Metabolites from the endophytic mitosporic Dothideomycete sp. LRUB20

Article abstract of DOI:10.1016/j.phytochem.2008.10.007

The endophytic mitosporic Dothideomycete sp. LRUB20 was found to produce pyrone derivatives, dothideopyrones A-D (1, 3, 4, and 5), together with seven known compounds, including questin (9), asterric acid (10), methyl asterrate (11), sulochrin (12), and e

Full text of DOI:10.1016/j.phytochem.2008.10.007

Phytochemistry 70 (2009) 121–127
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Metabolites from the endophytic mitosporic Dothideomycete sp. LRUB20
Porntep Chomcheon a,b, Suthep Wiyakrutta c, Nongluksna Sriubolmas d, Nattaya Ngamrojanavanich b,e
,
Chulabhorn Mahidol a,f, Somsak Ruchirawat a,f, Prasat Kittakoop a,
*
a Chulabhorn Research Institute and Chulabhorn Graduate Institute, Vipavadee-Rangsit Highway, Bangkok 10210, Thailand
b Program of Biotechnology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
c Department of Microbiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand
d Department of Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand
e Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
f Chulabhorn Research Centre, Institute of Science and Technology for Research and Development, Mahidol University, Thailand
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 June 2008
Received in revised form 17 August 2008
Available online 27 November 2008
The endophytic mitosporic Dothideomycete sp. LRUB20 was found to produce pyrone derivatives, dot-
hideopyrones A–D (1, 3, 4, and 5), together with seven known compounds, including questin (9), asterric
acid (10), methyl asterrate (11), sulochrin (12), and eugenitin (13), 6-hydroxymethyleugenitin (14), and
cis, trans-muconic acid (15). Dothideopyrone D (5) and its acetate derivative 6 exhibited moderate cyto-
toxic activity. This is the first report on a naturally occurring muconic acid, which is commonly known as
a biomarker in environments after exposure to benzene and phenol (or derivatives). Interestingly, the
LRUB20 fungus could produce muconic acid in relatively high yield (47.8 mg/L). The utility of endophytic
fungi in the field of white biotechnology is discussed.
Keywords:
Dothideomycete sp.
Endophytic fungi
Pyrone
Ó 2008 Elsevier Ltd. All rights reserved.
Muconic acid
Bioactive compounds
Cytotoxic activity
White biotechnology
Building blocks
1. Introduction
fight against plant parasitic nematodes. Endophytic fungi are prov-
ing to be rich sources of biologically active compounds with poten-
Fungal endophytes normally colonize living internal tissues of
plants without causing any obvious negative effects or external
symptoms. Endophytic fungi may offer either significant benefit
to their host plants by producing secondary metabolites that pro-
vide protection and survival advantage to the plants. Particular
secondary metabolites produced by endophytic fungi are believed
to benefit the host plants as they may be plant growth regulators,
antimicrobials, antivirals, and insecticidals, or even mediate resis-
tance to some types of abiotic stress (Stone et al., 2004). 3-Nitro-
propionic acid (3-NPA) is a good example of an endophytic
metabolite having both ecological and biological impacts. 3-NPA
is involved in nitrification processes in leguminous plants (Hipkin
et al., 2004); however, 3-NPA accumulated in leguminous plants
may be produced by associated endophytes (Chomcheon et al.,
2005). Fungal 3-NPA therefore possibly participates in the
nitrification process of the nitrogen cycle. 3-NPA (previously
reported as 3-hydroxypropionic acid) exhibited potent nematicidal
activity (Schwarz et al., 2004), which might benefit the hosts to
tial applications in agrochemical and pharmaceutical industries
(Gunatilaka, 2006; Pongcharoen et al., 2008; Rukachaisirikul
et al., 2008; Schulz et al., 2002; Strobel, 2006; Tan and Zou,
2001; Wiyakrutta et al., 2004).
We recently isolated the endophytic fungus LRUB20 from a Thai
medicinal plant, Leea rubra Blume ex Spreng (family Leeaceae).
Based upon analysis of the DNA sequences of the 18S and ITS re-
gion of the ribosomal RNA gene, the fungus LRUB20 is potentially
a new species (see Section 3). Interestingly, we have found that this
fungus can produce a large amount (gram scale) of 2-hydroxy-
methyl-3-methyl-cyclopent-2-enone, useful scaffold for organic
synthesis (Chomcheon et al., 2006). The term ‘‘white biotechnol-
ogy” has been recently introduced for microbial production of
building blocks, and various 2-oxocarboxylic acids and isoprenoids
are examples of chemical scaffolds from microbial fermentation
(Maury et al., 2005; Stottmeister et al., 2005). Moreover, the use
of microbial metabolites in organic syntheses provides fundamen-
tal advantages in comparison to chemical methods. Because the
LRUB20 fungus is potentially a new species, we have extended
our study on secondary metabolites by varying the culture media.
It was found that this fungus could produce various types of
* Corresponding author. Tel.: +66 86 9755777; fax: +66 2 5740622x1513.
E-mail address: prasatkittakoop@yahoo.com (P. Kittakoop).
0031-9422/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2008.10.007

122
P. Chomcheon et al. / Phytochemistry 70 (2009) 121–127
metabolites, and might be useful for the production of building
blocks for organic syntheses. Twelve secondary metabolites were
isolated from the fungus LRUB20, four of which (compounds 1, 3,
4, and 5) were new. In addition, cis, trans-muconic acid (15) was
found for the first time as a natural product. This paper reports
the isolation, characterization, and biological activity of the iso-
lated compounds from the endophytic fungus LRUB20 (Figs. 1
and 2).
+0.14
MeO
+0.02
O
+0.28
O
-0.01
-0.01
O
-0.05
OMTPA
2. Results and discussion
Fig. 2.
Dd Values [d(S)–d(R)] for the MTPA esters 7 and 8.
