FULL PAPER
DOI: 10.1002/chem.200901556
Discovery of New Natural Products by Intact-Cell Mass Spectrometry and
LC-SPE-NMR: Malbranpyrroles, Novel Polyketides from Thermophilic
Fungus Malbranchea sulfurea
Yu-Liang Yang,[a] Wen-Ying Liao,[b, c] Wan-Yun Liu,[a] Chih-Chuang Liaw,[b, d]
Chia-Ning Shen,[c] Zih-You Huang,[a] and Shih-Hsiung Wu*[a]
Dedicated to the late Professor Kuei-Yu Chen for her contribution to thermophilic fungi research in Taiwan
Abstract: Six photosensitive polyketides, malbranpyrroles A–F, were discovered from the thermophilic fungus
Malbranchea sulfurea by using intactcell desorption/ionization on silicon
mass (ICD-MS) and LC-SPE-NMR.
These two strategies facilitate the
searching and structural determination
of unstable natural products. The ICD-
MS indicated that only brown hyphae
of M. sulfurea can produce malbranpyrroles. The biosynthetic pathway of malKeywords: Malbranchea sulfurea ·
mass
spectrometry
·
natural
products · NMR spectroscopy ·
polyketides
Introduction
The generation of secondary metabolites by microorganism
shows strong correlations to the growth conditions, such as
media, pH values, temperatures, and so on. Incubating microorganism on different media in order to search for bioactive natural products is a common strategy in the pharmaceutical industry. For example, the antibiotic platensimycin
[a] Dr. Y.-L. Yang, W.-Y. Liu, Z.-Y. Huang, Prof. Dr. S.-H. Wu
Institute of Biological Chemistry
Academia Sinica, Taipei 115 (Taiwan)
Fax: (+ 866) 2-2653-9142
E-mail: [email protected]
[b] W.-Y. Liao, Dr. C.-C. Liaw
Graduate Institute of Pharmaceutical Chemistry
College of Pharmacy, China Medical University
Taichung 404 (Taiwan)
[c] W.-Y. Liao, Dr. C.-N. Shen
Genomics Research Center
Academia Sinica, Taipei 115 (Taiwan)
[d] Dr. C.-C. Liaw
Department of Life Sciences
National Chung-Hsing University, Taichung 402 (Taiwan)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200901556.
Chem. Eur. J. 2009, 00, 0 – 0
branpyrroles was evidenced by 13C isotope precursors and amino acid feeding
experiments. The cytotoxicity data revealed that the conformation of the
conjugated system in malbranpyrroles
does not affect cytotoxic potency
against cancer cell lines. In addition,
the chlorine atom was shown to be the
pharmacophore for cytotoxicity.
was discovered from 83 000 strains under three growth conditions.[1] By following biological function-guided fractionation and purification strategies, HPLC-appended systems,
such as LC-MS, LC NMR, and some off-line spectroscopic
techniques, such as quantitative NMR spectroscopy
(qNMR),[2] provide valuable chemical information in searching for new bioactive natural products. These techniques are
all applied to collecting structural information from bioactive extracts or fractions. However, because of the complex
extraction and fractionation methods it is sometimes difficult to observe unstable but active natural products. Also,
the different growth stages or morphologies of microorganisms might produce various secondary metabolites and/or
their biosynthetic intermediates. Intact-cell mass spectrometry techniques can provide a real-time screening strategy for
searching these compounds. Intact-cell MALDI mass spectrometry (ICM-MS) has been reported together with 16 S ribosomal DNA sequence analysis and bioassays as a screen
for bacteria from marine sources.[3] Otherwise, because of its
matrix-free character, desorption/ionization on silicon mass
(DIOS) opens a new technique for natural product investigation.[4] In the bioassay screening of thermophilic fungi, the
ethyl acetate extract of Malbranchea sulfurea showed strong
cytotoxicity against various cancer cell lines. In this study,
we used intact-cell DIOS mass (ICD-MS) together with LCsolid phase extraction-NMR spectroscopy (LC-SPE-NMR)
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to search the secondary metabolites of M. sulfurea. Both
techniques offered us valuable information on isolation and
structural determination of photosensitive polyketides, malbranpyrroles A–F (1–6).
