Unexpected talaroenamine derivatives and an undescribed polyester from the fungus Talaromyces stipitatus ATCC10500

Article abstract of DOI:10.1016/j.phytochem.2015.09.002

Chemical investigation of the fungus Talaromyces stipitatus ATCC 10500, whose genome has been sequenced, led to the isolation of four undescribed talaroenamines B-E along with the known talaroenamine A. Their structures were elucidated on the basis of spectroscopic studies including mass spectrometry, extensive 2D NMR and electronic circular dichroism (ECD). Interestingly, talaroenamine A had previously been isolated from the strain of T. stipitatus Δtrop C, a strain knocked out for the gene encoding a non-heme Fe(II)-dependent dioxygenase catalyzing the oxidative ring expansion leading to the tropolone, but never from a wild-type strain. All talaroenamines were evaluated for their antiplasmodial activity and Talaroenamine D exhibited the best inhibition against the chloroquine-resistant Plasmodium falciparum (FcB1 strain) without noticeable toxicity on HeLa and preadipose cell lines. In the course of the chemical investigation of T. stipitatus, an undescribed polyester was also isolated and its absolute configuration was determined by hydrolysis and transesterification followed by gas chromatography on chiral column.

Full text of DOI:10.1016/j.phytochem.2015.09.002

Phytochemistry 119 (2015) 70–75
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Unexpected talaroenamine derivatives and an undescribed polyester
from the fungus Talaromyces stipitatus ATCC10500
Yi Zang a, Grégory Genta-Jouve b, Tithnara Anthony Sun a, Xuwen Li a, Buisson Didier a, Stéphane Mann a,
Elisabeth Mouray a, Annette K. Larsen c, Alexandre E. Escargueil c, Bastien Nay a, , Soizic Prado a,
⇑
⇑
a Molécules de Communication et Adaptation des Micro-organismes, UMR 7245 CNRS/MNHN, Muséum National d’Histoire Naturelle, 57 rue Cuvier (CP54),
75231 Paris Cedex 05, France
b Laboratoire de Pharmacognosie, UMR 8638 CNRS, Faculté des Sciences Pharmaceutiques et Biologiques, Paris Descartes University, Sorbonne Paris Cité, 4 Avenue de l’Observatoire,
75006 Paris, France
c Cancer Biology and Therapeutics, Centre de Recherche Saint-Antoine, Institut Universitaire de Cancérologie, Université Pierre et Marie Curie, Sorbonne Universités et Institut
National de la Santé et de la Recherche Médicale (INSERM) UMR 938, Hôpital Sant-Antoine, 184 rue du Faubourg Saint Antoine, 75571 Paris Cedex 12, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 March 2015
Received in revised form 31 August 2015
Accepted 3 September 2015
Available online 18 September 2015
Chemical investigation of the fungus Talaromyces stipitatus ATCC 10500, whose genome has been
sequenced, led to the isolation of four undescribed talaroenamines B–E along with the known talaroe-
namine A. Their structures were elucidated on the basis of spectroscopic studies including mass spec-
trometry, extensive 2D NMR and electronic circular dichroism (ECD). Interestingly, talaroenamine A
had previously been isolated from the strain of T. stipitatus
Dtrop C, a strain knocked out for the gene
encoding a non-heme Fe(II)-dependent dioxygenase catalyzing the oxidative ring expansion leading to
the tropolone, but never from a wild-type strain. All talaroenamines were evaluated for their antiplas-
modial activity and Talaroenamine D exhibited the best inhibition against the chloroquine-resistant
Plasmodium falciparum (FcB1 strain) without noticeable toxicity on HeLa and preadipose cell lines. In
the course of the chemical investigation of T. stipitatus, an undescribed polyester was also isolated and
its absolute configuration was determined by hydrolysis and transesterification followed by gas chro-
matography on chiral column.
Keywords:
Talaromyces stipitatus
Fungal natural products
Polyketides
Polyesters
Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction
This strain has been previously investigated by Cox and coll.,
especially for the biosynthetic study of fungal tropolones
Filamentous fungi produce a wide variety of biologically active
compounds and some of them have proven useful in drug develop-
ment (Schueffler and Anke, 2014). Recent bioinformatic tools asso-
ciated with the increasing number of sequenced genome have
highlighted the rich biosynthetic potential of filamentous fungi
(Brakhage and Schroeckh, 2011; Scharf and Brakhage, 2013). In this
context, new strategies have been proposed to reveal this
biosynthetic potential, among them the One Strain-Many Com-
pounds (OSMAC) approach which is based on the dependence of
microbial biosynthetic gene expression on the composition of the
culture medium (Paranagama et al., 2007). Known to produce a
variety of structurally interesting polyketides such as tropolones
(Birkinshaw et al., 1942) or duclauxin (Shibata et al., 1965), the fun-
gus Talaromyces stipitatus ATCC 10500 (syn. Penicillium stipitatum)
was thus selected for the search of new secondary metabolites.
