Structural diversity of new C13-polyketides produced by Chaetomium mollipilium cultivated in the presence of a NAD+-dependent histone deacetylase inhibitor

Article abstract of DOI:10.1021/ol302539s

Cultivation of Chaetomium mollipilium with nicotinamide, a NAD ⁺-dependent HDAC inhibitor, stimulated its secondary metabolism, leading to the isolation of structurally diverse new C₁₃-polyketides, mollipilin A-E (1-5) as well as two known compounds (6 and 7). Spectroscopic methods, X-ray single crystal diffraction analysis, and VCD elucidated the absolute configurations of structures 1-6, and plausible biosynthetic pathways for 1-7 were proposed based on structural relationships. Mollipilins A (1) and B (2) exhibited moderate growth inhibitory effects on HCT-116 cells.

Full text of DOI:10.1021/ol302539s

ORGANIC
LETTERS
2012
Vol. 14, No. 21
5456–5459
Structural Diversity of New
C13-Polyketides Produced by Chaetomium
mollipilium Cultivated in the Presence of a
NADþ‑Dependent Histone Deacetylase
Inhibitor
Teigo Asai,*,† Shuntaro Morita,† Naoki Shirata,† Tohru Taniguchi,‡ Kenji Monde,‡
Hiroaki Sakurai,§ Tomoji Ozeki,§ and Yoshiteru Oshima*,†
Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba-yama, Aoba-ku,
Sendai 980-8578, Japan, Faculty of Advanced Life Science, Frontier Research Center for
Post-Genome Science and Technology, Hokkaido University, Kita 21 Nishi 11, Sapporo
001-0021, Japan, and Department of Chemistry and Materials Science, Tokyo Institute
of Technology, 2-12-1-H-63 O-okayama, Meguro-ku, Tokyo 152-8551, Japan
tasai@m.tohoku.ac.jp
Received September 14, 2012
ABSTRACT
Cultivation of Chaetomium mollipilium with nicotinamide, a NADþ-dependent HDAC inhibitor, stimulated its secondary metabolism, leading to the
isolation of structurally diverse new C13-polyketides, mollipilin AꢀE (1ꢀ5) as well as two known compounds (6 and 7). Spectroscopic methods,
X-ray single crystal diffraction analysis, and VCD elucidated the absolute configurations of structures 1ꢀ6, and plausible biosynthetic pathways
for 1ꢀ7 were proposed based on structural relationships. Mollipilins A (1) and B (2) exhibited moderate growth inhibitory effects on HCT-116 cells.
Histone deacetylases (HDACs) are an important class of
enzymes capable of removing the acetyl group from ε-N-
acetyl lysine residues in the amino-terminal tail of core
histones. HDACs suppress gene expression in most cases
and are crucial epigenetic factors.1 Based on the hydrolysis
mechanism, HDACs are classified into two families: Zn-
(II)-dependent and NADþ-dependent deacetylases.2 Re-
cent studies have shown that HDACs transcriptionally
regulate fungal secondary metabolite production.3,4 A dis-
ruption mutant of HdaA, a Zn(II)-dependent HDAC,
significantly increases the expression of secondary meta-
bolite gene clusters in Aspergillus nidulans, including those
producing sterigmatocystin and penicillin.3 Moreover, sub-
eroylanilide hydroxamic acid (SAHA), which is a represen-
tative Zn(II)-dependent HDAC inhibitor, suppresses the
HDAC activity in several fungi, and the increased accumu-
lation of their metabolites has resulted in the discovery of
several new compounds.5
† Tohoku University.
‡ Hokkaido University.
§ Tokyo Institute of Technology.
We have applied this type of HDAC inhibitor, mainly
suberoyl bis-hydroxamic acid (SBHA), to entomopathogenic
(1) Ruijter, A. J. M.; Gennip, A. H. V.; Caron, H. N.; Kemp, S.;
Kuilenburg, A. B. P. Biochim. J. 2003, 370, 737–749.