Results and Discussion
In the ICD-MS spectra, several highly abundant signals between 300–400 Da were observed and some of them were
halogenated compounds based on their specific isotope distribution. We found that these particular signals are only
produced by brown hyphae of M. sulfurea, which generated
much more spores than the white hyphae of M. sulfurea, as
observed by microscopy (Figure 1). Furthermore, we applied
LC-diode array detector-MS (LC-DAD-MS) to screen the
crude extracts of the brown hyphae of M. sulfurea. The results indicated that M. sulfurea can produce photosensitive
compounds, which are also the major signals observed in
ICD-MS spectra (Figures S1 and S2 in the Supporting Information). Two strategies were applied in this study in order
to determine the native structures of photosensitive components: 1) by using LC-SPE-NMR and LC-MS, which can
help us to elucidate the native structures of photosensitive
components because they are difficult to purify; 2) by purifying these compounds in the dark room for off-line NMR
spectroscopy and MS structural analysis. In this study, six
photosensitive polyketides, malbranpyrrole A–F (1–6;
Figure 2) were determined and five of them (1 and 3–6)
were purified. The photosensitive isomers of isolated com-
Figure 1. ICD-MS spectra of: A) brown, and B) white hyphae in two-week M. sulfurea cultures.
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compounds, auxarconjugatin A
and 12E-isorumbrin, possessed
potent cytotoxic properties
against an NS-1 cell line (LD99
2.3 and 0.41 mg mL 1). Meanwhile, the authors indicated
that the chloride metabolites
can be transferred into bromide or dechloride metabolites, which displayed significantly reduced cytotoxicity
when the fungi were incubated
in medium containing NaBr.
However, we have not observed any bromide or iodide
metabolites produced from
M. sulfurea incubated in PDA
medium containing KBr or KI
using ICD-MS; this was also
confirmed by LC-DAD-MS
analysis of crude extracts (Figure S3 in the Supporting InforFigure 2. Structures of malbranpyrroles A–F (1–6) and 1H-1H COSY (bold lines), HMBC (arrow) correlations
mation).
of malbranpyrroles A (1), C–F (3–6).
The molecular formula of
malbranpyrrole A (1) was confirmed to be C21H25NO3 with ten double bond equivalents
pounds are also shown in the HPLC profiles (Figure 3). In
2006, some similar structures were reported from Gymnoas(DBE) by HRESIMS ([M + + H]: m/z 340.1907). The strong
cus reessii.[5] Although the authors did not emphasize the
UV absorption at 385 nm implied that compound 1 is funcphotoisomerization of those polyketides, the halogenated
tionalized with a polyene group.[5] In the 1H NMR spectra,
nine olefinic protons, including
one trans disubstituted double
bond and one monosubstituted terminal double bond were
observed together with two allylic methyl groups, two quaternary methyl groups, one
methoxyl group, and one exchangeable amine proton. According to interpretation of
1
H-1H COSY and
HMBC
spectra (Figure 2) the signals
dH 6.94/dC 120.1, dH 6.23/dC
110.1, dH 6.46/dC 111.4, and
amine signal dH 10.23, were an
a-monosubstituted
pyrrole
ring, which showed long-range
correlations with the methylene (dH 6.74/dC 126.9) of trisubstituted double bond. The
other two substitutions of this
trisubstituted double bond,
one allylic methyl group at dH
2.18 and the trans disubstitutFigure 3. RP-HPLC profiles of purified malbranpyrroles. After UV irradiation: panel 1 is malbranpyrrole D
ed double bond, were also re(4); panel 2 is malbranpyrrole A (1) and F (6); panel 3 is malbranpyrrole C (3) and E (5); panel 4 is the mixvealed by 1H-1H COSY and
ture of malbranpyrroles A (1) and C–F (3–6). Before UV irradiation: panel 5 is the mixture of malbranpyrroHMBC analysis.
les A (1) and C–F (3–6). The isomers of malbranpyrroles were named according to the order of retention time
(e.g., A1, A2, A3).
