Seven new and two known lipopeptides as well as five known polyketides: The activated production of silent metabolites in a marine-derived fungus by chemical mutagenesis strategy using diethyl sulphate

Article abstract of DOI:10.3390/md12041815

AD-2-1 is an antitumor fungal mutant obtained by diethyl sulfate mutagenesis of a marine-derived Penicillium purpurogenum G59. The G59 strain originally did not produce any metabolites with antitumor activities in MTT assays using K562 cells. Tracing newly produced metabolites under guidance of MTT assay and TLC analysis by direct comparison with control G59 extract, seven new (1-7) and two known (8-9) lipopeptides were isolated together with five known polyketides 10-14 from the extract of mutant AD-2-1. Structures of the seven new compounds including their absolute configurations were determined by spectroscopic and chemical evidences and named as penicimutalides A-G (1-7). Seven known compounds were identified as fellutamide B (8), fellutamide C (9), 1.-O-methylaverantin (10), averantin (11), averufin (12), nidurufin (13), and sterigmatocystin (14). In the MTT assay, 1-14 inhibited several human cancer cell lines to varying extents. All the bioassays and HPLC-photodiode array detector (PDAD)-UV and HPLC-electron spray ionization (ESI)-MS analyses demonstrated that the production of 1-14 in the mutant AD-2-1 was caused by the activated production of silent metabolites in the original G59 fungal strain. Present results provided additional examples for effectiveness of the chemical mutagenesis strategy using diethyl sulphate mutagenesis to discover new compounds by activating silent metabolites in fungal isolates.

Full text of DOI:10.3390/md12041815

Mar. Drugs 2014, 12, 1815-1838; doi:10.3390/md12041815
OPEN ACCESS
marine drugs
ISSN 1660-3397
www.mdpi.com/journal/marinedrugs
Article
Seven New and Two Known Lipopeptides as well as Five Known
Polyketides: The Activated Production of Silent Metabolites in a
Marine-Derived Fungus by Chemical Mutagenesis Strategy
Using Diethyl Sulphate
1
,2
2
1,2,
Chang-Jing Wu , Chang-Wei Li and Cheng-Bin Cui
*
1
Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education,
School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University,
Shenyang 110016, China; E-Mail: wucj2009@163.com
2
Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China;
E-Mail: sdrlcw@126.com
*
Author to whom correspondence should be addressed; E-Mail: cuicb@126.com or cuicb@sohu.com;
Tel./Fax: +86-10-6821-1656.
Received: 24 December 2013; in revised form: 12 February 2014 / Accepted: 17 February 2014 /
Published: 28 March 2014
Abstract: AD-2-1 is an antitumor fungal mutant obtained by diethyl sulfate mutagenesis of
a marine-derived Penicillium purpurogenum G59. The G59 strain originally did not
produce any metabolites with antitumor activities in MTT assays using K562 cells. Tracing
newly produced metabolites under guidance of MTT assay and TLC analysis by direct
comparison with control G59 extract, seven new (1–7) and two known (8–9) lipopeptides
were isolated together with five known polyketides 10–14 from the extract of mutant
AD-2-1. Structures of the seven new compounds including their absolute configurations
were determined by spectroscopic and chemical evidences and named as penicimutalides
A–G (1–7). Seven known compounds were identified as fellutamide B (8), fellutamide C
(
9), 1′-O-methylaverantin (10), averantin (11), averufin (12), nidurufin (13), and
sterigmatocystin (14). In the MTT assay, 1–14 inhibited several human cancer cell lines to
varying extents. All the bioassays and HPLC-photodiode array detector (PDAD)-UV and
HPLC-electron spray ionization (ESI)-MS analyses demonstrated that the production of
1
–14 in the mutant AD-2-1 was caused by the activated production of silent metabolites
in the original G59 fungal strain. Present results provided additional examples for
effectiveness of the chemical mutagenesis strategy using diethyl sulphate mutagenesis to
discover new compounds by activating silent metabolites in fungal isolates.

Mar. Drugs 2014, 12
1816
Keywords: marine-derived fungus; lipopeptide; penicimutalide; Marfey analysis;
polyketide; Penicillium purpurogenum; DES mutagenesis
1
. Introduction
Activating silent secondary metabolites of fungal isolates have attracted considerable attention and
various strategies have been developed for this purpose in the past decade [1–4]. The one strain-many
compounds (OSMAC) strategy [5] and chemical epigenetics methodology [6] have been widely
used by microbial chemists to access cryptic secondary metabolites [7]. The ribosome engineering
strategy [8] provided another simple way to activate silent pathways by introducing drug-resistance
mutations in bacteria to discover new antibiotics [9]. We previously extended this strategy to
fungi to activate silent metabolites by introducing drug-resistance in a bio-inactive marine-derived
Penicillium purpurogenum G59 using dimethyl sulfoxide (DMSO) as accessorial agent [10,11]. As
further extension of that work, we have just recently developed a practical strategy to discover new
antitumor agents by activating silent fungal metabolite production using a modified method of diethyl
sulphate (DES) mutagenesis on P. purpurogenum G59 [12]. Using this strategy, several new antitumor
agents including some with novel structures have also been discovered [12,13]. As a continuation, we
report here our recent work on another bioactive mutant AD-2-1, including the discovery of seven new
lipopeptides from activated production of silent metabolites in the original G59 fungal strain.
P. purpurogenum G59 is a marine-derived, wild-type fungal strain initially isolated by our
group [14] and originally did not produce any metabolites with antitumor activity in repeated bioassay
using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and K562 cells [10–14].
AD-2-1 was obtained as an antitumor mutant by DES mutagenesis of the strain G59 and the ethyl
acetate (EtOAc) extract of its cultures inhibited K562 cells with an inhibition rate (IR%) value of
4
9.8% at 100 µg/mL [12]. However, the metabolites with antitumor activity in the EtOAc extract have
not been investigated so far. In the present work, we thus investigated the antitumor metabolites in the
EtOAc extract, which were newly produced by the mutant AD-2-1 compared to its parent G59 strain.
This has resulted in the discovery of seven new lipopeptides (1–7) and the identification of two known
lipopeptides (8 and 9) as well as five known polyketides (10–14) shown in Figure 1. All 1–14 inhibited
several human cancer cell lines to varying extents. All the bioassays and HPLC-photodiode array
detector (PDAD)-UV and HPLC-electron spray ionization (ESI)-MS analyses demonstrated that the
production of 1–14 in the mutant AD-2-1 was caused by the activated production of silent metabolites
in original fungal G59 strain.
2
. Results and Discussion
2
.1. Fermentation, Isolation of 1–14, and Identification of Known Compounds 8–14
Large-scale fermentation and extraction of the mutant AD-2-1 produced an EtOAc extract that
inhibited the K562 cells with an IR% value of 64.6% at 100 µg/mL. However, the control G59 EtOAc
extract that was obtained by fermentation of the G59 strain at the same time and same conditions did

Mar. Drugs 2014, 12
1817
not inhibit the K562 cells (an IR% of 2.5% at 100 µg/mL). In preliminary analyses of the mutant
AD-2-1 and the control G59 extracts by thin layer chromatography (TLC), lipopeptide components
that contained 1–9 were detected as white spots by spraying distilled water to the silica gel TLC plates
in the mutant extract but not in the control G59 extract. The polyketide components including 10–14
were also detected only in the mutant extract as spots on the silica gel TLC plates or on the polyamide
film by examination under sunlight or UV lights (254 and 360 nm) or by visualization using 5% FeCl
3
reagent, Vaughan’s reagent or 10% sulfuric acid reagent. Thus, the separation of the mutant extract
was performed tracing these newly produced metabolites under the guidance of MTT assay and TLC
analysis by direct comparison with the control G59 extract.
A column chromatography of the mutant extract on Sephadex LH-20 by monitoring the targeted
components gave two fractions that contained the aimed lipopeptides and polyketides, respectively.
The lipopeptide-containing fraction was separated by chromatographic means, including repeatedly
performed preparative TLC, column chromatography, and semi-preparative HPLC, to obtain 1–9
shown in Figure 1. The HPLC separations were performed on a chiral, reversed-phase Click β-CD
column. The polyketide-containing fraction was separated by repeated column chromatography on
silica gel, polyamide, and Sephadex LH-20 to afford 10–14 (Figure 1).
Figure 1. Structures of 1–14 newly produced by the mutant AD-2-1.
Among the obtained compounds 1–14, structures of seven new compounds including their absolute
configurations were determined by spectroscopic and chemical evidences and named penicimutalides
A–G (1–7). Structure determinations of these new compounds are described in detail in Section 2.2.
Further, seven known compounds including two lipopeptides 8–9 and five polyketides 10–14 were
identified as fellutamide B (8) [15], fellutamide C (9) [16], 1′-O-methylaverantin (10) [17], averantin
(
11) [18], averufin (12) [18,19], nidurufin (13) [18–20], and sterigmatocystin (14) [21], respectively,
by their physicochemical and spectroscopic data. The identification of fellutamide B (8) and
fellutamide C (9) was also based on the chemical evidences to confirm their absolute configurations.