(2) Grozinger, C. M.; Schreiber, S. L. Chem. Biol. 2002, 9, 3–16.
(3) Shwab, E. K.; Bok, J. W.; Tribus, M.; Galehr, J.; Graessle, S.;
Keller, N. P. Eukaryot. Cell 2007, 6, 1656–1664.
(4) Lee, I.; Oh, J. H.; Shwab, E. K.; Dagenais, R. T.; Andes, D.;
Keller, N. P. Fungal Genet. Biol. 2009, 46, 782–790.
r
10.1021/ol302539s
Published on Web 10/19/2012
2012 American Chemical Society

Figure 2. Structures of 1ꢀ7.
Figure 1. HPLC profiles of the EtOAc extracts of C. mollipilium
cultivated with (a) nicotinamide 100 μM and (b) control as
detected by UV absorption at 215 nm.
C. mollipilium was cultivated in a YM liquid medium
with100 μM nicotinamide for 16daysat25°C (Supporting
Information). The culture medium (13 L) was extracted
twice with ethyl acetate. The ethyl acetate extract (1.4 g)
was separated by Sephadex LH-20, silica gel column chro-
matography, and reversed-phase HPLC to afford diverse
C13-polyketides, mollipilin A (1, 17.3 mg), B (2, 13.7 mg),
C (3, 5.7 mg), D (4, 5.5 mg), E (5, 2.7 mg), F (6, 14.6 mg),
and (ꢀ)-aureonitol (7, 488 mg) (Figure 2).
fungi and successfully isolated numerous novel natural
products containing unprecedented carbon skeletal com-
pounds, including tenuipyrone and indigotides CꢀE.6
Although NADþ-dependent HDACs have been identi-
fied in a number of fungi7 and are involved in the formation
and spreading of heterochromatin for transcriptional silenc-
ing,8 a study of their effects on secondary metabolite pro-
duction in fungi has yet to be reported. We hypothesized
that the addition of a NADþ-dependent HDAC inhibitor in
a fungal culture medium would realize transcriptional up-
regulation of biosynthetic gene clusters and enhance the
production of fungal secondary metabolites. Consequently,
we applied three typical inhibitors (nicotinamide, sirtinol,
and splitomycin)9 to our natural product study and found
that Chaetomium mollipilium cultivated with nicotinamide
(100 μM) showed a notable change in the secondary meta-
bolites (Figure 1). Five new C13-polyketides, mollipilin AꢀE
(1ꢀ5), as well as known spiroketal 6 (named as mollipilin
F) and (ꢀ)-aureonitol (7) were isolated. Herein we discuss
the structures of 1ꢀ5 and the absolute stereochemistry of
6 as well as their plausible biosynthetic relationship and
cell growth inhibitory activity.
The HREIMS of mollipilin A (1) at m/z 236.1049 [M]þ
(calcd: m/z 236.1049) suggested that the molecular formula
was C13H16O4, which required six degrees of unsaturation.
The IR spectrum (3445, 1698, and 1674 cmꢀ1) indicated
the presence of a hydroxy group and two R,β-unsaturated
carbonyl groups. The 13C NMR and DEPT spectra showed
the presence of one keto carbonyl, one aldehyde, one
quaternary sp2 carbon, five tertiary sp2 carbons, two oxy-
methines, one oxymethylene, one methylene, and one methyl
1
group (Table S1). The H and 13C NMR and HMQC
spectra indicated the presence of an epoxide function based
on resonance signals at δC 50.4 (C-11) and 49.4 (C-12) and
δH 3.40 (ddd, J = 7.3, 5.0, 2.2 Hz, H-11), 3.06 (dd, J = 5.5,
5.0 Hz, Ha-12), and 2.69 (dd, J = 5.5, 2.2 Hz, Hb-12). The
1
1Hꢀ H COSY spectrum exhibited sequential correlations
from H3-1 to H-5 and H-9 to H2-12, while the HMBC
correlations from H-9 and H-10 to C-8 carbonyl implied the
presence of a 9-en-8-one function (Figure 3). In addition, the
long-range correlations of H-4/C-6, H-5/C-13, and H-7/C-
6, C-8 suggested C-5ꢀC-6ꢀC-13 and C-6ꢀC-7ꢀC-8 link-
ages (Figure 3), and the large coupling constants of H-3/
H-4 (15.6 Hz) and H-9/H-10 (15.8 Hz) indicated that C-3/
C-4 and C-9/C-10 olefins had trans geometries (Table S1).