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In the HMBC spectrum, the correlations between methyl
group dH 1.95 and carbonyl carbon dC 179.7, olefinic quaternary carbons dC 118.2 and 152.2 were observed. The cross
peaks between trans disubstituted double bond (dH 7.16 and
6.55) and dC 152.2 suggested the connection of this double
bond and a,b-unsaturated carbonyl moiety (dC 152.2, 118.2
and 179.7). Besides, a methoxyl group (dC 4.07) and an isoprenyl group containing a terminal double bond were determined, respectively, to link the quaternary carbons dC 161.5
and 108.6, which correspond to a tetrasubstituted double
bond. According to the molecular formula and DBE of
compound 1, one oxygen atom is inserted between the two
low-field carbons dC 152.2 and 161.5 to form a g-pyrone
ring. NOE correlation (Figure 4) between the methoxyl
group and the methylene (dH 7.16) confirmed the substitu-
Figure 4. NOESY correlations of malbranpyrroles A (1), C–F (3–6).
tion of a position (dC 161.5) in the g-pyrone ring as methoxyl group and the b position (dC 108.6) is connected with the
isoprenyl group. The other NOE correlations: dH 10.23/dH
6.74, dH 6.74/dH 7.16, dH 6.46/dH 2.18, dH 2.18/dH 6.55, and dH
6.55/dH 1.95, revealed the conformation of compound 1.
The molecular formula of malbranpyrrole C (3) was
found to be C21H24ClNO3 with 10 DBE by HRESIMS ([M +
+ H]: m/z 374.1532). The strong UV absorption at 381 nm
revealed that 3 is a polyene compound. The 1H and 13C
chemical shifts together with HMBC and 1H-1H COSY interpretations revealed that the plane structure of compound 3
is similar to malbranpyrrole A (1) except for the pyrrole
moiety. One exchangeable amine proton (dH 10.35), two
methylenes (dH 6.97/dC 121.8 and dH 6.20/dC 110.1), and two
quaternary carbons (dC 115.4 and 126.5) were assigned to
the pyrrole moiety of compound 3; this suggests that the
chlorine atom is substituted in the pyrrole ring. The NOE
correlation (Figure 4) between the NH and methyl group at
dH 2.22 indicates that the conformation of the pyrrole ring
in 3 is different from 1. The stereohindrance effect between
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the chlorine atom and methyl group in the biosynthetic
pathway might be the cause of the conformational change in
the pyrrole moiety.
Malbranpyrrole D (4), C20H22ClNO3, which was deduced
from HRESIMS at m/z 360.1360 [M + + H], showed a similar
UV absorption to malbranpyrrole C (3). Like compound 3,
malbranpyrrole D (4) has a chlorinated pyrrole ring. However, the pyrone moiety and its substituents of 4 are significantly different from those of 3. The methoxyl group and
the terminal double bond of isoprenyl group signals were
not observed in the NMR spectra. Compound 4 showed the
same DBE as compounds 1 and 3; this implies that there is
one more ring in the structure of 4 than in those of compounds 1 and 3. In the HMBC spectrum, the correlations
between methyl group dH 2.03 and dC 158.1, 104.5, and 170.0
were observed, which corresponds to a a-pyrone moiety
rather than a g-pyrone in compounds 1 and 3.[5] In compound 4, a dihydrofuran ring
adjacent to the a-pyrone is
formed by a nucleophilic
attack of g-OH in the apyrone ring on the terminal
double bond of isoprenyl
group, which was confirmed by
observing a tertiary methyl
group and an oxymethylene
NMR signal. The conformations of the conjugated double
bond system and pyrrole ring
in 4 were found to be the same
as those in 3 by NOESY analysis (Figure 4).
Because of its unstable
nature malbranpyrrole B (2)
could not be purified for offline structural determination. The spectroscopic data were
collected with a HPLC-appended systems (LC-SPE-NMR,
LC-DAD-MS) for structural elucidation. The molecular formula C20H23NO3, which represents compound 2, is a dechloro analogue of compound 4. Most NMR signals of 2 and 4
can be superposed with each other except for the pyrrole
ring; this indicates that compound 2 contains a a-monosubstituted pyrrole ring. The 13C signals of the a-monosubstituted pyrrole ring in compound 2, which were deduced indirectly from the HSQC spectrum by LC-SPE-NMR, are identical with those in compound 1. Furthermore, without the
stereohindrance effect between the chlorine atom and
methyl group, the conformation of the pyrrole moiety in 2
was presumed to be the same as that of 1 rather than 3 and
4.