2.1. Structural determination
trum of
1 showed absorption bands for hydroxyl group
(3373 cmꢀ1) and a conjugated carbonyl (1682 cmꢀ1). The UV spec-
trum exhibited a maximum absorption at 299 nm, characteristic of
a pyrone. The 1H NMR spectrum of 1 showed signals for an isolated
sp2 methine (dH 6.44), an oxygenated sp3 methine (dH 4.40), a
methoxy group (dH 3.92), an oxygenated methylene (dH 4.48),
and a hexyl moiety (dH 0.86–1.82). The 13C NMR spectrum of 1 con-
tained 14 lines, and the DEPT data established the presence of two
methyls, six methylenes, two methines, and four non-protonated
carbons. The 1H–1H COSY spectrum of dothideopyrone A (1) as-
sisted the assembling of a partial structure from C-10 to C-70. The
HMBC spectrum showed correlations from H-5 to C-10, C-4, and
C-6; from the 4-OMe protons to C-4; from H-10 to C-5 and C-6;
and from H2-100 to C-2, C-3 and C-4. These correlations established
the positions of the substituents on the pyrone ring. The side chain
in 1, 1-heptanol, was linked to the pyrone unit at C-6 as evident
from the HMBC correlations of H-10 and H2-20 to C-6. Acetylation
of dothideopyrone A (1) yielded a diacetate derivative 2, confirm-
A crude extract of the endophytic mitosporic Dothideomycete
LRUB20 cultured in Czapek yeast autolysate medium was sepa-
rated by Sephadex LH-20 and silica gel column chromatography,
preparative TLC, and semi-preparative C18 reversed-phase HPLC,
furnishing four new pyrone derivatives, dothideopyrones A–D (1,
3, 4, and 5), together with the known fungal metabolites questin
(9), asterric acid (10), methyl asterrate (11), sulochrin (12), and
eugenitin (13). However, the LRUB20 fungus cultured in the M1D
medium (Strobel et al., 1996) supplemented with malt extract pro-
duced two known compounds, 6-hydroxymethyleugenitin (14)
and cis, trans-muconic acid (15). Spectroscopic data of known com-
pounds 9–15 were identical to those reported in the literature
(Barba et al., 1992; Elvidge et al., 1950; Feng et al., 2002; Har-
greaves et al., 2002; Huneck, 1972; Kalidhar, 1989; Shimada
et al., 2003; Stermitz et al., 1973; Wijeratne et al., 2006).
Dothideopyrone A (1) was isolated as colorless oil, and APCI–
TOF MS suggested the molecular formula C14H22O5. The IR spec-
MeO
4
7'''
3'''
MeO
4
1''
1''
5
4'''
1'''
R2O
5
3
O
2'
6'
3
4'
6
5'''
2'
6'
4'
6
2'''
6'''
2
O
O
1
1'
7'
3'
5'
2
7'
O
O
1'
3'
5'
1
O
OR 1
OH
4
, R = H, R = H
1
2
3
2
1
, R = Ac, R = Ac
1
2
, R = H, R = Me
1
2
OR
1'''
5''''
3''''
1''''
7''''
2'''
MeO
4
O
O
5, R = H
6, R = Ac
7, R = (S)-MTPA
8, R = (R)-MTPA
1''
6'''
2''''
6''''
5
O
3
2'
6'
4'
4'''
6
1'''''
2
O
O
1
7'
MeO
1'
3'
5'
OR
MeO
O
MeO
O
OH
MeO
O
OH
OH
HO
RO
O
O
HO
HO
O
OH
MeO
12
OMe
O
9
10, R = H
11, R = Me
OH
O
R
MeO
COOH
O
COOH
13, R = Me
14, R = CH2OH
15
Fig. 1. Structures of the isolated compounds and their derivatives.

P. Chomcheon et al. / Phytochemistry 70 (2009) 121–127
123
Table 2
ing the presence of two hydroxyl groups in 1. On the basis of these
1H and 13C NMR spectroscopic data (CDCl3) for compounds 4 and 5.
data, the gross structure of dothideopyrone A (1) was established.
Assignments of all 1H and 13C NMR spectroscopic signals for 1 were
made by analysis of 1H–1H COSY and HMBC spectra (Table 1).
Position
4
5a
dH (J in Hz)
dC
dH (J in Hz)
dC
Dothideopyrone
B (3) possessed the molecular formula
2
3
4
5
–
–
–
164.6
101.3
169.2
92.6
169.2
70.8
35.5
25.1
29.0
–
–
–
164.9
101.3
169.4
92.9
169.4
70.6
35.3
25.1
29.0
31.7
22.5
14.0
61.3
56.9
–
C15H24O5, as deduced from APCI–TOF MS data. The 1H and 13C
NMR spectroscopic data of 3 were similar to those of 1, except
for the presence of additional methoxy signals in 3. In the HMBC
spectrum, 100-OMe protons (dH 3.33) showed correlation to C-100,
indicating that 3 was a 4-O-methyl derivative of 1. Protons and car-
bons in 3 were assigned by analysis of 1H–1H COSY and HMBC
spectra (Table 1).
6.40 (s)
–
6.35 (s)
–
4.32 (dd, 4.0, 8.1)
1.74 (m); 1.57 (m)
1.33 (m)
1.20 (m)
1.20 (m)
1.20 (m)
0.80 (t, 6.7)
4.32 (s)
3.85 (s)
–
–
–
–
–
–
–
6
10
4.41 (dd, 4.0, 8.1)
1.85 (m); 1.64 (m)
1.40 (m)
1.28 (m)
1.28 (m)
1.28 (m)
0.88 (t, 6.6)
4.37 (s)
3.92 (s)
–
20
30
40
50
32.0
Dothideopyrone C (4) has the molecular formula C21H30O6, as
indicated by APCI–TOF MS data. The 1H and 13C NMR spectroscopic
data of 4 were again similar to those of 1, suggesting that 4 was a
derivative of 1. Analysis of 1H and 13C NMR spectra of 4 indicated
additional signal characteristic of a 2-hydroxymethyl-3-methyl-
cyclopent-2-enone unit (Chomcheon et al., 2006). The HMBC spec-
trum of 4 showed correlations of H2-100 to C-6000, and H2-6000 to C-100,
indicating the presence of C-100/C-600 ether linkage between the 2-
hydroxymethyl-3-methyl-cyclopent-2-enone unit and a dothideo-
pyrone A (1) unit. The 1H–1H COSY and HMBC spectra assisted the
assignment of all proton and carbon signals for 4 (Table 2).