(Davison et al., 2012). The authors pointed out that knockout of
the gene tropC, encoding a non-heme Fe(II)-dependent dioxyge-
nase which catalyzes the oxidative ring expansion to the tropolone
(strain T. stipitatus
DtropC), not only led to the abolition of
tropolone biosynthesis but also to the unexpected production of
the new metabolite talaroenamine (1).
Unexpectedly, in the course of our chemical investigation of the
wild type T. stipitatus ATCC 10500 cultured from the potato dex-
trose broth (PDB) medium, we isolated and characterized the four
new talaroenamines BꢀE (5–8) along with the previously known
talaroenamine (1) which we proposed to rename talaroenamine
A (Fig. 1). A new linear polyester (9), named talapolyester G,
was also isolated (Fig. 4). Moreover, an antiplasmodial assay using
the chloroquine-resistant Plasmodium falciparum (FcB1 strain)
revealed the moderate inhibitory activity of compounds 5–9
(IC50 = 19–40
lM) without noticeable toxicity on HeLa cell as well
as on 3T3-L1 preadipose cell lines (Table 3).
⇑
Corresponding authors.
E-mail addresses: bnay@mnhn.fr (B. Nay), sprado@mnhn.fr (S. Prado).
http://dx.doi.org/10.1016/j.phytochem.2015.09.002
0031-9422/Ó 2015 Elsevier Ltd. All rights reserved.

Y. Zang et al. / Phytochemistry 119 (2015) 70–75
71
The 1H–1H COSY spectrum (DMSO-d6) allowed identifying three
main substructures as emboldened in Fig. 2: a monosubstituted
phenyl ring, the enamine spin system (@CHANHA) mentioned
above and the substructure [C(CH3)@CH]. Assembling of these sub-
structures was performed using the cross-peaks observed on HMBC.
HMBC correlations were indicative of the leptosphaeronyl moi-
ety previously described (Lin et al., 2008). Indeed, the methyl CH3-7
at dH 1.31 ppm showed cross peaks with the quaternary carbon C-2
linked to an oxygen (dC 80.4) and with the two carbonyls C-1 and
C-3 respectively at dC 200.3 and 198.2. Moreover, the HMBC corre-
lations of olefinic proton H-4 (dH 5.63) with C-2, CH3-8 and C-6 con-
firmed this partial structure. Cross peaks from the amine NH-10 to
the aromatic carbons C-10 (dC 200.3) and C-20/C-60 (dC 117.9) and to
the quaternary carbon C-6 (dC 140.5) suggested that the mono-sub-
stituted benzene ring and the leptosphaeronyl part were connected
by the enamine function. This was confirmed by the HMBC
correlations observed between H-9 and the carbons C-1, C-5, C-6
and C-10. Consequently, the structure of 5 was determined as
shown in Fig. 2 and named talaroenamine B by comparison to the
known talaroenamine A (1) (Davison et al., 2012), differing by a
decarboxylation at C-20. A NOESY correlation between H-9 and
the methyl CH3-8 was observed, suggesting the Z configuration of
the enamine. In order to conclude on the absolute configuration
at C-2, we took advantage of the well described electronic circular
dichroism prediction methods (Bondu et al., 2012; Genta-Jouve
et al., 2013). As described on Fig. 3, a good agreement was observed
between both experimental and predicted CD spectra suggesting an
R configuration at C-2. Moreover, measurement of the optical rota-
tion and CD spectrum of 5 showed a negative value similar to the
one described for talaroenamine A (1) in MeOH.
Fig. 1. Structure of compounds 1 and 5–8.
2. Results and discussion
Compound (5) was isolated as a yellow oil. Its HR-ESI-MS (neg-
ative mode) showed the quasi-molecular ion [MꢀH]ꢀ at m/z:
256.0980 corresponding to the molecular formula C15H15NO3
(calcd. for C15H14NO3: 256.0974) and indicative of nine degrees
of unsaturation. The IR spectrum exhibited absorption bands for
two carbonyls groups at mmax 1687 and 1616 cmꢀ1 and for
hydroxyl and amine functionalities at mmax 3419 and 3252 cmꢀ1
,
respectively.