Mar. Drugs 2014, 12
.2. Structure Determination of New Compounds 1–7
1818
2
2
D
5
25
D
Penicimutalide A (1), [α]
−18.0 (c 0.1, MeOH), and penicimutalide B (2), [α]
−17.0 (c 0.1,
MeOH), were obtained as amorphous powders from MeOH, and their molecular compositions were
+
assigned to be C28
Na [M + Na] 594.3843). Penicimutalides C (3), [α]
−24.8 (c 0.3, MeOH), amorphous powders (MeOH), were assigned the same molecular formula
H
53
N
5
O
7
+
by HRESIMS (measured 594.3834 [M + Na] for both 1 and 2, calcd for
25
C
28
2
H
53
N
5
O
7
D
−23.7 (c 0.3, MeOH), and D (4),
5
[α]
D
+
+
C
C
27
H
H
51
N
N
5
O
O
7
(HRESIMS: measured 580.3695 [M + Na] for 3 and 580.3684 [M + Na] for 4; calcd for
+
25
D
27
2
51
5
7
Na [M + Na] 580.3686). Penicimutalides E (5), [α]
−15.0 (c 0.04, MeOH), and F (6),
5
[α]
D
−18.8 (c 0.04, MeOH), amorphous powders (MeOH), had also the same molecular composition
+
+
C
C
27
H
H
50
N
N
6
O
O
7
(HRESIMS: measured 571.3819 [M + H] for 5 and 571.3805 [M + H] for 6, calcd for
+
25
D
27
51
6
7
[M + H] 571.3819). Penicimutalide G (7), an amorphous powder (MeOH), [α]
−7.5
(
39 5 6
c 0.1, MeOH), was assigned the molecular formula C21H N O by HRESIMS (measured 458.2970
+
+
[
M + H] , calcd for C21
The UV spectra of 1–7 exhibited end absorptions (see their absorption curves in Figure S1-A2 in
the Supplementary Information), and their IR spectra (see their IR spectra in the Supplementary
40 5 6
H N O [M + H] 458.2979).
−1
Information) showed absorptions due to OH/NH (around 3273 and 3205 cm ), CH
3
2
/CH (around 2930
−1
−1
and 2860 cm ), and amide (amide I bands around 1650 cm for amide carbonyl and amide II bands
−1
around 1535 cm for amide H
2
NCO and/or HNCO) groups. These data, coupled with the molecular
1
13
sizes and the molecular compositions of 1–7, revealed that they were peptides. In the H and C NMR
1
13
spectra (see their NMR spectra in the Supplementary Information), 1–7 showed H and C NMR
signals that closely resembled the signals from the known lipopeptides 8 and 9 except several signals
from leucine-derived moieties have slightly changed (Tables 1 and 2). These NMR data indicated that
1
–7 are all lipopeptides that resembled 8 and 9 but differed only in the leucine-derived moieties. This
1
1
was further confirmed by the detailed analyses of their DEPT, H- H COSY, HMQC, and HMBC
spectra (see Tables S1–S7 for 1–7 and see also their spectra, all in the Supplementary Information).
1
1
Interpretation of the H– H COSY and HMQC data established structural parts related to the proton
spin systems including OH and NH in 1–7. The structure moieties of 3-hydroxydodecaonic acid
(
HDA, C21→C32), asparagine (Asn, C15→N20), and glutamine (Gln, C8→N14) in 1–7 were
deduced by the HMBCs detected on H -22/C-21, H-23/C-21, HO-23/C-22, HO-23/C-23, and
HO-23/C-24 in HAD, on H-16/C-15, H-16/C-18, H
and H-20/C-15 in Asn, and on H-9/C-8, H -10/C-8, H
and H-14/C-8 in Gln moieties. In addition, the HMBCs on H
demonstrated the presence of an isopentyl amino group (N7→C2→C6) with an ethoxy group at C-2 in
–2 and a methoxy group at C-2 in 3–4. The HMBCs on H -1′/C-1, H -1′/C-2, H-2/C-1, and H -3/C-1
2
2
-17/C-15, H
-10/C-12, H
-1'/C-2 in 1–2 and H
2
-17/C-18, H
2
-19/C-17, H
2
2
-19/C-18,
-13/C-12,
2
2
2
-11/C-12, H
2
-13/C-11, H
2
3
-1'/C-2 in 3–4
1
2
2
2
of 5 and 6 established the leucine (Leu) amide residues in 5 and 6. The connectivity of the HAD, Asn
and Gln in 1–7 were determined by the HMBCs of H-16 and H-20 in Asn with C-21 in HDA and of
H-9 and H-14 in Gln with C-15 in Asn. The isopentyl amino groups in 1–4 and Leu amide residues in
5
and 6 were further connected to C-8 in Gln by the HMBCs of H-2 and H-7 with C-8 in 1–4 and 5–6.
The terminal Gln in 7 was evidenced by the HMBCs on H -7/C-8 and H -7/C-9.
2
2

Mar. Drugs 2014, 12
1819
1
a
Table 1. H NMR data of 1–9 in DMSO-d
6
(δ
H
, J in Hz) .
b
b
c
c
b
b
c
c
c
Proton
1
2
3
4
5
6
7
8
9
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
9.35 s
—
3.30 ddd (10.6, 5.6, 4.8)
3.23 ddd (10.6, 6.0, 4.8)
3.84–3.72 (overlapped)
1
—
2
3
5.07 dt (9.2, 6.9)
5.06 dt (9.1, 6.9)
4.97 dt (9.1, 6.9)
4.96 dt (9.4, 7.2)
4.07–4.02 m
1.59–1.50 m
1.48–1.42 m
1.59–1.50 m
0.80 d (6.6)
0.87 d (6.6)
4.15–4.07 m
1.59–1.49 m
1.49–1.40 m
1.59–1.49 m
0.79 d (6.2)
0.84 d (5.6)
4.08–4.00 m
1.48 dt (13.8, 6.9) 1.45 dt (13.8, 6.9) 1.49 dt (13.8, 6.9) 1.49–1.38 (2H) m
1.56–1.44 (2H) m 1.40–1.25 (overlapped)
1
.40–1.33 m
1.40 dt (13.8, 6.9)
1.60–1.52 m
0.81 d (6.6)
1.42–1.30 m
1.65–1.54 m
0.83 d (6.7)
0.85 d (6.7)
—
—
—
4
5
6
1.65–1.57 m
0.83 d (6.8)
0.86 d (6.7)
1.61–1.49 m
0.81 d (6.6)
0.84 d (6.6)
1.68-1.58 m
0.84 d (6.4)
0.89 d (6.8)
1.63–1.52 m
0.82 d (6.4)
0.86 d (6.4)
0.84 d (6.8)
7
7
.34 br s
.08 br s
7
9
8.02 d (9.2)
8.04 d (9.1)
8.03 d (9.1)
8.04 d (9.4)
7.86 d (8.4)
7.87 d (8.4)
8.27 d (7.2)
7.43 d (7.6)
4.13 td (7.8, 4.6)
4.13–4.05 m
1.97–1.90 m
1.78–1.70 m
2.13–2.03 m
7.20 br s
4.18–4.08 m
2.02–1.88 m
1.80–1.68 m
2.14–2.03 m
7.25 br s
4.13–4.05 m
2.00–1.89 m
1.81–1.69 m
2.13–2.05 m
7.24 br s
4.13–4.09 m
1.95–1.88 m
1.78–1.72 m
2.21–2.16 m
7.22 br s
4.15–4.07 m
2.00–1.92 m
1.77–1.67 m
2.08–2.00 m
7.18 br s
4.06 td (8.0, 4.6)
1.95 dtd (13.2, 7.5, 4.6)
1.70 ddt (13.2, 8.0, 7.5)
2.06 t (7.5)
4.21–4.16 m
2.02–1.90 m
1.82–1.68 m
2.14–2.04 m
7.21 br s
4.15–4.05 (overlapped)
1.97–1.85 m
1.78–1.65 m
2.11–2.02 m
7.20 br s
1
0
1.99–1.89 m
1
.78–1.68 m
11
2.12–2.04 m
1
3
7.21 br s
7.21 br s
6
.75 br s
6.74 br s
6.79 br s
6.78 br s
6.76 br s
6.71 br s
6.75 br s
6.76 br s
6.74 br s
1
1
1
4
6
7
7.94 d (7.8)
4.49 q (7.0)
8.06 d (7.6)
4.50 q (7.0)
8.05 d (8.0)
4.49 q (6.9)
8.12 d (7.6)
4.50 q (7.0)
8.10 d (7.2)
4.48 q (7.2)
2.58–2.42 m
2.58–2.42 m
7.46 br s
8.09 d (8.4)
4.45 q (6.9)
2.54 dd (15.6, 6.9)
2.43 dd (15.6, 6.9)
7.41 br s
7.98 d (8.0)
8.10 d (7.6)
4.50 q (7.2)
2.55 dd (15.5, 7.2)
2.44 dd (15.5, 7.2)
7.42 br s
8.00 d (8.0)
4.48 q (7.0)
4.48 br q (6.9)
2.55 dd (15.6, 6.9)
2.43 dd (15.6, 6.9)
7.42 br s
2.54 dd (15.6, 7.0) 2.55 dd (15.6, 7.0) 2.54 dd (15.5, 6.9) 2.55 dd (15.6, 7.0)
.43 dd (15.6, 7.0) 2.43 dd (15.6, 7.0) 2.43 dd (15.5, 6.9) 2.42 dd (15.6, 7.0)
2.57 dd (15.5, 7.0)
2.43 dd (15.5, 7.0)
7.42 br s
2
1
9
7.37 br s
.90 br s
7.40 br s
6.94 br s
7.41 br s
6.93 br s
7.43 br s
7.24 br s
6
6.95 br s
6.91 br s
6.96 br s
6.93 br s
6.94 br s
2
2
2
2
0
2
3
4
8.08 d (7.0)
2.26–2.16 m
3.82–3.75 m
1.40–1.33 m
8.05 d (7.0)
2.25–2.16 m
3.80–3.75 m
1.39–1.30 m
8.12 d (6.9)
2.27-2.16 m
3.84–3.73 m
1.42–1.30 m
8.08 d (7.0)
2.25–2.16 m
3.82–3.72 m
1.38–1.30 m
8.26 d (7.2)
2.24–2.16 m
3.80–3.74 m
1.40–1.18 m
8.15 d (7.2)
2.25–2.15 m
3.81–3.74 m
1.40–1.18 m
8.09 d (7.0)
8.09 d (7.2)
2.24–2.16 m
3.82–3.72 m
1.40–1.20 m
8.10 d (6.9)
2.25–2.14 m
2.25–2.17 m
3.84–3.72 (overlapped)
1.40–1.20 m
3.82–3.73 m
1.40–1.20 m