The other two a-pyrone analogues, malbranpyrroles E
and F (5, 6) were found to have the molecular formulae
C20H22ClNO3 and C19H20ClNO3, respectively. The a-pyrone
in 5 was substituted with one methoxyl and one isoprenyl
group, on the other hand, a dihydrofuran ring adjacent to a-
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pyrone was observed in 6. However, in compounds 2 and 4,
the allylic methyl group in the d position of the pyrone ring
was not observed in compounds 5 and 6. The methyl substituent on the conjugated double bond of 5 and 6, which
was deduced from 1H-1H COSY correlations of H-6/H-7, H7/H-8, H-8/H-15, is also different from 1–4. The HMBC correlations between the methyl group (H-15) and e-carbon (C10) of a-pyrone together with H-6 and C-4, C-5 confirmed
the above assignment. Both E and F have chlorine atoms
substituted in the pyrrole rings. The conformations were revealed by NOE correlations of NH/H-7, H-7/H-15, H-15/H11, and H-6/H-8 (Figure 4).
From the biosynthetic perspective, malbranpyrroles could
be regarded as three separate building blocks. The first one
is a polyketide intermediate directly derived from intact acetate units joined in a head-to-tail fashion of a polyketide
pathway. The second is an isoprenoid, which should be generated through the mevalonate or the methylerythritol phosphate pathway. The third part, pyrrole and chloropyrrole, is
formed through proline or its upstream precursor. We investigated the incorporation of [1-13C]acetate, [2-13C]acetate,
[13C2]acetate, and [2-13C]glycerol into 6. The results proved
that the isoprenoid building block is formed through the mevalonate pathway (Table S1 in the Supporting Information;
Figure 5). It is not surprising that the mevalonate pathway
has been revealed to be the only rout to contribute to the
biosynthesis of isoprenoid in Eumycetes.[6] The incorporation
pattern of [1-13C]acetate and [2-13C]acetate in the pyrrole
building block was consistent with the biosynthesis pathway
of proline, which can be derived from acetyl-CoA through
the TCA cycle.[7]
In order to understand if the other amino acids interfere
with the biosynthesis, amino acid feeding experiments were
FULL PAPER
carried out. Interestingly, 1 % methionine or lysine suppressed the production of malbranpyrroles (Figure S4 in the
Supporting Information). Methionine is the major precursor
of S-adenosyl-l-methionine (SAM) involved methylation in
the biosynthesis of natural products. Since the structure of
malbranpyrroles need the C- and O-methylation, the methionine should be the important precursor during biosynthesis. Some reports indicate that methionine can be precursor and inhibitor of methylation in some microorganisms
with low and high concentration, respectively.[8] In M. sulfurea > 0.1 % methionine can suppress malbranpyrroles production. However, 0.01 % of 13C-methyl methionine feeding
did not enrich the 13C signal of methylated carbon in the
structures deduced from NMR spectra (data not shown).
Both d- and l-ethionine, the inhibitor of SAM methylation
also suppressed malbranpyrroles production; this implies
SAM methylation was indeed involved in the biosynthesis
pathway.[9] This has yet to be confirm by 13C-methyl methionine feeding experiment in the 0.01 ~ 0.1 % concentration
range. In addition, the suppression caused by lysine hints
that M. sulfurea might not only use proline directly to produce the pyrrole ring, but also use the upstream precursor,
ornithine. The structure similarity of lysine and ornithine
might interfere with the ornithine cyclodeaminase to convert
ornithine into proline.[10]
From the cytotoxicity assay, the IC50 of isolated malbranpyrroles C–F (3–6) against PANC-1, HepG2, and MCF-7
cancer cells lines were about 3–11 mm (Table S2 in the Supporting Information). Compound 1 did not show cytotoxicity
against these cancer cell lines; this implies that the chloride
atom is the key substituent of the pharmacophore. Although
malbranpyrroles are photosensitive, the conformation seems
not to significantly change their cytotoxic potency. The IC50
Figure 5. The isotope map of [2-13C]glycerol in malbranpyrrole F (6).
