A tetrahydrobenzofuran derivative from the fermentation broth of Lentinus squarrosulus BCC 22366

Article abstract of DOI:10.1016/j.phytol.2010.12.002

A new tetrahydrobenzofuran derivative, (6S,7S)-6,7-dihydroxy-3,6-dimethyl- 2-isovaleroyl-4,5,6,7-tetrahydrobenzofuran (1), together with the known 1,2-dihydroxymintlactone (2), was isolated from the fermentation broth of the edible mushroom fungus Lentinus squarrosulus BCC 22366. The structure was elucidated by interpretation of NMR spectroscopic and mass spectrometry data. The absolute configuration of 1 was addressed by application of the modified Mosher's method.

Full text of DOI:10.1016/j.phytol.2010.12.002

Phytochemistry Letters 4 (2011) 106–108
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
A tetrahydrobenzofuran derivative from the fermentation broth of Lentinus
squarrosulus BCC 22366
*
Masahiko Isaka , Malipan Sappan, Pranee Rachtawee, Thitiya Boonpratuang
National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand Science Park, Phaholyothin Road, Klong Luang, Pathumthani 12120, Thailand
A R T I C L E I N F O
A B S T R A C T
new tetrahydrobenzofuran derivative, (6S,7S)-6,7-dihydroxy-3,6-dimethyl-2-isovaleroyl-4,5,6,7-
Article history:
A
Received 22 November 2010
Accepted 7 December 2010
Available online 21 December 2010
tetrahydrobenzofuran (1), together with the known 1,2-dihydroxymintlactone (2), was isolated from
the fermentation broth of the edible mushroom fungus Lentinus squarrosulus BCC 22366. The structure
was elucidated by interpretation of NMR spectroscopic and mass spectrometry data. The absolute
configuration of 1 was addressed by application of the modified Mosher’s method.
Keywords:
ß 2010 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Lentinus squarrosulus
Benzofuran
Mushroom
1. Introduction
10
Lentinus is a genus of mushroom fungi that include many
edible species such as ‘‘shiitake’’ (L. edodes). Investigations of
dried shiitake mushroom led to the identification of an
antifungal polysulfide lentionine (1,2,3,5,6-pentathiepane)
(Morita and Kobayashi, 1966) and related compounds (Morita
and Kobayashi, 1967; Chibata et al., 1969; Takazawa et al.,
1982). There have been several reports on the isolation of low
molecular weight secondary metabolites from cell cultures:
hirsutane sesquiterpenes from L. crinitus DR-5 (Abate and
Abraham, 1994) and L. connatus BCC 8996 (Rukachaisirikul
et al., 2005), chromanones from L. crinitus (Abraham and Abate,
1995), striguerollide A, striguellol A, and striguellone A from L.
striguellus (Zheng et al., 2009), and 2-methoxy-5-methyl-1,4-
benzoquinone from L. adhaerens (Lauer et al., 1991). As part of
our research program on novel fungal metabolites, we have
investigated the fermentation broth of L. squarrosulus BCC 22366
as the 1H NMR spectrum of an extract exhibited unique
resonance patterns. Chemical studies on a large-scale fermen-
tation broth of BCC 22366 afforded a new tetrahydrobenzofuran
derivative 1, along with the known 1,2-dihydroxymintlactone
(2) (Stadler et al., 1995). There was a report of two antibiotic
compounds from L. squarrosulus, although their structures have
not been elucidated (Sudirman et al., 1994).
4
7
CH3
CH3
5
3a
3
5
4
8
13
6
14
H3C
11
9
8
6
7
O
10 CH3
3
1
2
7a
9
H3C
2
O
O
HO
HO
OH
O
CH3
12
OH
1
2
2. Results and discussion
The molecular formula of compound 1 was established by
HRESIMS as C15H22O4. Inspection of 1H and 13C NMR, DEPT135, and
HMQC data revealed that 1 consisted of a conjugated ketone
resonating at dC 191.6, four sp2 quaternary carbons at dC 151.4,
149.2, 128.8, and 122.9, an oxygenated quaternary carbon at dC
71.8, an oxygenated methine at dC 69.3 (dH 4.40, d, J = 5.0 Hz), a
methine at
four methyl groups at
d, J = 6.7 Hz), and 9.8 (dH 2.28, 3H, s). The 1H NMR spectrum
exhibited resonances of a tertiary alcohol OH ( H 2.45, br s) and a
secondary alcohol OH ( H 2.67, br d, J = 5.0 Hz). The presence of an
d
C 24.8, three methylenes at
d
C 47.9, 32.7, and 17.5, and
d
C 24.4 (
d
H 1.31, 3H, s), 22.7 Â 2 (
d
H 0.97, 6H,
d
d
isobutyl group (C-9–C-12) was evident from the COSY correlations.