The 13C-J modulated NMR spectrum (DMSO-d6) exhibited the
resonance of 15 carbons including two methyls, seven sp2 methi-
nes, six quaternary carbons including two carbonyls at dC 200.3
and 198.2 and an oxygenated carbon at dC 80.4 (Table 1). Taking
into account the nine degrees of unsaturation, the molecule 5
should include two rings.
The structure of compound 6 was found to be closely related to
structure 5. Indeed, the molecular formula of 6 was determined as
The 1H NMR spectrum displayed typical signals of two methyls
(dH 1.31, s, H-7) and (dH 2.24, d, J = 0.96 Hz, H-8), two olefinic
methine (dH 5.63, d, J = 0.96 Hz, H-4) and (dH 8.04, d, J = 12.8 Hz, H-
9) as well as of five aromatic protons (dH 7.49, d, J = 8.7 Hz, H-20; dH
7.41, dd, J = 8.7, 7.4 Hz, H-30; dH 7.18, t, J = 7.4 Hz, H-40; dH 7.41, dd,
J = 8.7, 7.4 Hz, H-50; dH 7.49, d, J = 8.7 Hz, H-60). The downfield chem-
ical shift of the latter proton and its coupling constant suggested the
presence of a secondary enamine at dH 12.13 (d, J = 12.8 Hz, H-10)
(Table 1).
C
16H17NO4 by positive mode HR-ESI-MS (m/z 288.1169 [M+H]+)
and 13C NMR. The 1H and 13C NMR spectra of 6 were very similar
to those of 5, differing solely by the presence of an oxygenated
methylene at dH 4.59 (1H, d, J = 12.5 Hz) and 4.56 (1H, d,
J = 12.5 Hz) at C-20. The absolute configuration at C-2 was also
assigned as R by comparison of the CD spectrum with the one
acquired for 5 (SI, Fig. 1). The compound 6 was named talaroe-
namine C.
Table 1
NMR spectroscopic (DMSO-d6 298 K, 1H 600 MHz, 13C 150 MHz) data for Compounds 5–8.
5
6
7
8
Position
dC
dH, (J in Hz)
dC
dH, (J in Hz)
dC
dH, (J in Hz)
dC
dH, (J in Hz)
1
2
200.3
80.4
–
–
199.8
80.5
–
–
199.6
80.9
–
–
199.8
80.3
–
–
3
4
5
6
198.2
115.7
154.9
104.5
28.2
–
198.2
115.7
154.9
104.9
28.2
–
197.9
117.6
154.3
107.1
27.9
–
198.3
114.7
155.2
103.6
28.5
–
5.63 d (0.96)
–
–
5.62 d (0.96)
–
–
5.73 s
–
–
5.55 d (0.96)
–
–
7
1.31 s
1.32 s
1.31 s
1.28 s
8
9
20.0
146.1
–
139.2
117.9
129.6
124.9
129.6a
117.9a
–
2.24 d (0.96)
8.04 d (12.8)
12.13 d (12.8)
–
7.49 d (8.7)
7.41 dd (8.7, 7.4)
7.18 t (7.4)
7.41 dd (8.7, 7.4)
7.49 d (8.7)
–
20.0
146.3
–
138.6
130.9
129.0
124.6
128.8
117.1
61.3
2.24 d (0.96)
7.99 d (12.8)
12.50 d (12.8)
–
19.9
142.9
–
139.9
122.7
136.7
123.7
135.7
115.7
–
2.27 s
8.08 d (12.6)
13.37 d (12.7)
–
20.1
145.0
–
119.9
149.7
99.7
156.0
107.3
116.6
–
2.20 d (0.96)
8.01 d (12.8)
12.42 d (12.8)
–
10
10
20
30
40
50
60
–
–
–
7.34 dd (7.5, 1.4)
7.16 td (7.5, 1.4)
7.38 td (7.5, 1.4)
7.65 d (7.8)
4.59 d (12.5)
7.96 d (7.6)
7.37 ddd (8.6, 7.6, 1.6)
7.74 td (8.6, 1.6)
7.92 d (8.6)
–
6.54 d (2.4)
–
6.42 dd (8.6, 2.4)
7.50 d (8.6)
–
CH2OH
61.3
4.56 d (12.5)
–
–
–
–
CHO
–
–
–
–
–
–
194.7
–
10.04 s
–
–
56.0
–
3.86 s
OCH3
OH
–
–
–
–
9.60 s
a
Overlapped.

72
Y. Zang et al. / Phytochemistry 119 (2015) 70–75
Table 2
1H NMR (CD3OD, 600 MHz, 298 K) and 13C NMR (CD3OD, 150 MHz, 298 K) data of 9.