Mar. Drugs 2014, 12
1820
Table 1. Cont.
b
b
c
c
b
b
c
c
c
Proton
1
2
3
4
5
6
7
8
9
1
1
.40–1.33 m
.30–1.18 m
1.39–1.30 m
1.30–1.18 m
1.30–1.18 m
1.30–1.18 m
1.30–1.18 m
1.30–1.18 m
1.30–1.18 m
1.30–1.18 m
0.86 t (7.2)
1.42–1.30 m
1.30–1.18 m
1.30–1.18 m
1.30–1.18 m
1.30–1.18 m
1.30–1.18 m
1.30–1.18 m
1.30–1.18 m
0.85 t (7.2)
3.11 (3H) s
—
1.38–1.30 m
1.30–1.18 m
1.30–1.18 m
1.30–1.18 m
1.30–1.18 m
1.30–1.18 m
1.30–1.18 m
1.30–1.18 m
0.86 t (6.8)
3.12 (3H) s
—
1.40–1.18 m
1.40–1.18 m
1.40–1.18 m
1.40–1.18 m
1.40–1.18 m
1.40–1.18 m
1.40–1.18 m
1.40–1.18 m
0.85 t (6.9)
6.97 br s
1.40–1.18 m
1.40–1.18 m
1.40–1.18 m
1.40–1.18 m
1.40–1.18 m
1.40–1.18 m
1.40–1.18 m
1.40–1.18 m
0.85 t (6.9)
7.17 br s
1.40–1.20 m
1.40–1.20 m
1.40–1.20 m
1.40–1.20 m
1.40–1.20 m
1.40–1.20 m
1.40–1.20 m
1.40–1.20 m
0.86 t (6.8)
—
1.40–1.20 m
1.40–1.20 m
1.40–1.20 m
1.40–1.20 m
1.40–1.20 m
1.40–1.20 m
1.40–1.20 m
1.40–1.20 m
0.85 t (6.8)
—
1.40–1.20 m
1.40–1.20 m
1.40–1.20 m
1.40–1.20 m
1.40–1.20 m
1.40–1.20 m
1.40–1.20 m
1.40–1.20 m
0.86 t (6.8)
—
2
5
2
2
2
2
3
3
3
6
7
8
9
0
1
2
1.30–1.18 m
1.30–1.18 m
1.30–1.18 m
1.30–1.18 m
1.30–1.18 m
1.30–1.18 m
0.85 t (7.0)
1
'
3.44 dq (14.1, 7.0) 3.46 dq (14.1, 7.0)
.27 dq (14.1, 7.0) 3.28 dq (14.1, 7.0)
3
6.95 br s
6.97 br s
—
—
—
2
'
1.05 t (7.0)
—
1.05 t (7.0)
—
—
—
—
—
—
—
—
1
-OH
—
—
—
—
—
—
4.54 br t (4.8)
4.64 br d (4.8)
2
3-OH
4.64 d (4.8)
4.62 d (4.8)
4.68 d (4.8)
4.65 d (5.1)
4.61 d (5.4)
4.66 br s
4.65 d (5.1)
4.63 d (5.2)
a
1
1
Chemical shifts were recorded in δ values using the solvent DMSO-d
6
signal δ
H
2.50 as reference; Signal assignments were based on the results of DEPT, H– H COSY, HMQC, and
b
1
c
1
HMBC experiments; Recorded at 600 MHz H NMR; Recorded at 400 MHz H NMR.

Mar. Drugs 2014, 12
Table 2. C NMR data of 1–9 in DMSO-d
1821
1
3
a
6
(δ
C
, multiplicity) .
b
b
c
c
b
b
c
c
c
Carbon
1
2
3
4
5
6
7
8
9
1
2
3
4
5
6
—
—
—
—
174.2 s
49.9 d
40.0 t
174.3 s
48.7 d
40.2 t
—
—
—
—
—
—
201.6 s
56.6 d
36.3 t
63.7 t
48.8 d
40.2 t
24.1 d
22.1 q
23.4 q
77.2 d
43.3 t
24.0 d
22.3 q
22.4 q
77.3 d
43.1 t
23.9 d
22.2 q
22.5 q
78.9 d
43.0 t
24.1 d
22.4 q
22.5 q
78.9 d
42.8 t
24.0 d
22.2 q
22.7 q
24.2 d
21.2 q
23.0 q
24.1 d
21.8 q
23.4 q
23.9 d
21.3 q
23.1 q
8
9
171.5 s 171.6 s 171.7 s
171.8 s
53.9 d
27.5 t
170.9 s
53.2 d
27.1 t
170.5 s
52.6 d
27.7 t
173.3 s
52.3 d
27.5 t
171.9 s 170.5 s
52.6 d
27.6 t
31.4 t
52.8 d
27.5 t
31.4 t
52.8 d
27.6 t
31.5 t
52.4 d
27.5 t
31.4 t
52.7 d
27.8 t
31.5 t
10
1
1
31.5 t
31.4 t
31.5 t
31.5 t
1
2
173.7 s 173.7 s 173.9 s
173.8 s
173.8 s
173.9 s
173.8 s
173.8 s 173.9 s
1
1
1
1
5
6
7
8
171.0 s 171.1 s 171.3 s
171.2 s
49.8 d
36.9 t
170.9 s
49.9 d
36.9 t
170.9 s
49.8 d
36.8 t
170.8 s
49.8 d
36.9 t
171.1 s 170.9 s
49.9 d
36.9 t
49.7 d
37.0 t
50.0 d
36.9 t
49.8 d
37.0 t
49.9 d
37.0 t
171.6 s 171.7 s 171.8 s
171.9 s
171.8 s
171.8 s
171.9 s
171.8 s 171.8 s
2
2
2
2
2
2
2
2
2
3
3
3
1
2
3
4
5
6
7
8
9
0
1
2
171.2 s 171.1 s 171.2 s
171.1 s
43.5 t
67.5 d
37.1 t
25.2 t
29.2 t
29.2 t
29.1 t
28.8 t
31.4 t
22.2 t
14.0 q
54.4 q
—
171.2 s
43.4 t
67.4 d
37.0 t
25.0 t
29.1 t
29.1 t
28.9 t
28.7 t
31.2 t
22.0 t
13.9 q
—
171.2 s
43.4 t
67.4 d
36.9 t
25.1 t
29.1 t
29.0 t
28.9 t
28.7 t
31.3 t
22.1 t
13.9 q
—
171.1 s
43.5 t
67.5 d
36.9 t
25.1 t
28.7 t
29.0 t
29.1 t
29.1 t
31.3 t
22.1 t
14.0 q
—
171.2 s 171.2 s
43.4 t
67.4 d
36.8 t
25.1 t
29.1t
43.4 t
67.4 d
36.8 t
25.1 t
29.1t
43.5 t
67.5 d
37.0 t
25.2 t
29.2 t
29.1 t
29.1 t
28.8 t
31.4 t
22.2 t
14.0 q
54.3 q
—
43.5 t
67.5 d
36.9 t
25.1 t
29.1 t
29.1 t
29.0 t
28.7 t
31.3 t
22.1 t
14.0 q
—
43.5 t
67.4 d
36.9 t
25.1 t
29.1 t
29.1 t
29.0 t
28.7 t
31.3 t
21.8 t
14.0 q
—
29.1t
29.1t
29.0 t
28.7 t
31.3 t
22.0 t
13.9 q
61.8 t
15.0 q
29.0 t
28.7 t
31.3 t
22.0 t
13.9 q
61.8 t
15.1 q
1
′
′
2
a
—
—
—
—
—
Chemical shifts were recorded in δ values using the solvent DMSO-d
6 C
signal δ 39.52 as reference.
Multiplicities of the carbon signals were determined by the DEPT method and are shown as s (singlet), d
1
1
(
doublet), t (triplet), and q (quartet), respectively. Signals were assigned on the basis of DEPT, H– H COSY,
b
13
c
13
HMQC, and HMBC experiments; Recorded at 150 MHz C NMR; Recorded at 100 MHz C NMR.
The absolute configurations of Asn and Gln in 1–7 and Leu amide further in 5–6 were determined
by Marfey’s method [22] using D- and L-Leu amides, D- and L-Asn, and D- and L-Gln as standards. We
hydrolyzed 1–7 with 6 N HCl at 110 °C for 24 hours to produce hydrolysates containing aspartic and
glutamic acids in 1–7 and leucine further in 5–6. Each standard was hydrolyzed also in the same
conditions. The hydrolysates of 1–7 and the standards were combined with Marfey’s reagent to
derivatize the amino acids. HPLC analyses of the Marfey’s derivatives from 1–7, using the derivatives
of the L- and D-standards as references, established the L configurations of Asn (16S) and Gln (9S) in