The 5E configuration from the NOE of H-5/H-13 con-
firmed the planar structure of 1 (Figure 3).
The 1H and 13C NMR spectra of mollipilin B (2),
C13H18O4 [HREIMS: m/z 238.1194 [M]þ (calcd: m/z
238.1205)], agreed well with those of 1, except 2 had two
additional methylene signals and lacked the signals as-
signed to the C-9/C-10 trans-olefin in 1 (Table S1). These
observations suggested that the C-9/C-10 double bond in 1
was reduced to methylenes in 2, which was consistent with
the strong IR band at 1718 cmꢀ1 due to a saturated
(5) (a) Williams, R. B.; Henrikson, J. C.; Hoover, A. R.; Lee, A. E.;
Cichewicz, R. H. Org. Biomol. Chem. 2008, 7, 1895–1897. (b) Henrikson,
J. C.; Hoover, A. R.; Joyner, P. M.; Cichewicz, R. H. Org. Biomol. Chem.
2009, 7, 435–438.
(6) (a) Asai, T.; Yamamoto, T.; Oshima, Y. Tetrahedron Lett. 2011,
52, 7042–7045. (b) Asai, T.; Dan, L.; Obara, Y.; Taniguchi, T.; Monde,
K.; Yamashita, K.; Oshima, Y. Tetrahedron Lett. 2012, 53, 2239–2243.
(c) Asai, T.; Chung, Y. M.; Sakurai, H.; Ozeki, T.; Chang, F. R.;
Yamashita, K.; Oshima, Y. Org. Lett. 2012, 54, 513–515. (d) Asai, T.;
Yamamoto, T.; Oshima, Y. Org. Lett. 2012, 54, 2006–2009.
(7) (a) Graessle, S.; Dangl, M.; Haas, H.; Mair, K.; Trojer, P.;
Brandtner, E. M.; Walton, J. D.; Loidl, P.; Brosch, G. Biochim. Biophys.
Acta 2000, 1492, 120–126. (b) Trojer, P.; Brandtner, E. M.; Brosch, G.;
Loidl, P.; Galehr, J.; Linzmaier, R.; Haas, H.; Mair, K.; Tribus, M.;
Graessle, S. Nucleic Acids Res. 2003, 31, 3971–3981.
(8) Denu, J. M. Curr. Opin. Chem. Biol. 2005, 9, 431–440.
(9) HPLC analyses of the extracts obtained from the fungal culture
broths using sirtinol or splitomycin showed that no extract had distinct
enhancement of the secondary metabolite production. Moreover, it
showed that major constituents in the extracts were degradation and
hydroxylation products of sirtinol or splitomycin, suggesting that the
two inhibitors were unstable in the culture media of fungi. Therefore, the
two inhibitors seemed to be unsuitable for the experiments.