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values of malbranpyrroles with and without UV irradiation
are similar. In addition, the percentage of cells in the G0/G1
phase were slightly increased; this indicates that MCF-7 and
HepG2 cells might be arrested by malbranpyrrole treatment
at the G0/G1 phase (Table S3 in the Supporting Information). So far, there have been no reports that explain how
the halide substituent plays the important effect on cytotoxicity against cancer cell lines. On the other hand, photoprotection and photoreactivation are the major functions of the
photosensitive metabolites, such as carotenoids, scytonemin,
flavonoids, and so on, in microorganisms and plants.[11] The
biochemical and physiological details of these photosensitive
metabolites on M. sulfurea are still under investigation.
Conclusion
The ICM-MS techniques, including imaging mass spectrometry, in natural products research is gaining interest. So far,
most reports have applied ICM-MS techniques for searching
known secondary metabolites of plant,[12] microorganisms,[3]
cyanobacteria,[13] and sponges.[13] Our results show that ICDMS can provide more opportunities to discover new natural
products and/or their biosynthetic intermediates since they
might be omitted in the traditional isolation procedures. On
the other hand, based on the development of various new
LC NMR systems, such as stop-flow, loop storage, SPE, and
capillary mode, natural product chemists have gradually applied LC NMR to de novo structural determination. A previous report has also demonstrated the suitability of
LC NMR to investigate mixtures of natural product isomers
that were exposed to light.[14] However, most applications
deal with the characterization of plant metabolites, whereas
applications to study microorganism or marine natural products are still rare.[15] In this study, we have applied ICD-MS
technique and LC-SPE-NMR to discover photosensitive
polyketides from brown hyphae of M. sulfurea. ICD-MS (or
ICM-MS) together with LC-SPE-NMR provided us with
more information before we processed large-scale fermentation, purification of malbranpyrroles, and structural determination of malbranpyrrole B (2), which cannot be isolated as
a single pure sample for off-line structure analysis.
In terms of structures, isotope feeding pattern, and amino
acid feeding results, we believe that the biosynthetic gene
cluster of malbranpyrroles consists of three major regions:
the first one is a hybrid NRPS–PKS; the second is for isoprenoid biosynthesis-related enzymes, such as HMG-CoA
synthase, IPP isomerase, prenyltransferases, and the third
one is the enzymes for modification, such as halogenase, Oand C-methyltranferases. Several polyketide–isoprenoid
hybrid metabolites have been identified from microorganisms and many of them were reported to show biological activity.[16] In contrast to investigation of the biological activity
of those compounds there are not many reports on their biosynthetic genes and enzymes,[17] especially from fungi. Our
cytotoxicity results reveal that the chloride atom is the key
pharmacophore substituent, however, the function of the
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isopreny group is still unclear. The sequence and analysis of
the biosynthetic gene cluster of malbranpyrroles is ongoing
in our laboratory. In the future, we wish to generate many
analogues through bioengineering strategies for studying the
structure–activity relationship.
Experimental Section
General: 1H NMR (500 MHz), 13C NMR, 1H-1H COSY, 1H-1H TOCSY,
1
H-13C HSQC, 1H-13C HMBC, ROESY and NOESY spectra were obtained on a Bruker Avance II NMR spectrometer equipped with a
Bruker QNP-Z probe or a Bruker LC-SEI-Z probe. High-resolution
ESIMS were measured on a Bruker Bio-TOF III mass spectrometry.
MALDI micro-MX matrix-assisted laser desorption/ionization time-offlight mass spectrometer was used for intact-cell DIOS mass measurement. The Agilent 1100 series HPLC system equipped with Develosil
C30 columns (5 mm, 250 10 mm and 250 4.6 mm) were used for isolation and analysis. Low resolution LC-DAD-ESIMS were measured on a
Thermo LTQ XL linear ion trap mass spectrometer.
Fungal material: Thermophilic fungal strain, M. sulfurea, was isolated
from the soil of fumaroles in Sihchong River Hot Springs Zone, Pingtung
County, Taiwan. The fungal strain was identified by the late Prof. KueiYu Chen according to various morphological, biochemical and physiological characteristics described previously.[18] A voucher specimen (F-19) is
deposited in the Institute of Biological Chemistry, Academia Sinica,
Taiwan.