The methylene protons resonated downfield at dH 2.71 (H2-9)
showed HMBC correlation to the ketone carbon (C-8), which
indicated an isovaleroyl group (C-8–C-12). COSY data also
demonstrated the connection of two other methylenes (CH2-4
and CH2-5), which constituted a part of the six-membered ring.
HMBC correlations from the methyl protons (H3-14) to the tertiary
* Corresponding author. Tel.: +66 25646700x3554; fax: +66 25646707.
E-mail address: isaka@biotec.or.th (M. Isaka).
1874-3900/$ – see front matter ß 2010 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.phytol.2010.12.002

M. Isaka et al. / Phytochemistry L
[
etters 4 (2011) 106–108
107
-0.02
-0.02
Table 1
+0.02
NMR data for compound 1 in CDCl3.
-0.04
H H
CH3
H
Position
dC mult.
dH mult. (J in Hz)
HMBC
-0.03H
-0.16 H3C
HO
2
149.2 C
128.8 C
122.9 C
17.5 CH2
32.7 CH2
71.8 C
O
3
0.00
3a
4
+0.02 O
2.54 m; 2.31 m
2.03 m; 1.75 m
6, 7a
5
3a, 6, 7
O
6
7
69.3 CH
151.4 C
191.6 C
47.9 CH2
24.8 CH
22.7 CH3
22.7 CH3
9.8 CH3
24.4 CH3
4.40 d (5.0)
3a, 7a
MTPA
4a (S)-MTPA ester
4b (R)-MTPA ester
7a
8
9
2.71 dd (7.0, 1.5)
2.27 m
8, 10, 11, 12
10
11
12
13
14
6-OH
7-OH
Fig. 3. Dd-Values (dS
À
d
R) of the (S)- and (R)-MTPA esters 4a and 4b.
0.97 d (6.7)
0.97 d (6.7)
2.28 s
9
9
2, 3, 3a, 8
5, 6, 7
1.31 s
d
H 1.78 (H -5) and 2.33 (H -4), which indicated that these protons
were coplanar. The cross-peak of H-7 to H -5 further confirmed the
a a
2.45 br s
a
2.67 br d (5.0)
7
assigned relative configuration. The absolute configuration of 1 was
therefore established as 6S,7S. The same method was applied to the
assignment of the unknown absolute configuration of 1,2-dihydrox-
ymintlactone(2).Theabsoluteconfigurationofthe1,2-diolmoietyof
2 proved to be the same sense as 1 (Fig. 3).
Compound 1 is structurally related to the bisabolane sesqui-
terpenes, cheimonophyllons A–E and cheimonophyllal, which
were isolated along with the monoterpene 2 from the Basidiomy-
cete Cheimonophyllum candidissium TA 8644 (Stadler et al.,
1994a,b). Cheimonophyllon E and cheimonophyllal possess the
1,2-diol moiety similar to 1 and 2, with the same relative
configuration (Stadler et al., 1994a, 1995).
alcohol carbon (C-6), C-5, and the secondary alcohol carbon (C-7),
and from the methylene protons (H2-5) to C-6 and C-7 indicated
the linkage of C-5–C-6–C-7. The secondary alcohol proton (H-7)
showed HMBC correlations to sp2 quaternary carbons at dC 122.9
(C-3a) and 151.4 (C-7a), while H2-5 and H2-4 exhibited HMBC
correlations respectively to C-3a and C-7a. HMBC correlations from
the downfield methyl protons (dH 2.28, H3-13) to C-3a and two
other quaternary sp2 carbons at dC 149.2 (C-2) and 128.8 (C-3)
revealed the connection of C-3 and C-3a. The methyl protons (H3-
13) also exhibited weak 4J correlation to ketone C-8, which
demonstrated the attachment of the isovaleroyl group to C-2. The
molecular formula of 1 requested the formation of the furan ring,
which was consistent with the chemical shifts of four sp2
quaternary carbons (C-2, C-3, C-3a, and C-7a). Accordingly, the
planar structure of 1 was assigned as 6,7-dihydroxy-3,6-dimethyl-
2-isovaleroyl-4,5,6,7-tetrahydrobenzofuran (Table 1).
