
3
structure of 7 was elucidated by interpretation of 2D NMR
spectra including COSY, HMBC, and HMQC. The complete
assignment of all the protons and carbons was accomplished as
shown in Table 1. The presence of the benzene ring was
suggested by the COSY correlations (bold line in Figure 1) and
the HMBC correlations (H-2′/C-1′, H-2′/C-3′, H-2′/C-4′, H-
3
′/C-1′, H-3′/C-2′, H-4′/C-2′, H-4′/C-3′). The pentane-1,3,4-
triol moiety was elucidated by the COSY correlations (H-1/H-
B
dx.doi.org/10.1021/np500075m | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Note
Figure 3. Protective activity of 1−8 against ER stress-dependent cell death. Neuro2a cells were incubated with various concentrations of compounds
in the absence or presence of 10 nM thapsigargin (TG) for 24 h. The cell viabilities were analyzed by MTT assay, and the values were represented as
the mean ± SE of the relative percentage of surviving cells compared with the untreated cells (n = 10−12). (*) p < 0.01, Tukey−Kramer multiple
comparisons tests.
5
SL-II, Nacalai Tesque, Japan) and a reversed-phase HPLC column
(fractions 19-15-1 to 19-15-11), and then fraction 19-15-6 (92.7 mg)
was further separated by normal-phase HPLC (Senshu Pak AQ,
(
(
Develosil C30-UG-5, Nomura Chemical, Japan). Silica gel plates
Merck F254), ODS gel plates (Merck F254), and silica gel 60 N (Kanto
CHCl
19-15-6-15). Compound 7 (10.2 mg) was purified by a normal-phase
HPLC (Senshu Pak AQ, CHCl −MeOH, 96:4) from fraction 19-15-6-
10 (16.9 mg). Fraction 18 (374 mg) was separated by preparative silica
TLC (CH Cl −MeOH, 9:1) to obtain six fractions (fractions 18-1 to
−MeOH, 98:2) to obtain 15 fractions (fractions 19-15-6-1 to
3
Chemical, Japan) were used for analytical TLC and for flash column
chromatography, respectively.
Fungal Material. Mature fruiting bodies of Mycoleptodonoides
aitchisonii were collected at Narusawa Village, Yamanashi Prefecture, in
Japan, in August 2006, and identified by one of the authors (H.K.). A
voucher specimen of the organism is located in Shizuoka University
3
2
2
18-6), and then fraction 18-5 (72 mg) was further separated by
preparative ODS TLC (90% MeOH) to afford compound 6 (21.6
mg). Compound 3 (1.6 mg) was purified by normal-phase HPLC
(
voucher specimen no. SU2006MA).
(
Senshu Pak AQ, CHCl −MeOH, 99:1) from fraction 18-3 (37 mg).
Extraction and Isolation. The fresh fruiting bodies of M.
3
Similarly, the EtOAc-soluble part (25.0 g) was fractionated by silica gel
flash column chromatography (CH Cl , CH Cl −MeOH, 9:1, 7:3, 5:5,
aitchisonii (13.0 kg) were extracted with EtOH (21 L, three times)
and then acetone (10 L, once). After the solutions were combined and
concentrated under reduced pressure, the concentrate was partitioned
between CH Cl and H O and then between EtOAc and H O. The
CH Cl -soluble part (73.6 g) was fractionated by silica gel flash
column chromatography (hexane−CH Cl , 7:3, hexane−EtOAc, 9:1,
5
2
2
2
2
and MeOH, 1 L each), and 10 fractions were obtained (fractions 1 to
0). Fraction 6 (12.0 g) was further separated by silica gel flash
column chromatography (CH Cl , CH Cl −MeOH, 99:1, 98:2, 95:5,
1
2
2
2
2
2
2
2
2
2
2
8
:2, 1:1, 1 L each) to obtain nine fractions (fractions 6-1 to 6-9), and
2
2
then fraction 6-3 (53.9 mg) was further separated by normal-phase
:5, CH Cl −MeOH, 8:2, 5:5, 0:10, 2 L each) to obtain 25 fractions
2
2
HPLC (Cosmosil 5SL-II, CHCl −hexane, 9:1) to give compound 5
3
(
fractions 1−25), and fraction 19 (6.9 g) was further separated by
(2.2 mg). Compounds 1 (2.0 mg) and 2 (3.8 mg) were purified from
silica gel flash column chromatography (CH Cl −acetone, 9:1, 7:3,
3
2
2
fraction 6-6 (63.5 mg) by reversed-phase HPLC (Develosil C30-UG-5,
5% MeOH).
-Hydroxy-4-(1-hydroxyethyl)-3-methylfuran-2(5H)-one (1): col-
:7, acetone−MeOH, 5:5, and MeOH, 1 L each), and 24 fractions
2
were obtained (fractions 19-1 to 19-24). Fraction 19-12 (1.0 g) was
further separated by silica gel flash column chromatography (CH Cl −
acetone, 9:1, 8:2, CH Cl −MeOH, 9:1, 8:2, and EtOH, 1 L each) to
5
2
2
26
−1 1
orless oil; [α]D −16 (c 0.12, MeOH); IR (neat) 3290, 1749 cm ; H
2
2
13
+
and C NMR, see Table 1; HRESIMS m/z 159.0687 [M + H] (calcd
obtain nine fractions (fractions 19-12-1 to 1-12-9). Fraction 19-12-6
613.2 mg) was further separated by normal-phase HPLC (Develosil
Silica-HILIC, CHCl −MeOH, 98:2) to afford 21 fractions (fractions
for C H O , 159.0657).