Position
dC
14.6
dH, (J values in Hz)
1
2
1.41 t (7.1)
4.43 dq (10.8, 7.1)
62.5
4.40 dq (10.8, 7.1)
3
4
5
6
7
8
9
10
172.4
105.9
166.1
102.7
163.5
113.7
143.6
43.6
–
–
–
6.21 s
–
6.22 s
–
3.22 dd (13.3, 4.8)
3.02 dd (13.3, 8.5)
5.16 ddq (8.5, 6.2, 4.9)
1.26 d (6.2)
–
2.39 dd (14.8, 7.0)
2.30 dd (14.8, 6.2)
4.06 m
11
12
13
14
72.8
20.4
172.6
45.1
15
16
65.4
23.0
1.09 d (6.2)
aromatic low-field signals, and showed an additional methoxy
group dH 3.86 (3H, s), dC 56.0 and a hydroxyl group at dH 9.60
(1H, s). Three signals being part of an ABX spin coupling system
were observed at d 6.54 (1H, d, J = 2.4), 6.42 (1H, dd, J = 8.6, 2.4),
and 7.50 (1H, d, J = 8.6). This is compatible with the two
oxygenated aromatic carbons at dC 149.7 (C-20) and at dC 156.0
(C-40) observed on the 13C NMR spectrum, showing HMBC correla-
tions with the methoxy group (dH 3.86) and the OH (dH 9.60),
respectively. The position of the methoxy and the hydroxyl group
on the aromatic ring were confirmed by correlations between
C-20 and H-30, C-20 and NH, C-40 and H-50, and C-40 and H-60. In con-
clusion, all data were consistent with the structure 8 (Fig. 2), which
was named talaroenamine E.
Compound (9) was isolated as a yellow oil. Its molecular for-
mula was established as C16H22O7, based on its ESI-MS spectra
which shows a molecular ion at m/z 349.1282 [M+Na]+. The strong
IR absorption at 3408 and 1734 cmꢀ1 proved the presence of
hydroxyl and ester groups, respectively. The 1H and 13C NMR data
of 9 (Table 2) revealed that this compound was very similar to the
15G256v polyester (10) previously isolated from Talaromyces flavus
(Ito et al., 1992). The only difference was the nature of the ester at
C-4. The 1H–1H COSY correlation between CH3-1 and H-2 as well as
the key HMBC correlations from H-2 (dH 4.43 and 4.40) to C-1 (dC
14.6) and C-3 (dC 172.4) confirmed the planar structure of 9
(Fig. 4). The absolute configuration at C-11 was deduced as R from
Fig. 2. Key 1H–1H COSY (bold bonds) and 1H–13C HMBC correlations (arrows) for
compounds 5–8.
The molecular ion peak at m/z 284.0959 [MꢀH]ꢀ (calcd. for
C
16H14NO4: 284.0923) observed by HR-ESI-MS (negative mode)
and 13C NMR data for compound 7 indicated a molecular formula
of C16H15NO4. The spectroscopic data (Table 1) of 7 were similar
to those of 5 excepting an additional aldehyde group (dH 10.04,
dC 194.7) at C-20, which was consistent with the difference of
28 amu as compared to 5. The presence of the aldehyde group
was also confirmed by HMBC correlations between the aldehyde
proton (dH 10.04) and C-10, C-20, and C-30 (Fig. 2). Thus, we gave
the name talaroenamine D to compound 7 and as for the other
compound, the absolute configuration at C-2 was also assigned
as R by comparison of the CD spectrum (SI, Fig. 1).
Comparison of the NMR and MS data of compound 8 also
revealed that this compound is an analogue of talaroenamine A
(1). The molecular formula of 8 was deduced to be C16H17NO5 from
the ESI-MS experiments in negative mode (m/z 302.1065 [MꢀH]ꢀ)
and was supported by 13C NMR (9 unsaturated degrees) and 1H
NMR data (Table 1). The main differences in 1H NMR spectra
compared to the other compounds were also located on the
the negative rotation of the hydrolysate (ꢀ) (R) 6-hydroxymellein
20
(11) [
a]
D
(ꢀ44.0, c 0.1 in MeOH, litt. ꢀ49.3, c 1.15 in MeOH (Ito
et al., 1992)) obtained from alkaline hydrolysis followed by
lactonization in acidic condition as previously described by He
et al. (2014). The R configuration of C-15 was determined after
Table 3
Biological evaluations of compounds 1, 5–9.