Dothideopyrone D (5) possessed 1H and 13C NMR spectra simi-
lar to those of 1. The 13C NMR spectrum of 5 showed 14 lines. How-
ever, the APCI–TOF MS data indicated the molecular formula
C28H42O9, indicating that 5 had C2 symmetry. The HMBC spectrum
readily established the ether linkage of the two identical units at
the hydroxyl methylene, showing correlations from H2-100 to C-
100000 and from H2-100000 to C-100. Upon acetylation of 5, a diacetate
derivative 6 was obtained; only the hydroxyl groups at C-10 and
C-10000 were acetylated, which further confirmed the structure of
5. Proton and carbon signals for 5 were assigned by analysis of
1H–1H COSY and HMBC spectra (Table 2). Pyrone dimers are very
rare in nature. To our knowledge, multiforisin D is the only natural
bis-pyrone dimer, previously isolated from the fungus Gelasinos-
pora multiforis (Fujimoto et al., 1995).
60
22.6
14.1
61.6
56.8
208.6
136.9
177.5
31.7
34.5
60.9
70
100
4-OMe
1000
2000
3000
4000
5000
6000
7000
–
–
–
–
–
–
–
–
2.53–2.55 (m)
2.38–2.40 (m)
4.20 (s)
2.20 (s)
17.7
a
Compound 5 has C2 symmetry, so NMR data for the two halves are identical.
2.2. Biological activity and prospects on fungal metabolites in white
biotechnology
The isolated compounds and derivatives were evaluated for
cytotoxicity toward nine cancer cell lines (Table 3). Compounds
1–4 and 9–15 were either inactive (at 50
weak activity (14.8–45.0 g/mL) towards some cell lines (Table
3). Dothideopyrone D (5) and its acetate derivative 6 exhibited
moderate cytotoxic activity (8.6–25.0 g/mL). Previous reports
lg/mL) or showed only
l
l
have demonstrated that many pyrones are not cytotoxic (Zhan
et al., 2007). However, members of this class show phytotoxic
activity (Pedras and Chumala, 2005), inhibitory effects on NF-kap-
paB (Folmer et al., 2008), and interactions with the GABA-A recep-
tor (Boonen and Haberlein, 1998). Questin (9) was recently found
to be an inhibitor of Cdc25B phosphatase (Choi et al., 2007).
Interestingly, the LRUB20 fungus cultured in M1D medium
(supplemented with 0.5 g/L malt extract) produced large quanti-
ties of cis, trans-muconic acid (15), up to 47.8 mg/L. Generally,
cis, cis-muconic acid and trans, trans-muconic acid are known
metabolites from the metabolism of benzene and phenol (or deriv-
atives) in living organisms, including humans (Tsai et al., 2005;
Xiao et al., 2007; Yoshikawa et al., 1993), and thus are employed
as biomarkers for benzene contamination in living organisms
(Melikian et al., 2002; Navasumrit et al., 2008, 2005; Yardley-Jones
et al., 1991). To date, only a monomethyl ester of cis, cis-muconic
acid isolated from a marine sponge Plakortis simplex has been re-
ported as a natural product (Shen et al., 2001). To our knowledge,
the present work is the first report on naturally occurring muconic
acid. Interestingly, a prior report showed that genetically engi-
neered Escherichia coli can catalytically convert D-glucose to cis,
cis-muconic acid (Draths and Frost, 1994). Consequently, such
muconic acid production initiated an environmentally compatible
synthesis of adipic acid, an important ingredient for the production
of nylon 6.6, gelatin, jams, polyamides, polyurethanes, and lubri-
cants (Niu et al., 2002; Thomas et al., 2003). The current industrial
production of adipic acid involves nitric acid oxidation of interme-
diates, generating N2O as a byproduct, which contributes to both
ozone depletion and global warming. Overproduction of cis,
trans-muconic acid (15) through a genetic manipulation of the
LRUB20 fungus, by analogy to that for the genetically engineered
E. coli (Draths and Frost, 1994), is a challenging research approach
Dothideopyrones A–D (1, 3, 4, and 5) all process a chiral second-
ary alcohol unit, and all exhibited similar negative specific rota-
tions. The absolute configuration of dothideopyrones A–D was
assigned by application of the modified Mosher’s method (Dale
and Mosher, 1973), and dothideopyrone D (5) was selected as a
model compound. (S)-MTPA (7) and (R)-MTPA (8) esters of 5 were
prepared. The
Dd values [d(S)–d(R)] of the MTPA esters indicated the
S-configuration at C-10 (or C-10000) in 5 (Fig. 2). Based on the similar
negative specific rotations, the absolute configuration of C-10 in
dothideopyrones A–D (1, 3, 4, and 5) was also assumed to be S.
Table 1
1H and 13C NMR spectroscopic data (CDCl3) for compounds 1 and 3.
Position
1
3
dH (J in Hz)
dC
dH (J in Hz)
dC
2
3
4
5
–
–
–
165.2
103.6
167.7
92.8
169.1
70.5
35.3
24.9
28.9
31.6
22.5
13.9
54.3
56.7
–
–
–
–
164.6
101.3
169.1
92.4
169.2
70.8
35.5
25.0
28.9
31.6
22.5
14.0
63.2
56.8
58.4
6.44 (s)
–
6.34 (s)
–
6
10
4.40 (dd, 4.0, 7.9)
1.82 (m); 1.64 (m)
1.40 (m)
1.25 (m)
1.25 (m)
1.25 (m)
0.86 (t, 6.7)
4.48 (s)
4.36 (dd, 4.1, 8.1)
1.79 (m); 1.58 (m)
1.35 (m)
1.21 (m)
1.21 (m)
1.21 (m)
0.81 (t, 6.7)
4.25 (s)
3.87 (s)
3.33 (s)
20
30
40
50
60
70
100
4-OMe
100-OMe
3.92 (s)
–

124
P. Chomcheon et al. / Phytochemistry 70 (2009) 121–127
to be pursued. Based upon this investigation, endophytic fungi
could prove to be potential sources of industrial chemicals, and
may therefore offer potential utility in the field of white
biotechnology.