Culture conditions: The fungal strain was cultured at 40 8C in the dark
for 14 days on potato dextrose agar (PDA), KCl (0.5 %) in PDA, KBr
(0.5 %) in PDA, KI (0.5 %) in PDA, various amino acids (1 %) in PDA,
either on plates or tubes for extraction, isolation, and quantity analysis of
malbranpyrroles A–F. In the isotope feeding experiments, the strain was
cultured with 0.1 % [1-13C]acetate, [2-13C]acetate, [13C2]acetate, and
[2-13C]glycerol in PDA and then malbranpyrroles A–F were purified as
description below.
Extraction and isolation: The mass mycelium and medium of M. sulfurea
were extracted three times with EtOAc in the dark. The EtOAc extract
was purified by RP-HPLC (CH3CN/H2O, 74:26, C30 column) to obtain
malbranpyrroles A–F (1–6).
LC-DAD-ESIMS and LC-SPE-NMR analysis of malbranpyrroles: An
isocratic eluted system was applied for LC-DAD-ESIMS analysis: 70 %
MeCN, 1 mL min 1; Develosil C30 column, 250 4.6 mm. A gradient
eluted system was applied for LC-DAD-ESIMS analysis: 0 ~ 5 min, 60 %
MeCN, 1 mL min 1; 5 ~ 15 min, 72 % MeCN, 0.8 mL min 1; 15 ~ 25 min,
72 % MeCN, 0.8 mL min 1; 25 ~ 30 min, 80 % MeCN, 1.2 mL min 1; Develosil C30 column, 250 4.6 mm; positive mode ESI mass. An isocratic
eluted system was applied for LC-SPE-NMR analysis: 74 % MeCN,
0.8 mL min 1; Develosil C30 column, 250 4.6 mm; cartridge type: C18;
deuterated solvent: CD3CN.
Intact-cell desorption/ionization on silicon mass spectrometry: The
hyphae of M. sulfurea were suspended by using a Vortex mixer in MeCN
(50 %; 10 mL) aqueous solution containing TFA (0.1 %). An aliquot of
sample (1 mL) was dropped on DIOS plate directly. The sample was analyzed by using MALDI micro-MX matrix-assisted laser desorption/ionization time-of-flight mass spectrometer.
Cytotoxicity assay and cell cycle analysis: PANC-1 was purchased from
the American Type Culture Collection (ATCC); HepG2 and MCF-7 cell
lines were obtained from the Bioresource Collection and Research
Center (BCRC, Hsin-Chu, Taiwan). Cells were maintained either in Dulbeccos modified Eagles medium, Hams F12 medium or aMEM
medium supplemented with l-glutamine (2 mm; Sigma), penicillin
(100 unit mL 1), streptomycin (100 mg mL 1) and fetal bovine serum
(10 %; Invitrogen). Determination of cytotoxicity of malbranpyrroles was
carried out by using the MTT assay. MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltertrazolium bromide) was obtained from Sigma. Malbranpyrroles were dissolved in DMSO. HepG2, MCF-7, or PANC-1 cells (1–
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10 103 cells per well) were seeded into a 96-well plate and were treated
with a series of concentrations of malbranpyrroles for 48 h. The cells
were washed with 1 PBS (phosphate buffered saline) and then incubated with MTT solution (1 mg mL 1; 50 mL per well) at 37 8C for 2 h followed by addition of DMSO (150 mL per well) at room temperatures to
dissolve the blue–violet formazan deposit. Absorbance at 570 nm was
measured with an ELISA reader.
Cell cycle analysis was carried out utilizing propidium iodide (PI) staining followed by flow cytometric measurement of the PI fluorescence. Following malbranpyrrole treatment for 48 h (concentrations employed
were similar to those used for the MTT assay) cells were washed in 1 PBS, trypsinized, harvested in the culture medium, and centrifuged. The
pellet was washed in PBS, fixed in ice-cold ethanol (70 %), and stored at
20 8C, overnight. Before flow cytometric analysis, cells were washed
with 1 PBS and stained with PI solution containing Triton-X100 (1 %),
PI (200 mg mL 1) and RNAase (0.2 mg mL 1) and incubated at room temperature for 30 min. Flow cytometry analysis was performed by using
Calibur flow cytometer (BD Biosciences) at an excitation wavelength of
488 nm and emission wavelength of 610 nm. Data were collected for 3 104 cells and the percentages of cells in each phase of the cell cycle were
calculated by using the Modfit LT software package.