Compounds
IC50 (l
M, mean SEM)a
HeLa cell line
NIH-3T3-L1 cell line
P. falciparum
1
5
6
7
8
9
>50
NT
>100
>100
>100
>100
>100
>100
10 2.0
NT
>50
25.8 4.8
40.3 6.9
19.2 5.7
>50
32.0 1.7
NT
>100
>100
NT
28.0 12.3
5.6 2.0
NT
Cisplatin
Chloroquine
0.012 0.004
NT: Not tested.
a
Overlapped.
Fig. 3. Comparison between calculated and experimental CD spectra of 5 in MeOH.

Y. Zang et al. / Phytochemistry 119 (2015) 70–75
73
OH
4
OH
CH3
O
CH3
O
HO
O
OH
15
HO
O
OH
11
3
O
O
O
O
O
2
CH3
1
9
10
OH
OH
OH
O
O
HO
O
OH
HO
O
OH
O
O
O
O
11
12
Fig. 4. Structures of compounds 9–12. Key 1H–1H COSY (bold bonds) and 1H–13C HMBC correlations (arrows) for compound 9.
transesterification with sodium ethanolate of 9 to afford ethyl
3-hydroxybutyrate 12 which was analyzed by GC on chiral column.
The retention time of 12 was thus compared to racemic mixture
and to the (S) enantiomer obtained by baker’s yeast reduction of
ethyl acetoacetate (Wipf et al., 1983). The absolute configurations
of talapolyester G 9 was thus determined as (11R, 15R) which is
in accordance with the previously described 15G256 polyesters.
We thus isolated some new compounds from the strain
T. stipitatus ATCC10500, cultivated in PDB liquid medium, never
described until now. In our hands, out of all the liquid or solid
growth media we have tested, these compounds could only be iso-
lated from the PDB liquid medium. Thus, this work is another
example of the deep dependence of fungal metabolic expression
on culture conditions (the OSMAC strategy). Again, the plasticity
of the production of chemically diverse compounds by a given
strain highlights the biotechnological value of filamentous fungi.
All compounds were evaluated for their antiplasmodial activity
against the chloroquine-resistant P. falciparum (FcB1 strain).
Compound 7 displayed the best inhibition against P. falciparum
spectra were recorded in MeOH using a JASCO J-810. IR spectra were
taken on a Shimadzu FTIR-8400S Infrared spectrophotometer. UV
spectra were recorded on a Kontron Uvikon 9X3W Double Beam
UV/vis spectrophotometer (Bioserv, France). Mass spectra were
recorded on an API Q-STAR PULSAR i of Applied Biosystem. For the
CID spectra, the collision energy was 40 eV and the collision gas
was nitrogen. All NMR experiments were recorded on Bruker
Avance III HD 400 MHz and 600 MHz spectrometers (Wissembourg,
France) equipped with a BBFO Plus Smartprobe and a triple reso-
nance TCI cryoprobe, respectively. Chemical shifts are expressed
in d (ppm), and are referenced to the residual non-deuterated sol-
vent signals. Preparative HPLC was performed on an Agilent system
and an Agilent PrepHT XDB-C18 column (21.2 ꢁ 150 mm i.d.; 5
USA). Column chromatography (CC) was performed using silica gel
(Lichroprep RP-18, 40–63 m, Merck KGaA, Germany).
lm;
l
4.2. Fermentation, extraction and isolation
T. stipitatus ATCC 10500 strain was grown in sterilized liquid
media PDB at 27 °C under agitation on a rotary shaker (150 rpm).
After 14 days of cultivation, the culture was filtered to separate
the mycelium and the filtrate. The culture filtrate (3 L) was then
extracted by ethyl acetate (3 ꢁ 1 L) and the combined organic
phases were dried and gave, after removal of the solvent under
vacuum, 1.3 g crude extract.
with an IC50 value of 19 lM. Interestingly, this compound was
not cytotoxic on HeLa cells as well as on 3T3-L1 preadipose cell
lines model from mouse embryo.
3. Conclusion
This crude extract was subjected to preparative HPLC which
afforded compound 6 (5.2 mg) and 7 (4.5 mg) (gradient from 10%
to 60% acetonitrile in H2O for 42 min; flow rate: 20.0 mL/min; 6,
tR = 13.0 min; 7, tR = 14.0 min). Subfractions were further purified
by preparative HPLC (from 10% to 30% MeCN in H2O for 33 min,
flow rate: 20.0 mL/min) and afforded pure compounds 5 (2.1 mg,
tR = 15.4 min), 8 (1.2 mg, tR = 14.0), 1 (13.3, mg, tR = 14.0) and 9
(4.8 mg, tR = 21.9).