7
11
4
(
(
3R,4R)-4-((R)-1-Hydroxyethyl)-3-methyldihydrofuran-2(3H)-one
31
−1
3
(
4): colorless oil; [α]D +27 (c 0.50, CHCl ); IR (neat) 3446, 1763m ;
3
19-12-6-1 to 19-12-6-21). Compound 8 (2.5 mg) was purified from
1
H NMR (500 MHz, in CDCl ) δ 4.28 (1H, dd, J = 8.9, J = 8.9, H-5a),
3
fraction 19-12-6-16 (6 mg) by normal-phase HPLC (Develosil Silica-
3
.94 (1H, dd, J = 8.9, J = 8.9, H-5b), 3.84 (1H, m, H-1′), 2.56 (1H, m,
HILIC, CHCl −MeOH, 98:1). Fraction 19-8 (4.2 g) was further
3
H-4), 2.19 (1H, m, H-3), 1.33 (3H, d, J = 7.3, C-3−Me, 1.20 (3H, d, J
=
1
ESIMS m/z 167 [M + Na] .
3S,4R)-5-Phenylpentane-1,3,4-triol (7): colorless oil; [α] +63 (c
0.10, MeOH); IR (neat) 3364, 1456 cm ; H and C NMR, see
separated by silica gel flash column chromatography (CH Cl −
13
2
2
6.4, H-2′); C NMR (125 MHz, in CDCl ) δ 180.4 (C-2), 68.4 (C-
3
acetone, 9:1, 8:2, 5:5, CH Cl −MeOH, 9:1, 8:2, 1 L each) to obtain
2
2
′), 67.9 (C-5), 49.8 (C-3), 36.9 (C-4), 21.7 (C-2′), 16.3 (C-4−Me);
seven fractions (fractions 19-8-1 to 19-8-7), and then fraction 19-8-2
3.3 g) was further separated by normal-phase HPLC (Develosil Silica-
HILIC, CHCl −MeOH, 98:1) to obtain 11 fractions (fractions 19-8-2-
+
(
25
D
(
−1
1
13
3
1
to 19-8-2-11). Fraction 19-8-2-10 (27 mg) was separated by normal-
+
Table 1; HRESIMS m/z 219.1019 [M + Na] (calcd for C H NaO ,
1
1
16
3
phase HPLC (Senshu Pak AQ, CHCl −MeOH, 99:1) to afford
3
219.0997).
compound 4 (7.5 mg). Fraction 19-15 (751 mg) was separated by
silica gel flash column chromatography (CH Cl , CH Cl −MeOH,
Preparation of p-Bromobenzoates of (3R,4R)-4-((R)-1-Hy-
droxyethyl)-3-methyldihydrofuran-2(3H)-one and (3S,4R)-5-
Phenylpentane-1,3,4-triol. (3R,4R)-4-((R)-1-Hydroxyethyl)-3-
2
2
2
2
9
9:1, 95:5, 9:1, 1:1, and MeOH, 1 L each) to obtain 11 fractions
C
dx.doi.org/10.1021/np500075m | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Note
methyldihydrofuran-2(3H)-one (4) (2.1 mg) was dissolved in 0.25 mL
of anhydrous pyridine in a 4 mL vial, and p-bromobenzoyl chloride
(final concentration 0.5 μg/mL of TM or 10 nM TG) was applied to
the medium and incubated for additional 24 h. After treatment, the cell
21
(
16 mg) was added to the solution. After shaking at 50 °C for 3 days,
viability was measured by the MTT assay as described previously.
the reaction mixture was evaporated to dryness under reduced
Briefly, 0.25 mg/mL of MTT in D-MEM without FBS was added to
the cells and incubated for 2 h. The incubation was terminated by the
addition of 20% SDS (v/w) and 50% N,N-dimethylformamide in
water. The absorbance at 570 nm of the reaction mixture was
measured by a microplate reader (Molecular Devices, USA).