Mar. Drugs 2014, 12
1822
1
–7, the L configuration of Leu amide (2S) in 5, and the D configuration of the Leu amide (2R) in 6.
The 23R absolute configurations of 1–7 were determined as follows. The 23R absolute configurations
of the known 8 and 9 were determined by the chemical and Mosher’s methods in the literature [15,16].
Coproduction of 1–9 by the mutant AD-2-1 revealed the same absolute configuration 23R in 1–7 from
a biogenetic consideration. Coincidently, hydrolysis of 1–9 mixtures in 6 N HCl at 110 °C for 1 h,
2
D
5
25
D
as reported [23], produced 3R-hydroxydodecanoic acid ([α] −16.0 (c 0.05, CHCl
3
); lit. [α] −17.8
(
3
c 1.2, CHCl ) [24]) and established the 23R configuration in 1–7. Therefore, absolute configurations
have been defined for all chiral carbons in 1–7 except for the C-2 carbons in 1–4. Thus, 1/2 and 3/4
were each pairs of epimers at C-2.
Although several structurally related lipopeptides have been known to occur in Nature [15,16,23],
1
–7 are the first examples of new lipopeptides generated by mutagenic activation of silenced metabolic
pathways in fungi. Among the lipopeptides reported, fellutamides A (10-hydroxylated B) and B (8 in
Figure 1) were isolated in 1991 from Penicillium fellutanum and given their names by the mixed
characters from the species name fellutanum and the group name of compounds ―amide‖ [15]. Both
compounds carry 3-hydroxydodecaonic acid (HDA) side chains at N-20 and leucinal residues at N-7.
Later in 2010 [16], the lipopeptide 9 shown in Figure 1 was initially isolated as a new compound from
Penicillium vercicolor and named fellutamide C in view of the structural similarity to fellutamides A
and B (8). Fellutamide C (9) carries an L-leucinol residue, instead of the leucinal residue in 8. Later in
2
011, a new lipopeptide from an undescribed fungal species of Metulocladosporiella was reported by
Xu et al. giving the same name ―fellutamide C‖ [23]. The ―fellutamide C‖ differs from 8 in carrying a
-hydroxytetradecanoic acid (HTDA) side chain at N-20. However, the structure of this compound was
3
apparently different from the fellutamide C [16] (9) and this has thus confused the names of the same
class of compounds. Xu et al. [23] reported also another new lipopeptide in this class of compounds,
named fellutamide D, which differed from their ―fellutamide C‖ in the valinal residue at N-7, instead
of the leucinal residue in ―fellutamide C‖. We therefore propose here that the ―fellutamide C‖ should
be renamed, and fellutamide E was suggested for the name of this compound. Incidentally, we named
the new lipopeptides 1–7 as penicimutalides A–G (1–7), which differ from the fellutamides in the
leucine-derived moieties at N-7, and this would avoid similar confusion.
2
.3. HPLC-PDAD-UV/HPLC-ESI-MS Analyses of G59 and AD-2-1 Extracts for Detecting 1–14
The EtOAc extracts of the mutant AD-2-1 and the strain G59 were subjected to HPLC-PDAD-UV
and HPLC-ESI-MS analyses, using 1–14 as reference standards, to confirm the activated production of
1
1
–14 in the mutant AD-2-1 by the DES mutagenesis of original G59 strain. The reference standards
–6 were used as mixed pairs of 1/2, 3/4, and 5/6, respectively, because the peaks of two compounds
in these pairs had been known to appear at the same retention times in the given HPLC condition using
an achiral, reversed phase analytical column by pilot test of HPLC analysis.
In the HPLC-PDAD-UV analysis, 1–9 were eluted as peaks with retention times of 56.12 min for
1
/2, 54.57 min for 3/4, 50.62 min for 5/6, 46.52 min for 7, 52.77 min for 8, and 52.57 min for 9,
respectively. Because 1–9 showed only the end UV absorptions, they were hardly identified by the
HPLC-PDAD-UV analysis both in the mutant AD-2-1 and the control G59 extracts due to the lack of
the typical UV absorption curves and the baseline drift (see Figure S1-A1,A2 in the Supplementary

Mar. Drugs 2014, 12
1823
Information). On the other hand, 10–14 were eluted as peaks with retention times of 63.90 min for 10,
9.50 min for 11, 63.00 min for 12, 52.12 min for 13, and 50.25 min for 14, respectively. They were all
5
detected in the mutant AD-2-1extract but not in the parent G59 extract. The data for the detection of 14
is shown as a typical example in Figure 2. The detection of 10–14 was achieved by both the retention
times and the UV absorption curves (see Figure 2 and see also Figure S1-B1,B2,C1,C2 in the
Supplementary Information).
In the HPLC-ESI-MS analysis, retention times of the ion peaks of 1–14 were slightly shortened
(
see Experimental Section) than those in HPLC-PDAD-UV analysis because of the shortened flow
length from the outlet of the HPLC column to the inlet of MS in the HPLC-ESI-MS. By selective ion
+
−
−
(
[M + Na] for 1/2 and 14; [M − H] for 5/6, 7, 8, and 10–13; [M + Cl] for 3/4 and 9) monitoring with
both extracted ion chromatograms and related MS spectra, 1–14 were all detected in the mutant AD-2-1
extract, but none of these metabolites were detected in the G59 extract (see Figure S2A–K in the
Supplementary Information).
Figure 2. HPLC-PDAD-UV analysis of AD-2-1 and G59 EtOAc extracts for detecting 14.
These analyses indicated that the production of 1–14 in mutant AD-2-1 was caused by the
activation of silent metabolic pathways in the original G59 strain by the DES mutagenesis. Although
the silent pathways remained undefined, the structural features of 1–9 and 10–14 indicated they may
originate from the nonribosomal peptide synthetase (NRPS) systems and the polyketide synthase
(PKS) systems, respectively [25,26]. Various kinds of lipopeptides are known occurring in Nature.
However, 1–7 are new lipopeptides and several analogues including 8 and 9 were all discovered from
fungal metabolites [15,16,23]. Also, a lot of anthraquinone/xanthone-derived polyketides have been

Mar. Drugs 2014, 12
1824
isolated from fungal metabolites [25,26]. However, none of them were obtained by mutagenic
activation of their production, and 1–9 and 10–14 provide the first examples of the related lipopeptides
and polyketides produced by mutagenic activation of the silent metabolic pathways in fungi. Thus,
additional investigation into the activated metabolic pathways and their regulatory mechanisms
are warranted.
2
.4. Inhibitory Effects of 1–14 on Several Human Cancer Cell lines
Antitumor activities of 1–14 were assayed by the MTT method using human cancer K562, HL-60,
HeLa, BGC-823, and MCF-7 cells. Seven new lipopeptides 1–7 and the known one 9 weakly inhibited
these cells to varying extents with inhibition rate (IR%) values shown in Table 3, and the five known
polyketides 10–14 showed also similarly weak inhibitory effects on the K562 cells with the IR%
values of 11.6% (10), 51.9% (11), 37.9% (12), 25.5% (13) at 100 µg/mL, and 37.5% (14) at 50 µg/mL.
In contrast, the known C-terminal aldehyde lipopeptide fellutamide B (8) showed stronger activities
than 1–7 and 9–14 in inhibiting the tested cell lines with half-inhibitory concentration (IC50) values of
2
9.1 µg/mL (52.5 µM) for K562, 59.9 µg/mL (107.9 µM) for HL-60, 59.5 µg/mL (107.2 µM) for HeLa,
7.9 µg/mL (140.4 µM) for BGC-823, and 43.3 µg/mL (68.4 µM) for MCF-7 cells. The positive
7
control 5-flurouracel (5-FU) inhibited these cell lines with the IR% values of 48.5% (K562), 38.2%
HL-60), 37.4% (HeLa), 47.8% (BGC-823), and 47.4% (MCF-7) at 100 µg/mL. The above data were
(
taken with four new compounds that we have just recently reported [12], using 5-FU as the positive
control at the same time and same conditions, so that the same data for 5-FU [12] were also given as
references for 1–14.
Table 3. IR% values of 1–7 and 9 on human cancer cell lines by MTT assay at 100 µg/mL.
Compound
K562
37.6%
34.4%
40.7%
40.0%
17.0%
10.1%
16.1%
48.3%
HL-60
14.1%
28.0%
19.8%
29.6%
42.6%
38.8%
12.0%
33.4%
HeLa
12.8%
27.6%
19.1%
29.0%
42.0%
37.9%
11.5%
33.0%
BGC-823
19.9%
28.1%
13.5%
27.9%
43.9%
49.0%
10.3%
33.3%
MCF-7
33.0%
34.5%
27.0%
38.0%
12.5%
21.8%
16.5%
35.5%
1
2
3
4
5
6
7
9
Fellutamide A and B (8) were originally identified for their cytotoxic properties on murine leukemia
P388 and L1210, and human epidermoid carcinoma KB cells with IC50 values 0.1–0.8 µg/mL [15].
Later they were found to stimulate nerve growth factor (NGF) synthesis and secretion [27,28].
The similar fellutamides carrying C-terminal aldehyde group, fellutamide D and ―fellutamide C‖ that
we suggest to be renamed as fellutamide E, were also cytotoxic against human prostate carcinoma
PC-3 cells (IC50: 160 and 440 nM, respectively) and further showed antifungal activities on several
Candida species with MIC values ranging from 2 to 32 µg/mL [23]. Now, the C-terminal aldehyde
fellutamides are known to be members of peptide aldehyde proteasome inhibitors [23,29,30], and their
inhibition of the human proteasome, probably by formation of hemiacetal covalent adduct at the