Chemical investigation of the fungus T. stipitatus ATCC10500 in
PDB liquid medium led to the isolation of the four new talaroe-
namines BꢀE. Interestingly, talaroenamine A had previously been
isolated from the strain of T. stipitatus
DtropC but never from a
wild-type strain. In the course of this study, a new polyester was
also identified. All the compounds were evaluated for their
antiplasmodial activity and some of them displayed a modest
activity against P. falciparum (IC50: 19.2 lM) without noticeable
toxicity against preadipocytes and Hela cells (IC50 > 100 lM).
4.2.1. Talaroenamine B (5): Pale yellow oil; [a D20
MeOH); ECD
]
ꢀ19.7 (c 0.0033,
4. Experimental
(c 0.390 ꢁ 10ꢀ3 mol/L, MeOH, 20 °C) kmax
(De) 230 (0.16), 330
(1.3), 372 (ꢀ1.12); UV (MeCN) kmax nm (log
e): 240 (3.5), 375
4.1. General
(3.9); IR (NaCl) mmax cmꢀ1: 3419, 3252, 2983, 2926, 1687, 1616,
1566, 1504, 1456, 1398, 1338, 1307, 1184, 1080, 1024, 837, 738,
688; 1H and 13C NMR data (DMSO-d6) see Table 1; (ꢀ) HR-ESI-MS:
m/z 256.0980 [MꢀH]ꢀ.
Optical rotations were determined using a Perkin Elmer 341
Polarimeter and the [
values are given in deg cm2 gꢀ1. ECD
a]
D

74
Y. Zang et al. / Phytochemistry 119 (2015) 70–75
4.2.2. Talaroenamine C (6): Pale yellow oil; [a D20
MeOH); ECD
]
ꢀ12.4 (c 0.0033,
(0.5 lCi) was subsequently added to each well, and parasites were
maintained for an additional 24 h. After a cycle of freezing and
thawing, the cells were harvested from each well onto glass fiber
filters, and the dried filters were counted in a scintillation counter.
The growth inhibition for each well was determined by
comparison of the radioactivity incorporated into the treated
culture with that in the control culture maintained on the same
plate. Chloroquine was used as positive control. Experiments were
performed in triplicate.
(c 0.35 ꢁ 10ꢀ3 mol/L, MeOH, 20 °C) kmax
(De) 228 (1.32), 330
(2.03), 365 (ꢀ3.03); UV (MeCN) kmax nm (log
e): 240 (3.5), 375
(3.8); IR (NaCl) mmax cmꢀ1: 3333, 2929, 1668, 1622, 1570, 1552,
1496, 1456, 1315, 1201, 1180; 1H and 13C NMR data (DMSO-d6)
see Table 1; (+) HR-ESI-MS: m/z 288.1169 [M+H]+, (ꢀ) HR-ESI-
MS: m/z 286.1170 [MꢀH]ꢀ.
4.2.3. Talaroenamine D (7): Pale yellow oil; [a D20
]
ꢀ20.0 (c 0.0033,
MeOH); ECD
4.4.2. Growth inhibition assays on HeLa and preadipocyte cells
The cytotoxicity was determined by the MTT assay. Briefly,
HeLa cells or 3T3-L1 preadipose cells were seeded in 96-well tissue
culture plates at 2500 cells/well and incubated overnight. The
exponentially growing cells were then exposed to different drug
concentrations for four doubling times. Cellular viability was
determined by exposing cells to the MTT tetrazolium salt for 4 h
at 37 °C, and the formation of formazan was measured at 560 nm
by a microplate reader. The concentration inhibiting cell growth
by 50% compared with untreated controls was determined from
the curves plotting survival as a function of dose. All values are
averages of at least three independent experiments each done in
duplicate. CisplatinÒ was used as a control compound. Experiments
were performed in triplicate.
(c 0.35 ꢁ 10ꢀ3 mol/L, MeOH, 20 °C) kmax
(De) 232 (1.73), 338
(0.52), 377 (ꢀ0.91); UV (MeCN) kmax nm (log
e): 265 (3.5), 385
(3.9); IR (NaCl) mmax cmꢀ1: 3450 (br), 1716, 1610, 1568, 1456,
1348, 1311, 1208, 1199, 1114, 1080, 835; 1H and 13C NMR data
(DMSO-d6) see Table 1; (ꢀ) HR-ESI-MS: m/z 284.0959 [MꢀH]ꢀ.