Statistical Analysis. Data collected were analyzed statistically
using Tukey−Kramer multiple comparisons tests to determine
significant differences in the data among the groups. p values less
than 0.01 or 0.05 were considered significant. The values are expressed
as mean ± SE.
pressure. The products were then purified by normal-phase HPLC
(
(
Senshu Pak AQ, CHCl −MeOH, 96:4) to give the p-bromobenzoate
3
1
1.5 mg) of 4. p-Bromobenzoate of 4: H NMR (500 MHz, in CDCl )
3
δ 7.85 (2H, d, J = 8.5, p-Br−COC H ), 7.56 (2H, d, J = 8.5, p-Br−
6
4
COC H ), 5.26 (1H, m, H-1′), 4.39 (1H, dd, J = 8.2, J = 9.2, H-5a),
6
4
3.99 (1H, dd, J = 8.6, J = 9.2, H-5b), 2.60 (1H, m, H-3), 2.51 (1H, m,
H-4), 1.51 (3H, s, C-3−Me), 1.35 (3H, d, J = 6.7, H-2′); ESIMS m/z
+
3
49 [M + Na] . (3S,4R)-5-Phenylpentane-1,3,4-triol (7) (1.3 mg) was
dissolved in 0.25 mL of anhydrous pyridine in a 4 mL vial, and p-
bromobenzoyl chloride (7.3 mg) was added to the solution. After
shaking at 50 °C for 2 days, the reaction mixture was evaporated to
dryness under reduced pressure. The products were then purified by
ASSOCIATED CONTENT
■
*
S
Supporting Information
normal-phase HPLC (Senshu Pak AQ, hexane−CHCl , 1:1) to give
3
1
H, 13C, and 2D NMR spectra of compounds 1 and 7, X-ray
1
the p-bromobenzoate (1.9 mg) of 7. p-Bromobenzoate of 7: H NMR
(
5
500 MHz, in CDCl ) δ 7.81 (6H, m), 7.56 (6H, m), 7.28 (5H, m),
.72 (1H, m), 5.60 (1H, m), 4.54 (1H, m), 4.38 (1H, m), 3.14 (2H,
crystallographic data for p-bromobenzoates of 4 and 7, and
effect of sodium valproate on ER stress-dependent cell death
are available free of charge via the Internet at http://pubs.acs.
org.
3
m), 2.36 (2H, m).
X-ray Crystallography Analysis. Crystal data for p-bromoben-
zoate of 4: C H Br O , M = 327.17, orthorhombic, a = 6.2502(1) Å,
b = 7.1121(1) Å, c = 31.0868(6) Å, V = 1381.87(4) Å , T = 193 K,
P2 2 2 , Z = 4, λ = 1.541 87 Å, μ(λ = 1.541 87) = 4.14 mm , F(000)
=
14
15
1
4
3
AUTHOR INFORMATION
Corresponding Author
*Tel/Fax: +81-54-238-4885. E-mail: achkawa@ipc.shizuoka.ac.
−1
■
1
1 1
664. The size of the crystal used for measurements was 0.60 × 0.10
0.05 mm. Diffraction data were collected on a Rigaku R-AXIS-
×
RAPID diffractometer with imaging plate detector; 25 210 reflections
were collected in the range 4.3° < θ < 68.2°, of which 2543 were
unique (Rint =2 0.0420). The structure was refined by full-matrix least-
squares on F values using all unique reflections. The final R indices
jp.
Author Contributions
#
J.-H. Choi and T. Suzuki contributed equally to this work.
2
were R(F) = 0.0254, wR(F ) = 0.0612 (all reflections) with goodness
Notes
of fit = 1.089. The Flack parameter was −0.02(2) with 1022 Friedel
pairs. Crystallographic data have been deposited at the Cambridge
Crystallographic Data Centre and allocated the deposition number
CCDC 978569. Crystal data for p-bromobenzoate of 7: C H Br O ,
M = 745.26, monoclinic, a = 14.899(3) Å, b = 5.6970(11) Å, c =
1
4
measurements was 0.14 × 0.03 × 0.01 mm. Diffraction data were
collected on a Rigaku Saturn724 diffractometer with imaging plate
detector; 23 580 reflections were collected in the range θ < 29.0°, of
which 7620 were unique (Rin t2 = 0.0812). The structure was refined by
full-matrix least-squares on F values using all unique reflections. The
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
32
25
3
6
This work was partially supported by a Grant-in-Aid for
Scientific Research on Innovative Areas “Chemical Biology of
Natural Products” from MEXT (grant no. 24102513).
3
7.092(5) Å, V = 1447.7(6) Å , T = 173 K, Z = 2, μ(λ = 0.71075) =
−
1
.24 mm , F(000) = 740. The size of the crystal used for
REFERENCES
■
(
1) Lindholm, D.; Wootz, H.; Korhonen, L. Cell Death Differ. 2006,
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2
final R indices were R(F) = 0.0660, wR(F ) = 0.1287 (all reflections)
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with goodness of fit = 1.001. The Flack parameter was 0.008 with 3420
Friedel pairs. The single crystal was mounted on a loop using oil.
Diffraction data were collected at −100 °C under a cold nitrogen
stream on a Rigaku VariMax with a Satrun diffractometer using a
multilayer mirror monochromated Mo Ka radiation source (λ = 0.710
7
5 Å). Eighteen preliminary data frames were measured at 0.5°
increments of ω, to assess the crystal quality and preliminary unit cell
parameters. The intensity images were also measured at 0.5° intervals
of ω. The intensity images were integrated using the Crystal Clear
program package. The structure was solved by direct methods
(
SHELX-97). All non-hydrogen atoms were refined anisotropically by
full-matrix least-squares technique. Crystallographic data have been
deposited at the Cambridge Crystallographic Data Centre and
allocated the deposition number CCDC 978780. The data can be
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