Malbranpyrrole A (1): Brown powder; 1H NMR (500 MHz, [D6]acetone):
d = 10.23 (br s, NH), 6.94 (m, H-2), 6.23 (m, H-3), 6.46 (m, H-4), 6.74 (s,
H-6), 7.16 (d, J = 15.5 Hz, H-8), 6.55 (d, J = 15.5 Hz, H-9), 2.18 (s, H-15),
1.95 (s, H-16), 6.25 (dd, J = 17.1, 10.5 Hz, H-18), 4.76 (dd, J = 10.5, 1.6 Hz,
H-19a), 4.87 (dd, J = 17.1, 1.6 Hz, H-19b), 1.44 (s, H-20, 21), 4.07 (s,
OCH3); 13C NMR (125 MHz, [D6]acetone): see Table 1; HRMS ESI: m/z:
calcd for C21H26NO3 [M + + H]: 340.1914; found: 340.1907.
H-6), 7.27 (d, J = 15.6 Hz, H-8), 6.62 (d, J = 15.6 Hz, H-9), 2.22 (s, H-15),
1.96 (s, H-16), 6.25 (dd, J = 17.0, 10.6 Hz, H-18), 4.76 (dd, J = 10.6, 1.3 Hz,
H-19a), 4.87 (dd, J = 17.0, 1.3 Hz, H-19b), 1.45 (s, H-20, 21), 4.09 (s,
OCH3); 13C NMR (125 MHz, [D6]acetone): see Table 1; HRMS ESI: m/z:
calcd for C21H25ClNO3 [M + + H]: 374.1523; found: 374.1532.
Malbranpyrrole D (4): Dark orange powder; 1H NMR (500 MHz,
[D6]acetone): d = 10.33 (br s, NH), 6.97 (m, H-2), 6.20 (m, H-3), 6.71 (s,
H-6), 7.26 (d, J = 15.3 Hz, H-8), 6.52 (d, J = 15.3 Hz, H-9), 2.19 (s, H-15),
2.03 (s, H-16), 4.58 (q, J = 6.5 Hz, H-18), 1.40 (d, J = 6.5 Hz, H-19), 1.17
(s, H-20), 1.36 (s, H-21); 13C NMR (125 MHz, [D6]acetone): see Table 1;
HRMS ESI: m/z: calcd for C20H23ClNO3 [M + + H]: 360.1361; found:
360.1360.
Malbranpyrrole E (5): Brown powder; 1H NMR (500 MHz, [D6]acetone):
d = 10.75 (br s, NH), 6.93 (m, H-2), 6.17 (m, H-3), 6.84 (d, J = 14.8 Hz, H6), 7.14 (dd, J = 14.8, 11.4 Hz, H-7), 7.18 (dd, J = 11.4, 0.8 Hz, H-8), 6.46
(s, H-11), 2.07 (d, J = 0.8 Hz, H-15), 6.18 (dd, J = 17.5, 10.6 Hz, H-17),
4.88 (dd, J = 17.5, 1.1 Hz, H-18a), 4.78 (dd, J = 10.6, 1.1 Hz, H-18b), 1.46
(s, H-19, 20), 3.93 (s, OCH3); 13C NMR (125 MHz, [D6]acetone): see
Table 1; HRMS ESI: m/z: calcd for C20H23ClNO3 [M + + H]: 360.1361;
found: 360.1361.
Malbranpyrrole F (6): Brown powder; 1H NMR (500 MHz, [D6]acetone):
d = 10.77 (br s, NH), 6.94 (m, H-2), 6.17 (m, H-3), 6.87 (d, J = 15.1 Hz, H6), 7.15 (dd, J = 15.1, 11.6 Hz, H-7), 7.21 (dd, J = 11.6, 1.0 Hz, H-8), 6.26
(s, H-11), 2.05 (d, J = 1.0 Hz, H-15), 4.58 (q, J = 6.6 Hz, H-17), 1.38 (d, J =
6.6 Hz, H-18), 1.17 (s, H-19), 1.36 (s, H-20); 13C NMR (125 MHz,
[D6]acetone): see Table 1; HRMS ESI: m/z: calcd for C19H21ClNO3 ACHTUNGRE[M +
+ H]: 346.1205; found: 346.1201.