Mar. Drugs 2014, 12
1825
active site of the proteasome [29,30], induced the NGF synthesis that may result in the neurotrophic
effects [27–30]. Meanwhile, the inhibition of human proteasome by the C-terminal aldehyde
fellutamides is also likely to be the reason for their cytotoxicity [23]. Recently, fellutamide B (8) was
known also to potently inhibit the Mycobacterium tuberculosis proteasome with kinetic mechanisms
that differ from its inhibition of the human proteasome [31]. Fellutamide C (9), the only fellutamide
without C-terminal aldehyde group, was recorded to exhibit the cytotoxic activity on the human lung
cancer A549, human ovarian cancer SK-OV-3, human skin cancer SK-MEL-2, human CNS cancer
XF498, and human colon cancer HCT-15 cells with half-effective dosage (ED50) values ranging from
3
.1 to 33.1 µM [16]. Our bioassay results for 8 and 9 on the five human cancer cell lines in the present
study confirmed their cytotoxic properties previously recorded on the other cell lines [15,16], but their
inhibitory effects on the present cell lines are much lower than recorded. Furthermore, the seven new
and one known lipopeptides all without the C-terminal aldehyde group, penicimutalides A–G (1–7)
and fellutamide C (9), showed also much lower cytotoxicity than the C-terminal aldehyde lipopeptide,
fellutamide B (8), on the five tested cell lines in the present bioassays. These data have revealed that
the C-terminal aldehyde is an important functional group for their cytotoxic effects on these cell lines,
and suggested also that the inhibition of the human proteasome by the aldehyde group in C-terminal
fellutamides [23,29] might have contributed to the cytotoxicity of these fellutamides [15,23,29].
In summary, we have reported 14 metabolites 1–14 in the culture extract of an antitumor mutant
AD-2-1 that was obtained by the DES mutagenesis of originally inactive, parent G59 fungal strain. By
tracing newly produced metabolites in the mutant AD-2-1 extract under the guidance of MTT assay
and TLC analysis using the parent G59 extract as control, seven new lipopeptides 1–7 were isolated
together with two known lipopeptides 8 and 9 and the five known polyketides 10–14. Structures of the
new compounds including their absolute configurations were determined by spectroscopic and
chemical evidences, and named penicimutalides A–G (1–7). All 1–14 inhibited several human cancer
cell lines to varying extents. All the bioassays and HPLC-PDAD-UV and HPLC-ESI-MS analyses
demonstrated that the production of 1–14 in the mutant AD-2-1 was caused by the activation of the
silent metabolic pathways in the original fungal G59 strain by the DES mutagenesis. Present results
provided additional examples for the effectiveness of the chemical mutagenesis strategy using an
old chemical mutagen DES to discover new compounds by activating silent metabolites in fungal
isolates [12].
3
. Experimental Section
3
.1. General Experimental
Melting points were measured on a Beijing Tiandiyu X-4 exact micro melting point apparatus and
the temperatures were not corrected. Optical rotations were measured on an Optical Activity Limited
polAAr 3005 spectropolarimeter. ESIMS was recorded on an Applied Biosystems API 3000 LC-MS
spectrometer. HRESIMS was measured on an Agilent 6520 Q-TOF LC-MS spectrometer. IR spectra
were taken on a Bruker Hyperion ATR-objective spectrophotometer or Bruker Tensor-27 infrared
1
spectrophotometer. All 1D and 2D NMR spectra were obtained on a JEOL JNM-GX 400 (400 MHz H
1
3
1
13
and 100 MHz C NMR) or Bruker-600 (600 MHz H and 150 MHz C NMR) NMR spectrometer.

Mar. Drugs 2014, 12
1826
1
13
The chemical shifts of H and C NMR signals were recorded in δ values using solvent signals
CDCl : δ 7.26, δ 77.1; DMSO-d : δ 2.50, δ 39.5; and acetone-d : δ 2.05, δ 206.26) as
references, respectively.
(
3
H
C
6
H
C
6
H
C
Precoated silica gel GF254 plates (10 cm × 20 cm, 0.25 mm thickness for analytical TLC and
0 cm × 20 cm, 0.5 mm thickness for preparative TLC; Yantai Chemical Industrial Institute, Yantai,
2
China) and polyamide thin layers (10 cm × 20 cm, Taizhou Luqiao Sijia Biochemical plastic factory,
Taizhou, China) were used in TLC and spots were detected under sunlight and UV light (254 and
3
65 nm) or by using Vaughan’s reagent (24 g of ammonium molybdate tetrahydrate
NH Mo O) and 1 g of ceric sulfate Ce(SO dissolved in 500 mL of 10% H SO ), 5%
FeCl reagent (5 g of FeCl dissolved in 100 mL of 95% aqueous EtOH) or 10% sulfuric acid reagent.
(
4
)
6
7
O
24·4H
2
4
)
2
2
4
3
3
For lipopeptides, TLC spots or bands were also detected by spraying distilled water onto the plates. Silica
®
gel H (100–200 mesh, Yantai Chemical Industrial Institute, Yantai, China), YMC*GEL ODS-A-HG
(12 nm S-50 µm, YMC Co., Ltd., Kyoto, Japan), Sephadex™ LH-20 (GE Healthcare, Uppsala, Sweden),
and Polyamide (100–200 mesh, Taizhou Luqiao Sijia Biochemical plastic factory, Taizhou, China) were
used for column chromatography. Both analytical and semi-preparative HPLC were performed on Waters
HPLC systems equipped with Waters 600 controller, Waters 600 pump, Waters 2414 refractive index
detector, Waters 2996 (for analytical HPLC) or 2998 (for preparative HPLC) photodiode array detector,
and Waters Empower™ software. Venusil MP C18 (5 µm, 100 Å, 4.6 mm × 250 mm; Agela
Technologies, Tianjin, China) and reversed-phase chiral Click β-CD (4.6 mm × 150 mm; Dalian
Institute of Chemical Physics, Dalian, China) columns were used in analytical HPLC, and
reversed-phase chiral Click β-CD column (8 mm × 150 mm; Dalian Institute of Chemical Physics,
Dalian, China) was used in semi-preparative HPLC.
Marfey’s reagent, the 1-fluoro-2,4-dinitrophenyl-5-L-alanineamide (FDAA), was the Sigma product,
which was provided by Yongsheng Che (Beijing Institute of Pharmacology and Toxicology, Beijing,
China). The standard materials used in Marfey analysis were purchased from ShangHai HanHong
Chemical CO., Ltd. (Shanghai, China) Standard (abbr.), lot No: L-asparagine (L-Asn),
BH-00303-100101; D-asparagine (D-Asn), BH-00301-100801; L-glutamine (L-Gln), BH-00802-100101;
D-glutamine (D-Gln) BH-00801-100101; L-leucinol (L-Leo), BH-01338-100501; D-leucinol (D-Leo)
BH-01337-100501; L-leucine (L-Leu), BH-01302-110501; D-leucine (D-Leu), BH-01301-110101;
L-aspartic acid (L-Asp) BH-00403-110501; D-aspartic acid (D-Asp), BH-00401-110502; L-glutamic acid
(L-Glu), BH-00702-110501; D-glutamic acid (D-Glu), BH-00701-110501.
ZHWY-2102 rotary shakers (Shanghai ZhiCheng Analyzing Instrument Manufactory Co., Ltd.,
Shanghai, China) were used for the fermentation of fungal strains. The VERSAmax-BN03152 micro
plate reader (Molecular Device) was used in bioassays to read the optical density (OD) and the AE31
EF-INV inverted microscope (Motic China Group Co., Ltd., Xiamen, Fujian, China) was used for
morphological examination of tumor cells.
Human chronic myelogenous leukemia K562 cell line was provided by Prof. Dr. Lili Wang (Beijing
Institute of Pharmacology and Toxicology, Beijing, China). Human acute promyelocytic leukemia
HL-60, human cervical cancer HeLa, Human gastric adenocarcinoma BGC-823, and human breast
cancer MCF-7 cell lines were provided by Wenxia Zhou (Beijing Institute of Pharmacology and
Toxicology, Beijing, China). Fetal bovine serum was purchased from Tianjin Hao Yang Biological
manufacture Co., Ltd. (Tianjin, China). The RPMI-1640 medium was purchased from Gibco (lot

Mar. Drugs 2014, 12
1827
No. 1403238) and MTT from Amresco (lot No. 0793). Streptomycin (lot No. 071104) and penicillin (lot
No. X1103302) were both purchased from North China Pharmaceutical Group Corporation, China. The
5-fluorouracil (5-FU, lot No.5402) was purchased from Aladdin Chemistry Co., Ltd. (Shanghai, China).
3
.2. MTT Assay
EtOAc extracts and fractions were dissolved in MeOH at 10 mg/mL and the MeOH solutions were
used in MTT assays. Pure compounds and 5-FU were dissolved in MeOH and DMSO to prepare
0.0 mg/mL stock solutions, respectively, and serial dilutions were made for MTT assay. 5-FU was
1
used as positive control, and MeOH and DMSO were used as blank control, respectively.
Exponentially growing K562, HL-60, HeLa, BGC-823 and MCF-7 cells were suspended in fresh
RPMI-1640 medium containing 10% fetal bovine serum and 100 µg/mL penicillin and streptomycin at
4
the cell density of 5 × 10 cells/mL and seeded into 96-well plates each 200 µL/well. The suspension
cells, K562 and HL-60, were incubated at 37 °C for 2 h, whereas the adherent cells, HeLa, BGC-823
and MCF-7, were incubated at 37 °C for 12 h. Then, 2 µL of MeOH or DMSO for control and the
test sample solutions was added to each well, and the cells were cultured at 37 °C for 24 h. After
morphological examination of the cells under an inverted microscope, MTT (20 µL; 5 mg/mL in PBS)
was added into each well, incubated at 37 °C for 4 h, and centrifuged at 2000 rpm, 4 °C for
2
0 min. After removal of the supernatant by aspirating, 150 µL DMSO was added into each well, and
shaken for 5 min to dissolve formazan crystals. The OD value at 570 nm was read for each well using
the VERSAmax-BN03152 plate reader. Each three wells were set for control and test groups,
respectively, and the inhibition rate (IR%) was calculated using OD mean values according to the
formula, IR% = (ODcontrol − ODsample)/ODcontrol × 100%. The IC50 value for a sample was obtained from
the IR% values of the sample at different concentrations.
3
.3. Fermentation and EtOAc Extract Preparation
3
.3.1. Initial Fungal Strain and its Mutant the 1–14 Producing Strain
The initial fungal strain, Penicillium purpurogenum G59 that was used as control strain in the present
study, was isolated from a soil sample collected at the tideland of Bohai Bay around Lüjühe in Tanggu
district of Tianjin, China, in September 2004 [14]. P. purpurogenum G59 was identified by Professor
Dr. Liangdong Guo, Institute of Microbiology, Chinese Academy of Sciences, China. This strain has
been deposited at the China General Microbiological Culture Collection Center under the accession
number CGMCC No.3560. The strain P. purpurogenum G59 did not produce any secondary
metabolites with antitumor activities in repeated MTT assays using K562 cells at high concentrations
of 100 and 1000 µg/mL [10–14].
The producing strain used for 1–14 production in present study is a bioactive mutant AD-2-1 that
was obtained by DES mutagenesis of the strain G59 [12]. Fresh G59 spores in 50% (v/v) DMSO were
treated with 1% (v/v) DES at 4 °C for 1 day, and single colony isolation on the treated spores followed
by bioassays afforded the mutant. EtOAc extracts of the mutant cultures inhibited K562 cells (an IR%
of 49.8% at 100 µg/mL), whereas the EtOAc extract of strain G59 by the fermentation at the same time
and same conditions did not inhibit the K562 cells (an IR% of 5.6% at 100 µg/mL) [12]. The mutant