Org. Lett., Vol. 14, No. 21, 2012
5457

which required five degrees of unsaturation. The 1H NMR,
13C NMR, and HMQC spectra showed the presence of
an exo-olefin (C-11/C-12) and two trans-olefins (C-2/C-3
and C-9/C-10) (Table S2). The above functionality ac-
counted for 3 of the 5 degrees of unsaturation, suggesting 3
possessed a bicyclic core. In addition, the 13C NMR and
DEPT spectra exhibited signals for four oxymethines, one
oxymethylene, one methine, and one methyl. The analysis of
1
the 1Hꢀ H COSY spectrum supported the carbon sequence
in the molecule (Figure 3). The HMBC correlations of H2-13/
C-8 and H-4/C-7 revealed that the connectivity of C-8ꢀC-13
and C-4ꢀC-7 was through ether bonds, indicating the pre-
sence of hexahydrofuro[3,4-b]furan (Figure 3), and the mo-
lecular formula indicated the presence of a C-5 hydroxy
group. 1D-NOE experiments elucidated the relative stereo-
chemistry of the bicyclic core of 3. The NOE of H-6/H-7
showed that the two tetrahydrofuran rings were fused in the
cis configuration (Figure 3). Moreover, the NOEs (H-3/H-5,
H-6, H-7, H-4/5- OH, and H-7/H-9) unequivocally defined
the remaining relative stereochemistries (Figure 3), and the
advanced Mosher method confirmed that the absolute
stereochemistry at C-5 was R (Figure S1, Table S6).
1
Figure 3. Key 1Hꢀ H COSY, HMBC, and NOE correlations of
1, 3, and 4.
carbonyl group. Analysis of the COSY and HMBC spectra
showed that 2 was a 9,10-dihydro derivative of 1.
To determine the absolute stereochemistries at C-7 and
C-11 in 1, the VCD method using DFT calculations was
applied. The experimental IR and VCD spectra of 1 mea-
sured in CDCl3 agreed well with the calculated spectra for
7S,11S- and 7S,11R-1 (Figure 4), suggesting that 1 had a
7S configuration. Unfortunately, the VCD experiments
could not elucidate the C-11 configuration because the
calculated spectra of the two isomers were almost identical
(Figure 4). Treatment of 1 or 2 with 2,4-dinitrophenylhy-
drazine yielded corresponding hydrazone 1a or 2a, which
could be converted into the (S)- and (R)-MTPA esters (1b,
1c, 2b, and 2c), respectively. By comparing the Δδ(S‑R)
values with those of 1, the absolute stereochemistry at C-7
in 2 was determined to be S (Scheme S1). Moreover, crys-
tallized 2a, which was supplied for an X-ray analysis study,
confirmed that the absolute stereochemistry at C-11 was R
(Figure 5), and the biosynthetic relationship between 1 and
2 implied that 1 also had an 11R configuration.
The molecular formula of mollipilin C (3) was C13H18O3
[HREIMS: m/z 222.1226 [M]þ (calcd: m/z 222.1256)],
Figure 5. Structure and ORTEP stereo diagram of 2a.
The HREIMS of 4 showed an ion peak at m/z 224.1416
(C13H20O3, calcd: m/z 224.1412). The 13C NMR assisted
by HMQC spectra displayed 26 signals, including one keto
carbonyl, ten tertiary sp2 carbons, two secondary sp2 car-
bons, one acetal carbon, two oxymethines, two oxymethy-
lenes, two methines, four methylenes, and two methyls.
1
Analyses of the 1Hꢀ H COSY and HMBC spectra suggested
two structures, 4a and 4b (Figure 3). Treatment of 4 with
pivaloyl chloride afforded 4c as a single product [HREIMS:
m/z 308.1974 [M]þ (calcd for C18H28O4 308.1988)]. The 1H
NMR spectrum of 4c agreed well with that of 4a except for
the presence of a methyl signal due to the pivaloyl ester and
the downfield shift of the H2-13 signals, indicating that 4c
was the 13-O-pivaloylated derivative of 4a (Figure 2). This
observation confirms that 4 was an equilibrium mixture of 4a
and 4b. The NOEs of H-7/H-5 and H2-9 in 4b determined the
relative configurations at C-6, C-7, and C-8, and apply-
ing the advanced Mosher method to 4c indicated that the
absolute stereochemistry at C-7 was S (Figure S1, Table S7).
Figure 4. Comparison of IR (lower flame) and VCD (upper
flame) spectra: (a) observed for 1 with those of calculated for
7S,11R- and 7S,11S-1; (b) observed for 6 with those of calcu-
lated for 7S,8S,11S- and 7S,8S,11R-6.