Malbranpyrrole B (2): 1H NMR (500 MHz, CD3CN): d = 9.39 (br s, NH),
6.87 (m, H-2), 6.23 (m, H-3), 6.45 (m, H-4), 6.67 (s, H-6), 7.15 (d, J =
15.4 Hz, H-8), 6.39 (d, J = 15.4 Hz, H-9), 2.12 (s, H-15), 2.00 (s, H-16),
Table 1. 13C NMR spectroscopy data (125 MHz, [D6]acetone for 1, 3–6;
CD3CN for 2) of malbranpyrroles A–F (1–6). The signals of malbranpyrrole B (2) were deduced indirectly from HSQC by LC-SPE-NMR.
Compounds
4
Carbon
1
2
3
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
OCH3
120.1
110.1
111.4
130.0
126.9
129.1
139.2
114.0
152.2
118.2
179.7
108.6
161.5
13.1
8.8
38.5
148.9
107.1
27.0
27.0
55.5
120.0
110.0
111.5
ND
127.0
ND
140.2
113.4
ND[a]
ND
ND
ND
ND
13.2
8.1
ND
91.5
13.9
19.5
24.9
121.8
110.1
115.4
126.5
123.6
131.4
139.6
116.5
152.9
119.9
180.7
109.7
162.6
14.2
9.8
39.5
149.9
108.1
28.0
28.0
56.7
121.8
110.1
115.6
126.6
123.8
131.5
140.7
115.6
158.1
104.5
170.0
109.7
160.1
14.2
8.9
44.2
92.4
14.9
20.5
25.8
5
6
121.5
110.7
114.7
127.6
124.6
121.3
132.7
126.0
160.8
94.6
168.0
111.4
162.6
12.6
40.6
149.7
108.2
28.2
28.2
121.5
110.7
114.8
127.6
124.9
121.2
133.3
126.2
164.6
93.9
170.2
109.5
160.4
12.8
43.7
92.6
14.9
25.8
20.5
56.62
[a] ND: not determined.
4.54 (q, J = 6.6 Hz, H-18), 1.36 (d, J = 6.6 Hz, H-19), 1.14 (s, H-20), 1.33
(s, H-21);
13
C NMR (125 MHz, CD3CN): see Table 1; HRMS ESI: m/z:
calcd for C20H24NO3 [M + + H]: 326.1756; found: 326.1761.
Malbranpyrrole C (3): Dark orange powder;
1
H NMR (500 MHz,
[D6]acetone): d = 10.35 (br s, NH), 6.97 (m, H-2), 6.20 (m, H-3), 6.69 (s,
Chem. Eur. J. 2009, 00, 0 – 0
Acknowledgements
The authors thank Dr. Shu-Chuan Jao (Institute of Biological Chemistry,
Academia Sinica) for technique support in NMR spectroscopy, Dr. MaoYen Chen (Institute of Biological Chemistry, Academia Sinica) for fungal
incubation, and Pei-Hsuan Chuang (Genomics Research Center, Academia Sinica) for LC-MS measurement. This work was financially supported in part by National Science Council, Taiwan.
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2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!
Received: June 8, 2009
Published online: && &&, 2009
Chem. Eur. J. 0000, 00, 0 – 0
Discovery of New Polyketides from M. sulfurea
FULL PAPER
Fungal rhythm: Intact-cell desorption/
ionization on silicon mass and LCSPE-NMR techniques were applied to
the identification of photosensitive
polyketides, malbranpyrroles A–F,
from the thermophilic fungus Malbranchea sulfurea, as illustrated in the
figure.
Natural Products
Chem. Eur. J. 2009, 00, 0 – 0
www.chemeurj.org
Y.-L. Yang, W.-Y. Liao, W.-Y. Liu,
C.-C. Liaw, C.-N. Shen, Z.-Y. Huang,
S.-H. Wu* . . . . . . . . . . . . . . . . . . . . . . &&&&—&&&&
Discovery of New Natural Products by
Intact-Cell Mass Spectrometry and
LC-SPE-NMR: Malbranpyrroles,
Novel Polyketides from Thermophilic
Fungus Malbranchea sulfurea
2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&9&
These are not the final page numbers! ÞÞ