Mar. Drugs 2014, 12
1828
AD-2-1 has been deposited at the China General Microbiological Culture Collection Center under the
accession number CGMCC No.3561.
3
.3.2. Preparation of Spore Suspensions
The mutant AD-2-1 was inoculated onto potato dextrose agar (PDA) plates from a PDA slant stock
stored at 4 °C and incubated at 28 °C for 4 days. Fresh spores formed on the PDA plates were harvested
and suspended in 80 mL of sterilized, distilled water with several glass beads in a 100 mL cone-shaped
flask and scattered well by shaking enough to prepare a crude spore suspension. A 100 µL portion of this
crude spore suspension was added into a well of 96-well plates, diluted with water with its OD at 600 nm
measured using a VERSAmax-BN03152 plate reader, and the dilution ratio was recorded when the OD
value reached 0.35. Then, the remaining whole crude spore suspension was diluted with sterilized,
distilled water in the same proportion to obtain a mutant AD-2-1 spore suspension. This mutant spore
suspension was used for the producing fermentation in the following experiments.
Similarly, the G59 spore suspension was also prepared in the same manner as mentioned above
using fresh spores formed by cultivation of the G59 strain at 28 °C for 3 days on PDA plates. This G59
spore suspension was used as control strain in the following experiments.
3
.3.3. Fermentation and Extraction
Aliquot (200 µL) of the mutant AD-2-1 spore suspension was inoculated into 100 cone-shaped
00 mL flasks containing 200 mL of the liquid medium (glucose 2%, maltose 1%, mannitol 2%,
5
glutamic acid 1%, peptone 0.5% and yeast extract 0.3% in distilled water, adjusted to pH 6.0 prior to
sterilization) and fermented at 28 °C for 12 days on rotary shakers at 200 rpm to obtain approximate
2
0 L of fermentation broth. The whole broth (20 L) was filtered to separate into a filtrate (17 L) and a
mycelial cake. The filtrate was extracted three times with same volumes of EtOAc (3 × 17 L) to give
an EtOAc extract. The mycelial cake was extracted three times with 3 L acetone-water (2:1) by
ultrasonication for 2 h. The aqueous acetone solution obtained by filtration was evaporated under
reduced pressure to remove acetone. Then, the remaining water layer (3 L) was extracted three times
with same volumes of EtOAc (3 × 3 L) to give another EtOAc extract. The EtOAc extracts both from
the filtrate and mycelia gave same TLC spot patterns and thus were combined to afford total 20 g of the
EtOAc extract. This EtOAc extract showed an inhibitory effect on K562 cells with the IR% value of
6
4.6% at the 100 µg/mL, which was used for the isolation of 1–14 in the following experiments.
On the other hand, 200 µL of the G59 spore suspension was inoculated into one cone-shaped 500 mL
flask with 200 mL of the same liquid medium and fermented at the same time and same conditions to
obtain 200 mL of fermentation broth. The whole broth (200 mL) was extracted as described above for
mutant AD-2-1 to afford an EtOAc extract (98 mg), which did not show inhibitory effect on K562 cells
(
an IR% value of 6.8% at 100 µg/mL). This G59 extract was used in the MTT assays and TLC analyses
as negative control and also in the HPLC-PDAD-UV and HPLC-ESI-MS analyses for detecting 1–14
in the following experiments.

Mar. Drugs 2014, 12
.4. Isolation of Compounds 1–14
1829
3
In preliminary TLC analyses of the EtOAc extracts of the control G59 strain and mutant AD-2-1,
the lipopeptide components containing 1–9 were detected in the mutant extract as white spots on
the silica gel TLC plates that were developed using CH Cl (D)MeOH (M)ammonia (A)
90:20:2→90:30:2) as developing solvents, but not detected in the G59 extract. Spots of the
2
2
(
lipopeptide components were visualized by spraying distilled water onto the plates. The components of
polyketides including 10–14 were also detected only in the mutant extract as several spots on the silica
gel TLC plates developed by dichloromethane (D)acetone (100:1→1:1) or on the polyamide film
developed by MeOHWater (60:40→90:10). Spots of the polyketide components were examined
3
under sunlight and UV lights (254 and 360 nm) or by using the 5% FeCl reagent, Vaughan’s reagent
or 10% sulfuric acid reagent. Thus, the separation of the mutant extract was performed tracing these
newly produced metabolites under the guidance of MTT assay and TLC analysis by direct comparison
with the control G59 extract.
The EtOAc extract (20 g) of the mutant AD-2-1 was separated into MeOH soluble (15 g) and
insoluble (5 g) parts by dissolving in approximate 200 mL of MeOH. The MeOH insoluble part (5 g)
did not show inhibitory effect on K562 cells. The MeOH soluble part (15 g) was dissolved in 100 mL
EtOH and subjected to a Sephadex LH-20 column (bed 5 cm × 40 cm in EtOH). Elution of the column
using EtOH as eluent afforded five fractions in the order of elution: Fr-1 (3.6 g), Fr-2 (3.1 g), Fr-3
(2.7 g), Fr-4 (3.4 g) and Fr-5 (2.2 g). These fractions inhibited the K562 cells with the IR% values of
5
0.4% (Fr-1), 25.5% (Fr-2), 18.9% (Fr-3), 50.1% (Fr-4), and 30.4% (Fr-5) at the 100 µg/mL. TLC
analyses of the fractions indicated that the aimed metabolites newly produced by the mutant, compared
to the control G59 strain, existed in two fractions: The lipopeptide components including 1–9 in the
fraction Fr-1 and the polyketide components including 10–14 in the fraction Fr-4.
Fr-1 (3.6 g) was further subjected to vacuum liquid chromatography (VLC) on an ODS column
(bed 4.8 cm × 10 cm) dry-packed with 25 g of ODS. A stepwise elution with Water (W)–EtOH (E)
(100:0→5:95) gave five fractions in the order of elution: Fr-1-1 (0.32 g, elution of water), Fr-1-2
(0.15 g, eluted by WE 4:1→7:3), Fr-1-3 (0.2 g, eluted by WE 7:3), Fr-1-4 (2.3 g, eluted by WE
3
:2→1:1), and Fr-1-5 (0.6 g, eluted by WE 2:3→3:7). These fractions inhibited the K562 cells with
the IR% values of 24.9% (Fr-1-1), 11.5% (Fr-1-2), 40.3% (Fr-1-3), 70.3% (Fr-1-4), and 28.9%
Fr-1-5) at the 100 µg/mL. TLC analysis indicated that Fr-1-3 contained 7 and Fr-1-4 contained 1–6,
, and 9. Thus, Fr-1-3 (0.2 g) was subjected to preparative TLC (PTLC) on silica gel GF254 plates
20 cm × 20 cm, 0.5 mm thickness) using CH Cl (D)MeOH (M)ammonia (A) (90:30:2) as
developing solvent. Silica gels on a band with R value of 0.6, which was visualized by spraying
(
8
(
2
2
f
distilled water, were harvested and adsorbed substances were extracted by eluting with acetone.
Further purification of the substances by passing through a small Sephadex LH-20 column in MeOH
afforded compound 7 (30 mg) as a white amorphous powder from MeOH solution. Fr-1-4 (0.5 g) was
dissolved in 5 mL MeOH and subjected to PTLC using DMA (90:20:2) as developing solvent. Silica
gels on four bands (R
visualized by spraying distilled water. Then, substances adsorbed in the silica gels were extracted by
eluting with acetone to obtain four fractions: Fr-1-4-1 (60 mg, R ≈ 0.7), Fr-1-4-2 (210 mg, R ≈ 0.6),
Fr-1-4-3 (40 mg, R ≈ 0.5), and Fr-1-4-4 (30 mg, R ≈ 0.45). These fractions inhibited the K562 cells
f
values: 0.45, 0.5, 0.6, and 0.7) were harvested, respectively, which were
f
f
f
f