3. Experimental
3.1. General experimental procedures
Optical rotations were measured with a JASCO P-1030 digital
polarimeter. UV spectra were recorded on a GBC Cintra 404
spectrophotometer. FTIR spectra were taken on a Bruker VECTOR
22 spectrometer. NMR spectra were recorded on Bruker AV500D
and DRX400 spectrometers. ESI-TOF mass spectra were measured
with a Bruker micrOTOF mass spectrometer.
The absolute configuration of the secondary alcohol carbon (C-7)
was determined by application of the modified Mosher’s method
(Ohtani et al., 1991). The Ddvalues of the (S)- and (R)-MTPA esters 3a
and 3b, respectively, indicated the 7S configuration (Fig. 1). The
relative configuration of C-6/C-7 was addressed on the basis of the
NOESY correlations of the (S)-MTPA ester 3a (Fig. 2). Methyl group
3.2. Fungal material
HT -15 showed NOESY correlations to H-7 and methylene protons at
3
The fungus used in this study was collected on a dead wood in
Khao Yai National Park, Nakorn Rachasrima Province, Thailand. The
natural mushroom specimen was deposited in the BIOTEC Bangkok
Herbarium on July 10, 2005, as BBH 16924. The living culture was
deposited in the BIOTEC Culture Collection on July 28, 2005, as BCC
22366. Onthebasisofthemorphologybothmacro-andmicro-scopic
characteristics, this fungus was assigned to Lentinus squarrosulus
Mont. (family Polyporaceae) by one of the authors (T.B.).
0.00 +0.01
-0.07
H H
+0.03
H
-0.03 H
CH3
+0.03
0.00
-0.06
+0.02
+0.03
H3C
CH3
O
+0.01
HO
O
O
CH3
+0.01
MTPA
3a (
S
)-MTPA ester
)-MTPA ester
3.3. Fermentation, extraction, and isolation
3b (
R
Fig. 1. Dd-Values (dS
À
d
R) of the (S)- and (R)-MTPA esters 3a and 3b.
The fungus BCC 22366 was maintained on potato dextrose agar
at 25 8C. The agar was cut into small plugs and inoculated into
2Â 250 ml Erlenmeyer flasks containing 25 ml of potato dextrose
broth (PDB; potato starch 4.0 g/l, dextrose 20.0 g/l). After incuba-
tion at 25 8C for 7 days on a rotary shaker (200 rpm), each primary
culture was transferred into a 1000 ml Erlenmeyer flask containing
250 ml of the same liquid medium (PDB), and incubated at 25 8C
for 7 days on a rotary shaker (200 rpm). These secondary cultures
were pooled and each 25 ml portion was transferred into
20Â 1000 ml Erlenmeyer flasks containing 250 ml of malt extract
broth (MEB; malt extract 6.0 g/l, yeast extract 1.2 g/l, maltose
1.8 g/l, dextrose 6.0 g/l), and final fermentation was carried out at
[()TD$FIG]
H H
H
CH3
H
H3C
HO
CH3
CH3
O
H OMTPA
O
(S)
3a
Fig. 2. Key NOESY correlations for the (S)-MTPA ester 3a.