2.3. Concluding remarks
Four new pyrone derivatives, dothideopyrones A–D (1, 3, 4, and
5), and seven known compounds, questin (9), asterric acid (10),
methyl asterrate (11), sulochrin (12), and eugenitin (13), 6-hydrox-
ymethyleugenitin (14), and cis, trans-muconic acid (15) were iso-
lated from the endophytic mitosporic Dothideomycete sp.
LRUB20. This is the first report on a naturally occurring muconic
acid, a biomarker in environments after exposure to benzene and
phenol. Although the cytotoxic properties of the isolated metabo-
lites are not promising, the LRUB20 fungus can produce muconic
acid (15) (47.8 mg/L). The latter is an important reference com-
pound in environmental studies. The fungus LRUB20 could also
produce a gram scale of 2-hydroxymethyl-3-methyl-cyclopent-2-
enone, a useful scaffold for organic synthesis (Chomcheon et al.,
2006). The LRUB20 fungus is a potential fungal candidate for the
use in the field of white biotechnology.
3. Experimental
3.1. General experimental procedures
1H and 13C NMR spectra were recorded on a Bruker AM 400
NMR instrument (operating at 400 MHz for 1H and 100 MHz for
13C) and a Bruker AVANCE 600 NMR spectrometer (operating at
600 MHz for 1H and 150 MHz for 13C). FTIR data were obtained
using a universal attenuated total reflectance (UATR) attachment
on a Perkin–Elmer Spectrum One spectrometer. APCI–TOF MS were
determined using a Bruker MicroTOFLC spectrometer. Optical rota-
tions were measured with sodium D line (590 nm) on JASCO DIP-
370 digital polarimeter.
3.2. Fungal material
The LRUB20 fungus used in this study was isolated as an endo-
phyte from a Thai medicinal plant, Leea rubra Blume ex Spreng. (fam-
ily Leeaceae) collected from a forest area in Pitsanulok Province,
Thailand. DNA sequence analysis of the 18S ribosomal RNA gene
demonstrated that this fungus was closely related (96% homology)
to Acrospermum compressum UME 31704 and A. gramineum UME
31190 which are currently classified in the Dothideomycetes incer-
tae sedis, subphylum Pezizomycotina (Eriksson, 2000; Lumbsch and
Huhndorf, 2007). However, microscopic morphology of the LRUB20
differs from that of Dactylaria and Virgariella, the known anamorphs
of A. compressum and A. gramineum, respectively (Tubaki, 1958;
Webster, 1956). Additionally, the ITS1–5.8S–ITS2 DNA sequence
was found to be unique, with the highest homology of only 78% to
Mycoleptodiscus terrestris, a member of the mitosporic Magnaporth-
aceae, Sordariomycetes incertae sedis, subphylum Pezizomycotina.
Accordingly, the LRUB20 fungus may represent a new species and
it was characterized as mitosporic Dothideomycete LRUB20 (Chom-
cheon et al., 2006). The 18S rRNA gene sequence and ITS sequence of
LRUB20 have been submitted to GenBank with accession number
DQ381536 and DQ384608, respectively.
3.3. Extraction and isolation
3.3.1. Metabolites obtained from Czapek yeast autolysate medium
The fungus LRUB20 was cultured in Czapek yeast autolysate
medium (5 L) for 21 days at 25 °C. Fungal cells and broth were

P. Chomcheon et al. / Phytochemistry 70 (2009) 121–127
125
separated by filtration, and the filtrate was extracted with an equal
volume of EtOAc three times to obtain a crude broth extract (1.4 g).
Fungal cells were extracted sequentially with MeOH and CH2Cl2,
yielding a crude extract (900 mg). The extract was subjected to
Sephadex LH-20 column chromatography (CC) (3 ꢁ 85 cm), eluted
with MeOH, to yield 14 fractions (A1–A14). Fraction A9 was re-
crystallized from MeOH to afford asterric acid (10; 147 mg). Frac-
tion A6 was further purified by Sephadex LH-20 CC (3 ꢁ 48 cm),
eluted with MeOH, and ten fractions (B1–B10) were obtained. Frac-
tion B6 was again subjected to chromatography purification on
Sephadex LH-20 CC (2.5 ꢁ 52 cm) using MeOH as a mobile phase
to provide fourteen fractions (B61–B614). Fraction B66 was sub-
jected to semi-preparative HPLC (C18 reversed-phase column;
MeCN:H2O (1:1, v/v) as eluent; flow rate of 8.0 mL/min) to furnish
dothideopyrone C (4; 28.8 mg; tR 8 min). Fraction B4 and B5 were
combined and subjected to further CC over silica gel (2.5 ꢁ 40 cm),
eluting with a mixture of EtOAc/CH2Cl2 (2:8, v/v) to yield ten frac-
1694, 1643, 1563, 1466, 1390, 1269, 1227, 1049, 823 cmꢀ1; ESI–
TOF–MS: m/z 401.1936 [M + Na]+ (calcd. for C15H24NaO5,
401.1940); for 1H and 13C NMR spectroscopic data (see Table 2).
3.4.4. Dothideopyrone D (5)
White amorphous solid; ½a D25
ꢀ 72 (c 0.22, CHCl3); UV (MeOH)
ꢂ
kmax (log e) 248 (3.0), 301 (5.0) nm; IR tmax 3398, 2927, 1687,
1640, 1561, 1466, 1393, 1270, 1229, 1033, 823 cmꢀ1; ESI–TOF–
MS: m/z 545.2715 [M+Na]+ (calcd. for C28H42NaO9, 545.2727); for
1H and 13C NMR spectroscopic data (see Table 2).
3.5. Acetylation of dothideopyrone A (1) and dothideopyrone D (5)
A solution containing dothideopyrone A (1; 22 mg), pyridine
(1 mL), and Ac2O (3 mL) was stirred at room temperature for 2 h.