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Org. Lett., Vol. 14, No. 21, 2012

Scheme 1. Plausible Biosynthetic Relationship among the Isolated Polyketides (1ꢀ7)
Compounds 5 and 6 had the same molecular formula
C13H20O4, and the analyses of 1D and 2D NMR showed
that they were geometric isomers at the C-3/C-4 double
bond (Figure S2, Table S4). A comparative study of the 1H
and 13C NMR spectra with the reported data further
identified 6 as 3-hydroxymethyl-8Z-(20E-pentenyliden)-
2,6-dioxaspiro[4.4]nonanol-9 (Table S4).10 The NOEs of
H-7/H2-9 suggested 6 had a 7S*,8S* relative configuration
(Figure S3), and the advanced Mosher method for 12-O-
pivaloylated derivative 6a substantiated the 7S configura-
tion in 6 (Figure S1, Table S8). Although the relative
stereochemistry of 6 was proposed as 7S*,8S*,11R* from
its NMR data,9 stereomodeling suggested itwas difficult to
determine the relative stereochemistry at C-11 from NMR
analysis. Consequently, a VCD study was conducted to
clarify the absolute configuration at C-11 and the absolute
stereochemistry of the molecule. The experimental VCD
spectrum agreed well with the spectrum of the 7S,8S,11S
isomer rather than that of 7S,8S,11R (Figure 4). Based on
their biosynthetic relationship, 5 and 6 had the same
stereochemistry. Therefore, the structures of 5 and 6 were
determined as shown in Figure 2.
The cell growth inhibitory activities of polyketides 1ꢀ7
were evaluated in human HCT 116 cells. Mollipilin A (1)
and B (2) exhibited moderate inhibitory effects on the cell
growth with GI50 values of 1.8 and 3.7 μM, respectively. The
Western blot with an antibody against acetylated histone H4
showed that nicotinamide treatment of C. mollipilium induced
hyperacetylated histone H4 in a dose dependent manner
(Figure S3). In our study of C. mollipilium, nicotinamide
significantly affected its secondary metabolite production at
concentrations of 50 and 100 μM, while the influence of
10 μM was less than that of 100 μM (data not shown).
Additionally, employing isonicotinamide (100 μM), an
analog of nicotinamide without NADþ-dependent HDAC
inhibitory activity, did not affect secondary metabolite
production. These observations suggested that the HDAC
inhibitory activity of nicotinamide was correlated with
secondary metabolite production.
In this study, we demonstrated that the addition of
nicotinamide to the culture medium of C. mollipilium signi-
ficantly stimulated secondary metabolite production. A
previous feeding experiment suggested a biosyntheticroute
of 7 from linear C13-polyene 8 via an epoxide rearrange-
ment.11 Our findings herein, which were based on structur-
al considerations, also indicated that 1ꢀ6 were biosynthe-
sized from 8 (Scheme 1). The structural diversity in the C13-
polyenes was realized by an epoxide rearrangement of 8
followed by tautomerization, sequential oxidations, and
rearrangements. This study is the first to demonstrate that
a NADþ-dependent HDAC inhibitor can effectively ex-
plore novel fungal secondary metabolites.
Acknowledgment. This work was supported in part by a
Grant-in-Aid for Scientific Research (Nos. 23710248
and 23590582) from the Ministry of Education, Science,
Sports and Culture of Japan; A-STEP (FS-stage) (No.
AS231Z01347G) from Japan Science and Technology
Agency (JST); the Uehara Memorial Foundation; and
the Japan Association for Chemical Innovation.
Supporting Information Available. Tables, figures,
schemes, experimental methods, NMR spectra of new com-
pounds, and the cif file for the 2,4-dinitrophenylhydrazone
derivative of mollipilin B (2a). This material is available free
of charge via the Internet at http://pubs.acs.org.
(10) Abraham, W. R.; Arfmann, H. A. Phytochemistry 1992, 31,
2405–2408.
(11) Saito, M.; Seto, H.; Yonehara, H. Agric. Biol. Chem. 1983, 47,
2935–2937.
The authors declare no competing financial interest.
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