4.2.4. Talaroenamine E (8): Pale yellow oil; [a D20
]
ꢀ10.6 (c 0.0033,
MeOH); ECD
(c 0.33 ꢁ 10ꢀ3 mol/L, MeOH, 20 °C) kmax
(De) 249 (0.6), 333
e): 395 (3.6); IR (NaCl)
(2.02), 385 (ꢀ0.26); UV (MeCN) kmax nm (log
m
max cmꢀ1: 3410, 1666, 1620, 1566, 1516, 1454, 1338, 1296, 1184,
1026, 999, 825, 763, 1H and 13C NMR data (DMSO-d6) see Table 1;
(ꢀ) HR-ESI-MS: m/z 302.1065 [MꢀH]ꢀ, 605.2133 [2MꢀH]ꢀ.
4.2.5. Talapolyester G (9): Pale yellow oil; [a D20
UV (MeCN) kmax nm (log
265 (3.8), 298 (3.5), 395 (2.9); IR (NaCl)
1734, 1716, 1647, 1616, 1589, 1448, 1317, 1261, 1195, 1174, 850,
800; 1H NMR data (CD3OD) see Table 2; (ꢀ) ESI-MS: m/z 325.1251
[MꢀH]ꢀ, 651.2572 [2MꢀH]ꢀ.
]
ꢀ44.0 (c 0.1, MeOH);
Acknowledgments
e)
m
max cmꢀ1: 3408, 2980,
The ‘‘China Scholarship Council (CSC)” is acknowledged for the
PhD fellowship to Y.Z. The authors wish to thank L. Dubost for the
mass spectra, A. Deville for NMR spectra and C. Bance for technical
assistance. 400 MHz and 600 MHz NMR spectrometers used in this
study were funded jointly by the Région Ile-de-France, the MNHN
(Paris, France) and by the CNRS (France). The Centre National de
la Recherche Scientifique (interdisciplinary call Physic-Chemistry-
Biology 2011) is acknowledged for research funding.
4.3. Absolute configurations
For compound 5, the structure geometry was optimized using
the density functional theory (DFT) at the 6–311 + g(d,p) level.
After the check for non imaginary frequencies, the rotational
strengths were then calculated using the time dependent (TD)
DFT at the same level of theory on the most stable conformer. All
calculation were performed using the Gaussian 09W Rev. D.01
package (Frisch et al., 2013). Compound 9 (3.9 mg) was stirred in
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.phytochem.2015.
09.002.
1 N NaOH (2.1 mg in 200 lL H2O) for 12 h at room temperature.
The reaction was quenched by acidification with 1 N HCl solution.
Reaction mixture was thus extracted with ethyl acetate and the
organic phase was dried and evaporate under vaccum to afford
11 (86%, 2 mg).
References
Birkinshaw, J.H., Chambers, A.R., Raistrick, H., 1942. Studies in the biochemistry of
micro-organisms: stipitatic acid, C(8)H(6)O(5),
Penicillium stipitatum Thom. Biochem. J. 36, 242–251.
a metabolic product of
Similarly 9 (1.7 mg) was dissolved in EtOH (0.3 mL) and was
stirred at room temperature 24 h after addition of EtONa (60 lL).
The reaction was quenched by acidification with 1 N HCl methano-
lic solution and subjected to GC analysis on chiral column.
Bondu, S., Genta-Jouve, G., Leiros, M., Vale, C., Guigonis, J.M., Botana, L.M., Thomas,
O.P., 2012. Additional bioactive guanidine alkaloids from the Mediterranean
sponge Crambe crambe. RSC Adv. 2, 2828–2835.
Brakhage, A.A., Schroeckh, V., 2011. Fungal secondary metabolites – strategies to
activate silent gene clusters. Fungal Genet. Biol. 48, 15–22.
Davison, J., Al Fahad, A., Cai, M., Song, Z., Yehia, S.Y., Lazarus, C.M., Bailey, A.M.,
Simpson, T.J., Cox, R.J., 2012. Genetic, molecular, and biochemical basis of fungal
tropolone biosynthesis. Proc. Natl. Acad. Sci. U.S.A. 109, 7642–7647.
Desjardins, R.E., Canfield, C.J., Haynes, J.D., Chulay, J.D., 1979. Quantitative
assessment of antimalarial activity in vitro by a semiautomated microdilution
technique. Antimicrob. Agents Chemother. 16, 710–718.
Column CP-chirasil-Dex CB 25 m 0.32 mm, 25 lm, (carrier gas He
10 psi, oven temperature 65 °C (20 min), Injector 190 °C, gradient
8 °C/min 80 °C). Retention times (S)-ethyl 3-hydroxybutyrate,
21.1 min, (R)-ethyl 3-hydroxybutyrate 21.5 min.