H2O (6 mL) was added to the reaction mixture, which was subse-
quently extracted with CHCl3. The organic layer was evaporated
to give an oily mixture (33 mg), which was purified by preparative
TLC eluted with a mixture of CH2Cl2:EtOAc (9:1), to afford the ace-
tate 2 (17.8 mg). Acetylation of dothideopyrone D (5; 19.6 mg) was
performed in a similar manner, yielding acetate derivative 6
(9.1 mg).
tions (C1–C9). Fraction C4 contained dothideopyrone
A (1)
(126 mg), while fraction C6 gave dothideopyrone B (3) (12 mg).
Fraction C8 was further purified by preparative TLC using hex-
ane:acetone (2:1, v/v) as eluent to furnish dothideopyrone D (5)
(48 mg). Fraction A11 was re-crystallized from MeOH, and sub-
jected to preparative TLC eluted with a hexane:CH2Cl2:acetone
(1:1:1, v/v), yielding sulochrin (12) (10 mg). Fraction A13 was puri-
fied by preparative TLC, using acetone:hexane (1:1, v/v) as eluent,
to yield questin (9) (25 mg). Crude cell extract was separated by
Sephadex LH-20 CC (3 ꢁ 85 cm), eluted with MeOH, yielding
twelve fractions (D1–D12). Fractions D7 and D8 were combined
and subjected to further CC over silica gel (2 ꢁ 30 cm), eluting with
EtOAc/hexane (2:3, v/v), to yield eight fractions (E1–E8). Fraction
E3 gave methyl asterrate (11) (98 mg). Fraction D4 was subjected
to Sephadex LH-20 CC (2 ꢁ 100.5 cm) to give nine fractions (F1–
F9). Fraction F7 was separated on a preparative TLC using hex-
ane:acetone (7:3, v/v) as eluent, yielding eugenitin (13) (12 mg).
Compound 2: colorless oil; 1H NMR (CDCl3, 400 MHz) d 6.16
(1H, s, H-5), 5.37 (H, t, J = 6.7, H-10), 4.90 (2H, s, H-100), 3.88 (3H,
s, 4-OMe), 2.07 (3H, s, 10-CO2Me), 1.99 (3H, s, 100-CO2Me), 1.83
(2H, m, H-20), 1.21 (8H, m, H-30, H-40, H-50 and H-60), 0.81 (3H, t,
13
J = 6.7, H-70); C NMR (CDCl3, 100 MHz) d 171.0 (C, 100-CO2Me),
169.9 (C, 10-CO2Me), 168.4 (C, C-6), 163.8 (C, C-4), 162.7 (C, C-2),
100.5 (C, C-3), 94.4 (CH, C-5), 72.3 (CH, C-10), 56.8 (CH3, 4-OMe
or 4000-OMe), 56.1 (CH2, C-100), 32.1 (CH2, C-20), 31.5 (CH2, C-50),
28.7 (CH2, C-40), 24.9 (CH2, C-30), 22.5 (CH2, C-60), 20.9 (CH3, 100-
CO2Me), 20.8 (CH3, 10-CO2Me), 13.9 (CH3, C-70); ESI–TOF–MS: m/z
389.1382 [M + Cl]ꢀ (calcd. for C18H26Cl O7, 389.1367).
Compound 6: colorless oil; 1H NMR (CDCl3, 400 MHz) d 6.20
(2H, s, H-5 or H-5000), 5.42 (2H, t, J = 6.7, H-10 or H-10000), 4.44 (4H,
s, H-100 or H-100000), 3.92 (6H, s, 4-OMe or 4000-OMe), 2.12 (6H, s, 10-
CO2Me or 10000-CO2Me), 1.87 (4H, m, H-20 or H-20000), 1.28 (16H, m,
H-30 or H-30000, H-40 or H-40000, H-50 or H-50000, and H-60 or H-60000),
0.88 (6H, t, J = 6.7, H-70 or H-70000); 13C NMR (CDCl3, 100 MHz) d
169.9 (C, 10-CO2Me or 10000-CO2Me), 168.3 (C, C-6 or C-6000), 163.9
(C, C-4 or C-4000), 162.8 (C, C-2 or C-2000), 102.9 (C, C-3 or C-3000),
94.9 (CH, C-5 or C-5000), 72.3 (CH, C-10 or C-10000), 61.5 (CH2, C-100 or
C-100000), 56.7 (CH3, 4-OMe or 4000-OMe), 32.1 (CH2, C-20 or C-20000),
31.5 (CH2, C-50 or C-50000), 28.8 (CH2, C-40 or C-40000), 24.9 (CH2, C-30
3.3.2. Metabolites obtained from M1D medium (supplemented with
malt extract)
The fungus LRUB20 was grown in M1D medium (supplemented
with 0.5 g/L malt extract; 3.2 L) for 21 days at 25 °C. Mycelia were
separated from the culture broth by filtration, and the broth was
subsequently extracted three times with an equal volume of EtOAc,
yielding a crude extract (630 mg). The extract was purified by
MPLC (C18 reversed-phase column, 3.6 ꢁ 46 cm), with a stepwise
elution starting with a mixture of MeOH/H2O (1:1, v/v) to MeOH,
to afford cis, trans-muconic acid (15; 153 mg, tR 22 min) and 6-
hydroxymethyleugenitin (14; 72.3 mg, tR 66 min).
or C-30000), 22.5 (CH2, C-60 or C-60000), 20.8 (CH3, 10-CO2Me or 10000
-
CO2Me), 13.9 (CH3, C-70 or C-70000); ESI–TOF–MS: m/z 641.2723
[M + Cl]ꢀ (calcd. for C32H46Cl O11, 641.2729).
3.4. Spectroscopic data of compounds
3.6. Preparation of (R)- and (S)-MTPA esters of 5
3.4.1. Dothideopyrone A (1)
Colorless oil; ½a D25
ꢂ
ꢀ 77 (c 0.22, CHCl3); UV (MeOH) kmax (log
e
)
The reaction mixture containing dothideopyrone D (5; 3 mg),
248 (3.2), 299 (4.1) nm; IR tmax 3373, 2926, 1682, 1600, 1466,
1392, 1252, 1138, 999, 795 cmꢀ1; ESI–TOF–MS: m/z 271.1540
[M + H]+ (calcd. for C14H23O5, 271.1546); for 1H and 13C NMR spec-
troscopic data (see Table 1).