108
M. Isaka et al. / Phytochemistry Letters 4 (2011) 106–108
25 8C for 49 days under static conditions. The cultures were filtered
to separate broth (filtrate) and mycelia (residue). Culture broth
was extracted with EtOAc (3Â 4.5 l) and the combined organic
phase was concentrated to obtain a brown gum (1.37 g). This
extract was subjected to column chromatography (CC) on Sephadex
LH-20(4.5 Â 60 cm,MeOH)toobtainsevenpooledfractions;fraction
1 (56 mg), 2 (955 mg), 3 (158 mg), 4 (5 mg), and 5 (101 mg). Fraction
2 was subjected to CC on silica gel (3.5 Â 15 cm, EtOAc/CH2Cl2, step
gradient elution from 0:100 to 30:70) to obtain nine fractions;
fraction 2-1–2-9. Fraction 2-7 (427 mg) was fractionated by
preparative HPLC using a reverse phase column (Prep NovaPak HR
3.5. Synthesis of Mosher esters 4a and 4b
(S)-MTPA ester 4a (1.7 mg) was synthesized from 2 (1.0 mg)
and (À)-(R)-MTPACl (5
ml) using the same procedure as described
above for 3a and 3b. However, similar reaction of 2 with (+)-(S)-
MTPACl did not occur under the same conditions, probably due to
the relatively larger steric hindrance. (R)-MTPA ester 4b (2.1 mg)
was prepared by treatment of 2 (1.0 mg) with (+)-(S)-MTPACl
(15
ml) and N,N-dimethylaminopyridine (DMAP; 15 mg) in CH2Cl2
(0.15 ml) at room temperature for 15 h.
C
18, 25 Â 100 mm; MeCN/H2O = 30:70, flow rate 8 ml/min) toobtain
3.5.1. (S)-MTPA ester derivative 4a
a fraction containing 2 (95 mg, tR 12 min) and pure compound 1
(48.8 mg, tR 18 min).Compound2wasfurtherpurifiedbyCConsilica
gel (1.5 Â 15 cm, EtOAc/CH2Cl2, step gradient elution from 0:100 to
90:10) to a colorless amorphous (25.8 mg). Fraction 2-8 (176 mg)
was subjected to CC on silica gel (2.5 Â 15 cm, EtOAc/CH2Cl2, step
gradient elution from 0:100 to 90:10) to furnish 2 (15.3 mg).
Colorless gum; 1H NMR (400 MHz, CDCl3)
d 7.63 (2H, m, phenyl
of MTPA), 7.46–7.43 (3H, m, phenyl of MTPA), 5.029 (1H, br d,
J = 9.3 Hz, H-3), 4.864 (1H, d, J = 9.3 Hz, H-2), 3.61 (3H, br s, –OCH3
of MTPA), 2.641 (1H, m, H -5), 2.592 (1H, m, H -5), 2.078 (1H, m,
a
b
H -6), 1.858 (3H, br s, H-10), 1.530 (1H, dt, J = 5.6, 13.5 Hz, H -6),
b
a
1.114 (3H, s, H-7); HRESI-MS (m/z): 437.1198 [M+Na]+ (calc. for
C20H21O6F3Na, 437.1188).
3.3.1. (6S,7S)-6,7-Dihydroxy-3,6-dimethyl-2-isovaleroyl-4,5,6,7-
tetrahydrobenzofuran (1)
3.5.2. (R)-MTPA ester derivative 4b
26
MeOH
Colorless solid; [a D
]
+51 (c 0.10, MeOH); UV lmax
nm
Colorless gum; 1H NMR (400 MHz, CDCl3)
d 7.62 (2H, m, phenyl
(log ): 284 (4.22); IR
e
n
max KBr cmÀ1 3471, 3373, 1649, 1538, 1055;
of MTPA), 7.47–7.41 (3H, m, phenyl of MTPA), 5.013 (1H, br d,
J = 9.2 Hz, H-3), 4.862 (1H, d, J = 9.2 Hz, H-2), 3.55 (3H, br s, –OCH3
of MTPA), 2.660 (1H, m, H -5), 2.610 (1H, m, H -5), 2.120 (1H, m,
1H (500 MHz, CDCl3) and 13C (125 MHz, CDCl3): Table 1; HRESI-MS
(m/z): 289.1418 [M+Na]+ (calc. for C15H22O4Na, 289.1410).
a
b
H -6), 1.841 (3H, br s, H-10), 1.562 (1H, m, H -6), 1.273 (3H, s, H-
b
a
3.3.2. 1,2-Dihydroxymintlactone (2)
7); HRESI-MS (m/z): 437.1190 [M+Na]+ (calc. for C20H21O6F3Na,
437.1188).
Colorless solid; [
a]
25 +51 (c 0.50, CHCl3); 1H (500 MHz, CDCl3)
D
and 13C (125 MHz, CDCl3) NMR spectroscopic data were consistent
with those reported in the literature (Stadler et al., 1995); HRESI-
MS (m/z): 221.0873 [M+Na]+ (calc. for C10H14O4Na,
289.141221.08740).