Mar. Drugs 2014, 12
1830
with the IR% values of 40.5% (Fr-1-4-1), 43.0% (Fr-1-4-2), 70.8% (Fr-1-4-3), and 37.3% (Fr-1-4-4)
at the 100 µg/mL. Fr-1-4-1 (60 mg) was further separated by repeated semi-preparative HPLC
(
column: a reversed-phase Click β-CD column, 8 mm × 150 mm, column temperature: 40 °C; mobile
phase: MeOH–H O 42:58, flow rate: 2 mL/min; detecting wave length: 210 nm) to give compounds 1
3 mg, t = 48 min), 3 (5 mg, t = 54 min), 2 (2.5 mg, t = 67 min), and 4 (7 mg, t = 76 min).
Fr-1-4-2 (60 mg) and Fr-1-4-3 (40 mg) were subjected also to repeated semi-preparative HPLC
column: a reversed-phase Click β-CD column, 8 mm × 150 mm, column temperature: 26 °C; mobile
phase: MeOH–H O 48:52, flow rate: 2 mL/min; detecting wave length: 210 nm) to obtain compound 8
13 mg, t = 54 min) from Fr-1-4-2 and compound 9 (10 mg, t = 42 min) from Fr-1-4-3, respectively.
On the other hand, repeated semi-preparative HPLC separation (column: a reversed-phase Click β-CD
column, 8 mm × 150 mm, column temperature: 26 °C; mobile phase: MeOH–H O 45:55, flow rate:
mL/min; detecting wave length: 210 nm) of Fr-1-4-4 (30 mg) afforded compound 5 (1 mg, t = 42 min)
and 6 (2 mg, t = 52 min).
2
(
R
R
R
R
(
2
(
R
R
2
2
R
R
Fr-4 (3.4 g) was subjected to VLC on a silica gel column (dry-packed with 25 g silica gel, bed
cm × 10 cm). Stepwise elution of the column with b.p. 60–90 °C Petroleum ether
2
(
P)–Dichloromethane (D) (100:0→0:100) and then DM (90:10→0:100) as eluting solvents gave five
fractions: Fr-4-1 (10 mg, eluted by PD 1:1→9:1), Fr-4-2 (0.8 g, eluted by PD 9:1→1:9), Fr-4-3
1.3 g, eluted by D), Fr-4-4 (1.2 g, eluted by DM 98:2→92:8), and Fr-4-5 (0.12 g, eluted by DM
2:8→4:1). These fractions inhibited the K562 cells with IR% values of 16.9% (Fr-4-1), 49.0%
Fr-4-2), 35.9% (Fr-4-3), 53.9% (Fr-4-4), and 13.2% (Fr-4-5) at the 100 µg/mL. Among these, three
(
9
(
fractions, Fr-4-2, Fr-4-3 and Fr-4-4, were known to contain targeted polyketide components by TLC
examination. Fr-4-2 (0.8 g) was subjected to Polyamide column (bed 2 cm × 30 cm) chromatography
and stepwise elution with WM (100:0→0:100) afforded compounds 10 (24 mg) from a fraction eluted
by WM (3:7) and 12 (18 mg) from a fraction eluted by WM (1:4). Fr-4-3 (1.3 g) was separated by
Sephadex LH-20 column (bed 2.5 cm × 60 cm in DM 1:1) chromatography (eluting solvent, DM 1:1)
to obtain three fractions: Fr-4-3-1 (0.3 g), Fr-4-3-2 (0.61 g), and Fr-4-3-3 (0.24 g). These fractions
inhibited the K562 cells with the IR% values of 19.0% (Fr-4-3-1), 41.6% (Fr-4-3-2), and 10.5%
(
Fr-4-3-3) at the 100 µg/mL. Recrystallization of Fr-4-3-2 (0.61 g) in DM (1:1) solution gave
compound 14 (400 mg) as yellowish needles. Fr-4-4 (1.2 g) was separated by Polyamide column (bed
cm × 30 cm) chromatography using WM (100:0→0:100) as eluting solvent to give a fraction (0.6 g)
2
eluted by WM (3:7→1:9), which contained targeted polyketides. This fraction (0.6 g) was further
separated by VLC on a silica gel column (bed 2.5 cm × 8 cm) using PD (1:3→1:5) as eluting solvent
to obtain compounds 11 (13 mg) from PD 1:3 eluent and 13 (20 mg) from PD 1:5 eluent.
3
.5. Physicochemical and Spectral Data for Compounds 1–14
Penicimutalide A (1): White amorphous powder (MeOH), [α]
2
D
5
−18.0 (c 0.1, MeOH). Positive
+
−
−
−
ESI-MS m/z: 594 [M + Na] ; negative ESI-MS m/z: 570 [M − H] , 606 [M + Cl] , 616 [M + HCOO] , 660
−
+
[M + HC
2
+
O
4
] . Positive HR-ESI-MS m/z: measured 594.3834 [M + Na] , calcd for C28
H
53 5 7
N O Na
+
+
[M + Na] 594.3843, measured 610.3626 [M + K] , calcd for C28H N O Na [M + K] 610.3582.
53 5 7
−
1
IR νmax cm (KBr): 3422, 3283, 2958, 2922, 2852, 1663, 1626, 1552, 1536, 1516, 1443, 1384, 1311,

Mar. Drugs 2014, 12
1831
1
13
1
261, 1095, 1018, 928, 879. H and C NMR data: see Tables 1 and 2 and see also Table S1 in the
Supplementary Information.
2
D
5
Penicimutalide B (2): White amorphous powder (MeOH), [α]
−17.0 (c 0.1, MeOH). Positive ESI-MS
+
−
−
−
m/z: 594 [M + Na] ; negative ESI-MS m/z: 570 [M − H] , 606 [M + Cl] , 616 [M + HCOO] , 660
−
+
[
M + HC
2
+
O
4
] . Positive HR-ESI-MS m/z: measured 594.3847 [M + Na] , calcd for C28
H
53 5 7
N O Na
+
+
[
M + Na] 594.3843, measured 610.3612 [M + K] , calcd for C28H N O K [M + K] 610.3582.
53 5 7
−
1
IR νmax cm (Diamond ATR crystal): 3283, 3215, 2934, 2862, 1654, 1540, 1419, 1320, 1290, 1255,
1
13
1
147, 1122, 1063, 883, 865. H and C NMR data: see Tables 1 and 2 and see also Table S2 in the
Supplementary Information.
2
D
5
Penicimutalide C (3): White amorphous powder (MeOH), [α]
−23.7 (c 0.3, MeOH). Positive
+
+
−
ESI-MS m/z: 558 [M + H] , 580 [M + Na] ; negative ESI-MS m/z: 592 [M + Cl] . Positive HR-ESI-MS
+
+
−1
m/z: measured 580.3695 [M + Na] , calcd for C27
H
51
N
5
O
7
Na [M + Na] 580.3686. IR νmax cm
(
Diamond ATR crystal): 3268, 2934, 2862, 1650, 1536, 1418, 1325, 1282, 1256, 1196, 1145, 1114,
1
13
1
062, 979, 949. H and C NMR data: see Tables 1 and 2 and see also Table S3 in the
Supplementary Information.
2
D
5
Penicimutalide D (4): White amorphous powder (MeOH), [α]
−24.8 (c 0.3, MeOH). Positive
+
+
+
−
ESI-MS m/z: 558 [M + H] , 580 [M + Na] , 596 [M + K] ; negative ESI-MS m/z: 556 [M − H] ,592
−
+
[
M + Cl] . Positive HR-ESI-MS m/z: measured 580.3684 [M + Na] , calcd for C27
H
51
N
5
O
7
Na
+
+
+
[
M + Na] 580.3686, measured 596.3426 [M + K] , calcd for C28H N O K [M + K] 596.3426.
53 5 7
−
1
IR νmax cm (Diamond ATR crystal): 3274, 2930, 2859, 1650, 1632, 1537, 1417, 1325, 1281, 1255,
1
13
1
197, 1139, 1112, 1056, 946, 865. H and C NMR data: see Tables 1 and 2 and see also Table S4 in
the Supplementary Information.
2
5
Penicimutalide E (5): White amorphous powder (MeOH), [α] −15.0 (c 0.04, MeOH). Positive
D
+
−
ESI-MS m/z: 571 [M + H] ; negative ESI-MS m/z: 569 [M − H] . Positive HR-ESI-MS m/z: measured
+
+
+
5
71.3819 [M + H] , calcd for C27
H
51
N
6
O
7
[M + H] 571.3819; measured 593.3634 [M + Na] , calcd for
+
−1
C
27
H
50
N
6
O
7
Na [M + Na] 593.3639. IR νmax cm (Diamond ATR crystal): 3273, 2931, 2859, 1642,
1
13
1
546, 1537, 1418, 1324, 1283, 1156, 1134, 1074, 864, 783. H and C NMR data: see Tables 1 and 2
and see also Table S5 in the Supplementary Information.
2
5
Penicimutalide F (6): White amorphous powder (MeOH), [α] −18.8 (c 0.04, MeOH). Positive
D
+
−
ESI-MS m/z: 571 [M + H] ; negative ESI-MS m/z: 569 [M − H] . Positive HR-ESI-MS m/z: measured
+
+
−1
5
71.3805 [M + H] , calcd for C27
51 6 7
H N O [M + H] 571.3819. IR νmax cm (Diamond ATR crystal):
1
13
3
273, 2933, 2860, 1653, 1628, 1540, 1418, 1177, 1132, 1024. H and C NMR data: see Tables 1 and 2
and see also Table S6 in the Supplementary Information.
2
5
Penicimutalide G (7): White amorphous powder (MeOH), [α] −7.5 (c 0.1, MeOH). Positive ESI-MS
D
+
+
−
−
m/z: 458 [M + H] , 480 [M + Na] ; negative ESI-MS m/z: 456 [M − H] , 492 [M + Cl] . Positive
+
+
HR-ESI-MS m/z: measured 458.2970 [M + H] , calcd for C21
H
40
N
5
O
6
[M + H] 458.2979; measured
+
+
+
4
80.2798 [M + Na] , calcd for C21
H
39
N
5
O
6
Na [M + Na] 480.2798; measured 496.2526 [M + K] , calcd
+
−1
for C27
H
5
N
5
O
7
K [M + K] 496.2537. IR νmax cm (Diamond ATR crystal): 3274, 3205, 2929, 2857,