(R)-(ꢀ)-
a-methoxy-a-(trifluoromethyl)phenylacetic acid (MTPA;
13.9 mg), N-[3-(dimethyllaminopropyl)-N0-ethylcarbodiimide hy-
drochloride (12.6 mg), and a catalytic amount of N,N-(dimethyl-
amino)pyridine was dissolved in 4 mL of (CH2)2Cl2, and heated
until reflux began, this being maintained for 4 h. The reaction mix-
ture was added to H2O (5 mL) and extracted with CHCl3 (5 mL). The
mixture was purified by preparative TLC, using EtOAc–CH2Cl2 (1:4,
v/v) as an eluent, to give the (R)-MTPA ester (4.5 mg). The (S)-
MTPA ester was prepared in the same manner; dothideopyrone D
3.4.2. Dothideopyrone B (3)
Yellow viscous oil; ½a D25
ꢀ 45 (c 0.15, CHCl3); UV (MeOH) kmax
ꢂ
(log e) 233 (3.0), 299 (3.7) nm; IR tmax 3400, 2925, 1687, 1561,
1466, 1395, 1230, 1083, 800 cmꢀ1; ESI–TOF–MS: m/z 307.1516
[M + Na]+ (calcd. for C15H24NaO5, 307.1521); for 1H and 13C NMR
spectroscopic data (see Table 1).
(5) was reacted with (S)-(ꢀ)-
a-methoxy-a-(trifluoromethyl)-
phenylacetic acid (MTPA), to give the (S)-MTPA ester (5.0 mg).
(S)-MTPA ester (Data for half of a molecule); 1H NMR (CDCl3,
400 MHz) d 7.53 (2H, m, aromatic signals of MTPA), 7.43 (3H, m,
aromatic signals of MTPA), 6.23 (1H, s, H-5), 5.67 (1H, t, J = 6.4,
H-10), 4.54 (2H, s, H-100), 3.83 (3H, s, 4-OMe), 3.52 (3H, br d, OMe
3.4.3. Dothideopyrone C (4)
Yellow viscous oil; ½a D25
ꢀ 64 (c 0.10, MeOH); UV (MeOH) kmax
ꢂ
(log e) 207 (4.4), 228 (4.0), 299 (3.9) nm; IR tmax 3403, 2927,

126
P. Chomcheon et al. / Phytochemistry 70 (2009) 121–127
of MTPA), 1.94 (1H, m, H-20a), 1.60 (1H, m, H-20b), 1.25 (4H, m, over-
lapping signals of H-30 and H-40), 1.23 (2H, m, H-40), 1.23 (2H, m, H-
50), 1.23 (2H, m, H-60), 0.86 (3H, t, J = 6.9, H-70); ESI–TOF–MS: m/z
955.3620 [M + H]+(calcd. for C48H57F6O13, 955.3703). (R)-MTPA es-
ter (Data for half of a molecule); 1H NMR (CDCl3, 400 MHz) d 7.51
(2H, m, aromatic signals of MTPA), 7.44 (3H, m, aromatic signals of
MTPA), 5.95 (1H, s, H-5), 5.72 (1H, t, J = 6.1, H-10), 4.52 (2H, s, H-
100), 3.69 (3H, s, 4-OMe), 3.58 (3H, br d, OMe of MTPA), 1.95 (1H, m,
H-20a), 1.60 (1H, m, H-20b), 1.29 (4H, m, overlapping signals of H-30
and H-40), 1.25 (2H, m, H-40), 1.25 (2H, m, H-50), 1.25 (2H, m, H-60),
0.88 (3H, t, J = 6.7, H-70); ESI–TOF–MS: m/z 955.3610 [M + H]+ (calcd.
for C48H57F6O13, 955.3703).
Choi, S.G., Kim, J., Sung, N.D., Son, K.H., Cheon, H.G., Kim, K.R., Kwon, B.M., 2007.
Anthraquinones, Cdc25B phosphatase inhibitors, isolated from the roots of
Polygonum multiflorum Thunb. Nat. Prod. Res. 21, 487–493.
Chomcheon, P., Sriubolmas, N., Wiyakrutta, S., Ngamrojanavanich, N., Chaichit, N.,
Mahidol, C., Ruchirawat, S., Kittakoop, P., 2006. Cyclopentenones, scaffolds for
organic synthesis produced by the endophytic fungus, mitosporic
Dothideomycete sp. LRUB20.. J. Nat. Prod. 69, 1351–1353.
Chomcheon, P., Wiyakrutta, S., Sriubolmas, N., Ngamrojanavanich, N., Isarangkul, D.,
Kittakoop, P., 2005. 3-Nitropropionic acid (3-NPA), a potent antimycobacterial
agent from endophytic fungi: Is 3-NPA in some plants produced by
endophytes? J. Nat. Prod. 68, 1103–1105.
Dale, J.A., Mosher, H.S., 1973. Nuclear magnetic resonance enantiomer
reagents. Configurational correlations via nuclear magnetic resonance
chemical shifts of diastereomeric mandelate, O-methylmandelate, and
methoxy- -trifluoromethylphenylacetate (MTPA) esters. J. Am. Chem. Soc.
95, 512–519.
a-
a
Doyle, A., Griffiths, J.B., 1997. Mammalian Cell Culture: Essential Techniques. John
Wiley and Sons.
Draths, K.M., Frost, J.W., 1994. Environmentally compatible synthesis of adipic acid
from D-glucose. J. Am. Chem. Soc. 116, 399–400.
3.7. Assay of cytotoxic activity
The MTT assay (Carmichael et al., 1987; Doyle and Griffiths,
1997; Mosmann, 1983; Tominaga et al., 1999) was applied for
the evaluation of cytotoxicity against HeLa, HuCCA-1, HepG2,
T47D, MDA–MB231, S102, and A549 cancer cell lines. Cells were
Elvidge, J.A., Linstead, R.P., Peter, S., Orkin, B.A., 1950. The third isomeric (cis-trans)-
muconic acid. J. Chem. Soc., 2235–2241.