Acknowledgement
Financial support from the Bioresources Research Network,
National Center for Genetic Engineering and Biotechnology
(BIOTEC), is gratefully acknowledged.
3.4. Synthesis of Mosher esters 3a and 3b
References
Compound 1 (1.0 mg) was treated with (À)-(R)-MTPACl (5
ml)
in pyridine (0.2 ml) at room temperature for 16 h. The mixture was
diluted with EtOAc and washed with H2O and 1 M NaHCO3, and the
organic layer was concentrated in vacuo. The residue was purified
by CC on silica gel to afford a (S)-MTPA ester derivative 3a (0.8 mg).
Similarly, (R)-MTPA ester derivative 3b (1.3 mg) was prepared
from 1 and (+)-(S)-MTPACl. Assignments of protons of the Mosher
ester derivatives 3a and 3b were established on the basis of COSY
and NOESY data.
Abate, D., Abraham, W.-R., 1994. Antimicrobial metabolites from Lentinus crinitus. J.
Antibiot. 47, 1348–1350.
Abraham, W.-R., Abate, D., 1995. Chromanones from Lentinus crinitus (Basidiomy-
cetes). Z. Naturforsch. 50c, 748–750.
Chibata, I., Okumura, K., Takeyama, S., Kotera, K., 1969. Lentinacin: a new hypo-
cholesterolemic substance in Lentinus edodes. Experientia 25, 1237–1238.
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Methoxy-5-methyl-1,4-benzoquinone, a thromboxan A2 antagonist from Len-
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its analogs. Chem. Pharm. Bull. 15, 988–993.
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8996. J. Nat. Prod. 68, 1674–1676.
3.4.1. (S)-MTPA ester derivative 3a
Colorless gum;1H NMR (400 MHz, CDCl3)
d7.55(2H, m, phenyl of
MTPA), 7.43–7.36 (3H, m, phenyl of MTPA), 5.920 (1H, s, H-7), 3.58
(3H, br s, –OCH3 of MTPA), 2.624 (1H, m, Ha-9), 2.612 (1H, m, Hb-9),
2.571 (1H, m, H -4), 2.330 (1H, m, H -4), 2.281 (3H, s, H-13), 2.212
b
a
(1H, m, H-10), 1.923 (1H, m, H -5), 1.784 (1H, m, H -5), 1.284 (3H, s,
b
a
H-14), 0.941 (3H, d, J = 6.9 Hz, H-11), 0.925 (3H, d, J = 6.8 Hz, H-12);
HRESI-MS (m/z): 505.1803 [M+Na]+ (calc. for C25H29O6F3Na,
505.1808).
Stadler, M., Anke, H., Sterner, O., 1994a. Six new antimicrobial and nematicidal
bisabolanes from the Basidiomycete Cheimonophyllum candidissimum. Tetrahe-
dron 50, 12649–12654.
Stadler, M., Anke, H., Sterner, O., 1994b. New nematicidal and antimicrobial
compounds from the Basidiomycete Cheimonophyllum candidissimum (Berk &
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new nematicidal monoterpene isolated from the Basidiomycete Cheimonophyl-
lum candidissimum (Berk & Curt.). Sing. Z. Naturforsch. 50c, 473–475.
3.4.2. (R)-MTPA ester derivative 3b
Colorless gum; 1H NMR (400 MHz, CDCl3)
d 7.58 (2H, m, phenyl
of MTPA), 7.43–7.38 (3H, m, phenyl of MTPA), 5.891 (1H, s, H-7),
3.56 (3H, br s, –OCH3 of MTPA), 2.590 (2H, d, J = 6.9 Hz, H-9), 2.559
(1H, m, H -4), 2.330 (1H, m, H -4), 2.248 (3H, s, H-13), 2.203 (1H,
`
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elucidation and apoptosis-inducing activity of new compounds from the edible
fungus Lentinus striguellus. Nat. Prod. Comm. 4, 501–506.
b
a
m, H-10), 1.989 (1H, m, H -5), 1.810 (1H, m, H -5), 1.339 (3H, s, H-
b
a
14), 0.943 (3H, d, J = 6.7 Hz, H-11), 0.916 (3H, d, J = 6.7 Hz, H-12);
HRESI-MS (m/z): 505.1801 [M+Na]+ (calc. for C25H29O6F3Na,
505.1808).