4.4. Biological assays
Frisch, M.J., Trucks, G.W., Schlegel, H.B., Scuseria, G.E.R., Robb, M.A., Cheeseman, J.R.,
Scalmani, G., Barone, V., Mennucci, B., Petersson, G.A., Nakatsuji, H., Caricato, M.,
Li, X., Hratchian, H.P., Izmaylov, A.F., Bloino, J., Zheng, G., Sonnenberg, J.L., Hada,
M., Ehara, M., Toyota, K., Fukuda, R., Hasegawa, J., Ishida, M., Nakajima, T.,
Honda, Y., Kitao, O., Nakai, H., Vreven, T., Montgomery Jr., J.A., Peralta, J.E.,
Ogliaro, F., Bearpark, M.J., Heyd, J., Brothers, E.N., Kudin, K.N., Staroverov, V.N.,
Kobayashi, R., Normand, J., Raghavachari, K., Rendell, A.P., Burant, J.C., Iyengar, S.
S., Tomasi, J., Cossi, M., Rega, N., Millam, N.J., Klene, M., Knox, J.E., Cross, J.B.,
Bakken, V., Adamo, C., Jaramillo, J., Gomperts, R., Stratmann, R.E., Yazyev, O.,
Austin, A.J., Cammi, R., Pomelli, C., Ochterski, J.W., Martin, R.L., Morokuma, K.,
Zakrzewski, V.G., Voth, G.A., Salvador, P., Dannenberg, J.J., Dapprich, S., Daniels,
A.D., Farkas, Ö., Foresman, J.B., Ortiz, J.V., Cioslowski, J., Fox, D.J., 2013. Gaussian
09. Gaussian Inc., Wallingford, CT, USA.
4.4.1. Antiplasmodial assay
The antiplasmodial activity was evaluated against the chloro-
quine-resistant FcB1/Colombia strain of P. falciparum. The assay
was performed using the method of Desjardins (Desjardins et al.,
1979). Extracts and pure compounds were diluted in DMSO and
add to the culture medium (final concentration: 20 lg/mL) in
96-well microplates. Asynchronous parasite cultures were then
added (1% parasitemia and 1% final hematocrite), and plates were
maintained for 24 h at 37 °C in a candle jar. [3H]hypoxanthine

Y. Zang et al. / Phytochemistry 119 (2015) 70–75
75
Genta-Jouve, G., Weinberg, L., Cocandeau, V., Maestro, Y., Thomas, O.P., Holderith, S.,
2013. Revising the absolute configurations of coatlines via density functional
theory calculations of electronic circular dichroism spectra. Chirality 25, 180–184.
He, J.Z., Li, X.X., Li, Y., Liu, X.Z., Yao, X.S., 2014. New polyesters from Talaromyces
flavus. Tetrahedron 70, 4425–4430.
Ito, M., Tsuchida, Y., Mizoue, K., Hanada, K., 1992. NG-011 and NG-012, novel
potentiators of nerve growth factor. II. The structure determination of NG-011
and NG-012. J. Antibiot. 45, 1566–1572.
production by Paraphaeosphaeria quadrisptata and Chaetomium chiversii. J. Nat.
Prod. 70, 1939–1945.
Scharf, D.H., Brakhage, A.A., 2013. Engineering fungal secondary metabolism: a
roadmap to novel compounds. J. Biotechnol. 163, 179–183.
Schueffler, A., Anke, T., 2014. Fungal natural products in research and development.
Nat. Prod. Rep. 31, 1425–1448.
Shibata, S., Ogihara, Y., Tokutake, N., Tanaka, O., 1965. Duclauxin, a metabolite of
Penicillium duclauxi (delacroix). Tetrahedron Lett. 6, 1287–1288.
Wipf, B., Kupfer, E., Bertazzi, R., Leuenberger, H.G.W., 1983. Production of
(+)-(S)-ethyl 3-hydroxybutyrate and (-)-(R)-ethyl 3-hydroxybutyrate by
microbial reduction of ethyl acetoacetate. Helv. Chim. Acta 66, 485–488.
Lin, Z., Zhu, T., Fang, Y., Gu, Q., Zhu, W., 2008. Polyketides from Penicillium sp. JP-1,
an endophytic fungus associated with the mangrove plant Aegiceras
corniculatum. Phytochemistry 69, 1273–1278.
Paranagama, P.A., Wijeratne, E.M.K., Gunatilaka, A.A., 2007. Uncovering biosynthetic
potential of plant-associates fungi: effect of culture conditions on metabolites