Eriksson, O., 2000. Notes on ascomycete systematics. Myconet 5, 1–35.
Feng, Y., Blunt, J.W., Cole, A.L., Munro, M.H., 2002. The isolation of two new
chromone derivatives from the New Zealand fungus Tolypocladium extinguens. J.
Nat. Prod. 65, 1681–1682.
Folmer, F., Harrison, W.T., Tabudravu, J.N., Jaspars, M., Aalbersberg, W., Feussner, K.,
Wright, A.D., Dicato, M., Diederich, M., 2008. NF-kappaB-inhibiting
naphthopyrones from the Fijian echinoderm Comanthus parvicirrus. J. Nat.
Prod. 71, 106–111.
Fujimoto, H., Satoh, Y., Nakayama, M., Takayama, T., Yamazaki, M., 1995. Isolation of
some immunosuppressive components from an ascomycete, Gelasinospora
multiforis. Chem. Pharm. Bull. 43, 547–552.
Gunatilaka, A.A.L., 2006. Natural products from plant-associated microorganisms:
distribution, structural diversity, bioactivity, and implications of their
occurrence. J. Nat. Prod. 69, 509–526.
Hargreaves, J., Park, J.O., Ghisalberti, E.L., Sivasithamparam, K., Skelton, B.W., White,
A.H., 2002. New chlorinated diphenyl ethers from an Aspergillus species. J. Nat.
Prod. 65, 7–10.
plated in a 96-well microplate (Costar No. 3599, USA, 100 lL/well
at a density of 5 ꢁ 103–2 ꢁ 104 cells/well), and incubated for 24 h
at 37 °C under 5% CO2 and 95% humidity. The tested compounds
at various concentrations were added to cell lines, which were
incubated further for 48 h. Cell viability was determined by stain-
ing with MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoli-
um bromide]. The MTT stock solution (5 mg/mL) was prepared in
phosphate buffered saline (PBS), which was diluted (1:10) with a
culture medium prior to use. After removing the culture medium,
the diluted MTT solution (50
and plates were incubated at 37 °C under 5% CO2 and 95% humidity
for 2–4 h. Subsequently, DMSO (100 L) was added to dissolve the
lL) was added to the adhesive cells,
Hipkin, C.R., Simpson, D.J., Wainwright, S.J., Salem, M.A., 2004. Nitrification by
plants that also fix nitrogen. Nature 430, 98–101.
l
resulting formazan by sonication. The plates were read on a micro-
plate reader (Molecular Devices, CA, USA), using a test wavelength
of 550 nm and a reference wavelength of 650 nm.
The cytotoxic activity against non-adhesive cells, HL-60 and
MOLT-3 cell lines, was evaluated using the XTT assay (Doyle and
Griffiths, 1997). Cells were plated in a 96-well microplate as men-
tioned above, and the tested compounds at various concentrations
were added to cell lines, which were incubated further for 48 h. A
dye solution was prepared by mixing 5 mL of XTT (2,3-bis-(2-
methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide)
Huneck, S., 1972. 6-Hydroxymethyleugenitin, ein neues chromon aus Roccella
fuciformis. Phytochemistry 11, 1489–1490.
Kalidhar, S.B., 1989. Structural elucidation in anthraquinones using 1H NMR
glycosylation and alkylation shifts. Phytochemistry 28, 3459–3463.
Lumbsch, H.T., Huhndorf, S.M., 2007. Outline of Ascomycota – 2007. Myconet 13, 1–
58.
Maury, J., Asadollahi, M.A., Moller, K., Clark, A., Nielsen, J., 2005. Microbial
isoprenoid production: an example of green chemistry through metabolic
engineering. Adv. Biochem. Eng. Biotechnol. 100, 19–51.
Melikian, A.A., Qu, Q., Shore, R., Li, G., Li, H., Jin, X., Cohen, B., Chen, L., Li, Y.,
Yin, S., Mu, R., Zhang, X., Wang, Y., 2002. Personal exposure to different
levels of benzene and its relationships to the urinary metabolites S-
phenylmercapturic acid and trans, trans-muconic acid. J. Chromatogr. B 778,
211–221.
solution (1 mg/mL) with 100
lL of phenazine methosulfate solu-
Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival:
application to proliferation and cytotoxicity assays. J. Immunol. Methods 65,
55–63.
tion (0.383 mg/mL). The dye solution (50
l
L) was added to cells,
which were further incubated for 4 h. The plates were read on a
microplate reader (Molecular Devices, CA, USA) at the wavelengths
of 492 and 690 nm. Etoposide and doxorubicin were used as the
reference drugs (Table 3).
Navasumrit, P., Arayasiri, M., Hiang, O.M., Leechawengwongs, M., Promvijit, J.,
Choonvisase, S., Chantchaemsai, S., Nakngam, N., Mahidol, C., Ruchirawat, M.,
2008. Potential health effects of exposure to carcinogenic compounds in incense
smoke in temple workers. Chem. Biol. Interact. 173, 19–31.
Navasumrit, P., Chanvaivit, S., Intarasunanont, P., Arayasiri, M., Lauhareungpanya,
N., Parnlob, V., Settachan, D., Ruchirawat, M., 2005. Environmental and
occupational exposure to benzene in Thailand. Chem. Biol. Interact. 153–154,
75–83.
Acknowledgements
P.K. thanks The Thailand Research Fund (Grant No.
DBG5180014) and the Center of Excellence on Environmental
Health, Toxicology and Management of Chemicals (ETM) for finan-
cial support. S.W. and N.S. acknowledge a research grant from
Mahidol University. We thank CRI colleagues from the Laboratories
of Immunology, Pharmacology, and Biochemistry, and the Inte-
grated Research Unit for cytotoxicity tests.
Niu, W., Draths, K.M., Frost, J.W., 2002. Benzene-free synthesis of adipic acid.
Biotechnol. Prog. 18, 201–211.
Pedras, M.S., Chumala, P.B., 2005. Phomapyrones from blackleg causing
phytopathogenic fungi: isolation, structure determination, biosyntheses and
biological activity. Phytochemistry 66, 81–87.
Pongcharoen, W., Rukachaisirikul, V., Phongpaichit, S., Kuhn, T., Pelzing, M.,
Sakayaroj, J., Taylor, W.C., 2008. Metabolites from the endophytic fungus
Xylaria sp. PSU-D14. Phytochemistry 69, 1900–1902.
Rukachaisirikul, V., Sommart, U., Phongpaichit, S., Sakayaroj, J., Kirtikara, K., 2008.
Metabolites from the endophytic fungus Phomopsis sp. PSU-D15.
Phytochemistry 69, 783–787.
References
Schulz, B., Boyle, C., Draeger, S., Rommert, A.K., Krohn, K., 2002. Endophytic fungi: a
source of novel biologically active secondary metabolites. Mycological Res. 106,
996–1004.
Barba, B., Diaz, J.G., Herz, W., 1992. Anthraquinones and other constituents of two
Senna species. Phytochemistry 31, 4374–4375.
Schwarz, M., Kopcke, B., Weber, R.W., Sterner, O., Anke, H., 2004. 3-
hydroxypropionic acid as
Phytochemistry 65, 2239–2245.
a nematicidal principle in endophytic fungi.
Boonen, G., Haberlein, H., 1998. Influence of genuine kavapyrone enantiomers on
the GABA-A binding site. Planta Med. 64, 504–506.
Shen, Y.C., Prakash, C.V., Kuo, Y.H., 2001. Three new furan derivatives and a new
fatty acid from a Taiwanese marine sponge Plakortis simplex. J. Nat. Prod. 64,
324–327.
Carmichael, J., DeGraff, W.G., Gazdar, A.F., Minna, J.D., Mitchell, J.B., 1987.
Evaluation of
a
tetrazolium-based semiautomated colorimetric assay:
assessment of chemosensitivity testing. Cancer Res. 47, 936–942.

P. Chomcheon et al. / Phytochemistry 70 (2009) 121–127
127
Shimada, A., Shiokawa, C., Kusano, M., Fujioka, S., Kimura, Y., 2003.
Hydroxysulochrin, tea pollen growth inhibitor from the fungus
Aureobasidium sp.. Biosci. Biotechnol. Biochem. 67, 442–444.
Stermitz, F.R., Schroeder, H.A., Geigert, J., 1973. Asterric acid from Scytalidium.
Phytochemistry 12, 1173.
Tsai, S.C., Tsai, L.D., Li, Y.K., 2005. An isolated Candida albicans TL3 capable of
degrading phenol at large concentration. Biosci. Biotechnol. Biochem. 69, 2358–
2367.
Tubaki, K., 1958. Studies on Japanese hyphomycetes. J. Hattori Bot. Lab. 20, 142–
244.
a
Stone, J.K., Polishook, J.D., White, J.F., 2004. Endophytic fungi. In: Mueller, G.M., Bills,
G.F., Foster, M.S. (Eds.), Biodiversity of Fungi: Inventory and Monitoring
Methods. Elsevier Academic Press, China, pp. 241–270.
Stottmeister, U., Aurich, A., Wilde, H., Andersch, J., Schmidt, S., Sicker, D., 2005.
White biotechnology for green chemistry: fermentative 2-oxocarboxylic acids
as novel building blocks for subsequent chemical syntheses. J. Ind. Microbiol.
Biotechnol. 32, 651–664.
Webster, J., 1956. Conidia of Acrospermum compressum and A. Gramineum. Trans. Br.
Mycol. Soc. 39, 361–366.
Wijeratne, E.M.K., Paranagama, P.A., Gunatilaka, A.A.L., 2006. Five new isocoumarins
from Sonoran desert plant-associated fungal strains Paraphaeosphaeria
quadriseptata and Chaetomium chiversii. Tetrahedron 62, 8439–8446.
Wiyakrutta, S., Sriubolmas, N., Panphut, W., Thongon, N., Danwisetkanjana, K.,
Ruangrungsi, N., Meevootisom, V., 2004. Endophytic fungi with anti-microbial,
anti-cancer and anti-malarial activities isolated from Thai medicinal plants.
World J. Microbiol. Biotechnol. 20, 265–272.
Strobel, G., 2006. Harnessing endophytes for industrial microbiology. Curr. Opin.
Microbiol. 9, 240–244.
Strobel, G., Yang, X., Sears, J., Kramer, R., Sidhu, R.S., Hess, W.M., 1996. Taxol from
Pestalotiopsis microspora, an endophytic fungus of Taxus wallachiana.
Microbiology 142, 435–440.
Xiao, Y., Zhang, J.J., Liu, H., Zhou, N.Y., 2007. Molecular characterization of a novel
ortho-nitrophenol catabolic gene cluster in Alcaligenes sp. strain NyZ215. J.
Bacteriol. 189, 6587–6593.
Tan, R.X., Zou, W.X., 2001. Endophytes: a rich source of functional metabolites. Nat.
Prod. Rep. 18, 448–459.
Thomas, J.M., Raja, R., Johnson, B.F., O’Connell, T.J., Sankar, G., Khimyak, T., 2003.
Bimetallic nanocatalysts for the conversion of muconic acid to adipic acid.
Chem. Commun., 1126–1127.
Yardley-Jones, A., Anderson, D., Parke, D.V., 1991. The toxicity of benzene and its
metabolism and molecular pathology in human risk assessment. Br. J. Ind. Med.
48, 437–444.
Yoshikawa, N., Ohta, K., Mizuno, S., Ohkishi, H., 1993. Production of cis, cis-muconic
acid from benzoic acid. Bioprocess Technol. 16, 131–147.
Tominaga, H., Ishiyama, M., Ohseto, F., Sasamoto, K., Hamamoto, T., Suzuki, K.,
Watanabe, M., 1999. A water-soluble tetrazolium salt useful for colorimetric
cell viability assay. Anal. Commun. 36, 47–50.
Zhan, J., Gunaherath, G.M., Wijeratne, E.M., Gunatilaka, A.A., 2007. Asperpyrone D
and other metabolites of the plant-associated fungal strain Aspergillus
tubingensis. Phytochemistry 68, 368–372.