Mar. Drugs 2014, 12
1832
1
13
1
651, 1632, 1540, 1414, 1320, 1281, 1254, 1200, 1133, 1095, 1070, 1042, 958, 866. H and C NMR
data: see Tables 1 and 2 and see also Table S7 in the Supplementary Information.
2
D
5
Fellutamide B (8): White amorphous powder (MeOH), [α]
−28.0 (c 0.5, MeOH). Positive ESI-MS
+
−
1
13
m/z: 556 [M + H] ; negative ESI-MS m/z: 554 [M − H] . H and C NMR data: see Tables 1 and 2.
2
D
5
Fellutamide C (9): White amorphous powder (MeOH), [α]
−33.0 (c 0.2, MeOH). Positive ESI-MS
+
+
−
m/z: 558 [M + H] , 580 [M + Na] ; negative ESI-MS m/z: 592 [M + Cl] . Positive HR-ESI-MS m/z:
+
+
measured 558.3862 [M + H] , calcd for C27
H
52
N
5
O
7
[M + H] 558.3867; measured 580.3692
+
+
+
[
M + Na] , calcd for C27
H
51
N
5
O
7
Na [M + Na] 580.3686; measured 596.3413 [M + K] , calcd for
+
1
13
C
27
H
51
N
5
O
7
K [M + K] 596.3426. H and C NMR data: see Tables 1 and 2.
2
5
1
′-O-Methylaverantin (10): Crystalline orange-red powder (CH
2
Cl
, 400MHz) δ: 12.78 (1H, s, 1-OH),
2.18 (1H, s, 8-OH), 10.10 (1H, br s, 6-OH), 9.69 (1H, br s, 3-OH) 7.23 (1H, d, J = 2.4 Hz, H-5), 7.13
1H, s, H-4), 6.64 (1H, d, J = 2.4 Hz, H-7), 4.97 (1H, dd, J = 7.9, 5.1 Hz, H-1′), 3.49 (3H, s, 1′-OCH ),
.95–1.75 (2H, m, H -3′), 1.58–1.38 (2H, m, H -2′), 1.38–1.24 (4H, m, H -4′, H -5′), 0.89 (3H, t,
J = 6.8 Hz, H -6′).
Averantin (11): Crystalline orange-red powder (CH
2
), mp 214216 °C, [α]
D
+1.8
+
1
(
c 0.4, MeOH), ESI-MS m/z: 387 [M + H] . H NMR (acetone-d
6
1
(
3
1
2
2
2
2
3
2
5
2
Cl
MeOH). ESI-MS m/z: 373 [M + H] . H NMR (acetone-d
1H, s, 8-OH), 9.00 (2H, br s, 1-OH, 3-OH), 7.09 (1H, d, J = 2.4 Hz, H-5), 7.00 (1H, s, H-4), 6.50 (1H,
d, J = 2.4 Hz, H-7), 5.29 (1H, dd, J = 7.8, 4.5 Hz, H-1′), 1.80–1.63 (2H, m, H -3′), 1.50–1.30 (2H, m,
-6′). C NMR (acetone-d , 100 MHz)
δ: 190.7 (C-9), 181.8 (C-10), 166.0 (C-8), 165.9 (C-1), 165.0 (C-6),161.7 (C-3), 136.4 (C-4a), 134.5
C-10a), 122.0 (C-2), 110.3 (C-5), 110.2 (C-9a), 109.4 (C-8a), 108.9 (C-7), 108.8 (C-4), 69.9 (C-1′),
2
), mp 279280 °C, [α]
D
−130.0 (c 0.52,
+
1
6
, 400MHz) δ: 12.63 (1H, s, 1-OH), 12.08
(
2
1
3
H
2
-2′), 1.30–1.10 (4H, m, H
2
-4′, H
2
-5′), 0.76 (3H, t, J = 6.8 Hz, H
3
6
(
3
6.9 (C-2′), 32.3 (C-3′), 25.6 (C-4′), 23.2 (C-5′), 14.3 (C-6′).
Averufin (12): Crystalline orange-red powder (CH Cl ), mp 280282 °C , [α]
H NMR (acetone-d , 400MHz) δ: 12.51 (1H, s, 1-OH), 12.19 (1H, s, 8-OH), 10.09 (1H, s, 6-OH), 7.22
1H, d, J = 2.5 Hz, H-5), 7.09 (1H, s, H-4), 6.63 (1H, d, J = 2.5 Hz, H-7), 5.29 (1H, d, J = 2.8 Hz, H-1′),
2
D
5
2
2
3
−1.7 (c 0.3, CHCl ).
1
6
(
1
3
2
1
2 3 6
.15–1.25 (6H, m, H -2′,3′,4′), 1.55 (3H, s, H -6′). C NMR (acetone-d , 100MHz) δ: 190.7 (C-9),
81.6 (C-10), 166.0 (C-8), 165.8 (C-1), 161.5 (C-6), 159.8 (C-3), 136.3 (C-4a), 134.4 (C-10a), 117.1
(
(
C-2), 110.2 (C-5), 109.5 (C-9a), 109.4 (C-8a), 108.9 (C-7), 108.4 (C-4), 102.2 (C-5′), 67.4 (C-1′), 36.3
C-2′), 28.0 (C-4′), 27.9 (C-6′), 16.5 (C-3′).
2
5
Nidurufin (13): Crystalline orange-red powder (CH
2
Cl
, 400MHz) δ: 12.58 (1H, s, 1-OH), 12.22 (1H, s,
-OH), 10.17 (1H, s, 6-OH), 7.26 (1H, d, J = 2.5 Hz, H-5), 7.14 (1H, s, H-4), 6.67 (1H, d, J = 2.5 Hz,
H-7), 5.17 (1H, br s, H-1′), 3.95 (1H, br s, H-2′), 2.30–1.30 (4H, m, H -3′, H -4′), 1.58 (3H, s, H -6′).
Sterigmatocystin (14): Pale yellow needles (CH Cl ), mp 246 °C (dec.), [α] −384 (c 0.5, CHCl
ESI-MS m/z: 325 [M + H] , 347 [M + Na] . H NMR (400 MHz, CDCl ) δ: 13.23(1H, s, 3-OH), 7.47
1H, dd, J = 8.4, 8.0 Hz, H-5), 6.80 (1H, d, J = 7.0 Hz, H-14), 6.79 (1H, dd, J = 8.0, 1.0 Hz, H-6), 6.72
1H, dd, J = 8.4, 1.0 Hz, H-4), 6.50 (1H, t, J = 2.5 Hz, H-17), 6.40 (1H, s, H-11), 5.43 (1H, t, J = 2.5 Hz,
2
), mp 188190 °C, [α]
D
3
+0.3 (c 0.2, CHCl ).
+
1
ESI-MS m/z: 385 [M + H] . H NMR (acetone-d
6
8
2
2
3
2
5
2
2
D
3
),
+
+ 1
3
(
(
1
3
3 3
H-16), 4.76 (1H, dt, J = 7.0, 2.5 Hz, H-15), 3.98 (3H, s, H -18). C NMR (100 MHz, CDCl ) δ: 181.2

Mar. Drugs 2014, 12
1833
(
C-1), 164.4 (C-10), 163.1 (C-12), 162.1 (C-3), 154.8 (C-7), 153.8 (C-8), 145.2 (C-17), 135.6 (C-5),
1
13.2 (C-14), 111.1 (C-4), 108.7 (C-2), 106.4 (C-6), 105.8 (C-9), 105.7 (C-13), 102.4 (C-16), 90.4
(
C-11), 56.7 (C-18), 47.9 (C-15).
3
.6. Marfey Analysis for 1–9
3
.6.1. Hydrolysis and Derivatization with FDAA
To nine glass tubes that one terminal has been sealed was added each 50 µL of 1–9 solutions in
MeOH at 1 mg/mL, respectively. After blown inside the tubes with nitrogen gas to dryness, 100 µL of
aqueous 6 N HCl solution was added into each tube. After the open terminal of the tubes was sealed by
blast burner, the tubes were kept at 110 °C for 24 h to hydrolyze 1–9. Then, one sealed terminal of the
tubes was cut out, reaction solutions were blown with nitrogen gas to dryness, and the tubes were
maintained in vacuo overnight to clean up remained HCl. At the same time, each 500 µg of standards
in 500 µL of aqueous 6 N HCl solution, L-Asn, D-Asn, L-Gln, D-Gln, L-Leu, D-Leu, L-Leu amide and
D-Leu amide, was also hydrolyzed in the same manner and same conditions. At this condition, the
L-Leu amide and D-Leu amide were hydrolyzed to L-Leu and D-Leu, respectively. Both hydrolyzates
of the standards and 1–9 were dissolved in each 10 μL of distilled water, mixed with 10 μL of 10 mM
L-FDAA in acetone and 10 μL of 1 N NaHCO
.5 h. The reaction mixtures were neutralized with 5 μL of 2 N HCl, respectively. Then, the reaction
mixtures were filtered, and the filtrates were subjected to HPLC analysis.
3
aqueous solution, respectively, and reacted at 45 °C for
1
3
.6.2. Oxidization of Leucinal in 8 to Leucine, then Hydrolysis and Derivatization with FDAA
Because we had the L-/D-leucine standards in hand but lacked the L-/D-leucinal standards, in order
to determine the absolute configuration of leucinal in 8, the leucianl in 8 was oxidized into leucine at
first. The 100 µg of 8 was suspended in 100 µL distilled water, then 2 µg KMnO was added, and
4
reacted by stirring at room temperature for 30 min to oxidize the leucianl in 8 into the leucine. The
reaction mixture was extracted with n-BuOH (250 μL × 2) to obtain an oxidized product of 8 from the
n-BuOH solution. Then, the total of this oxidized product was hydrolyzed using 100 μL of aqueous
6
N HCl at 110 °C for 24 h, derivatized as mentioned above with FDAA at the same time and same
conditions, and then subjected to the HPLC analysis, in the same manner as mentioned.
3
.6.3. HPLC Analysis of FDAA Derivatives of 1–9 to Determine Absolute Configurations
HPLC analysis of the FDAA derivatives was performed on a Venusil MP C18 column (5 µm, 100 Å,
4
2
.6 mm × 250 mm) at room temperature, using mixed A (H O containing 1% HCOOH) and B
(
acetonitrile) solutions in a linear gradient (15% B at initial time 0 min→50% B at 60 min→100% B at
0 min; flow rate, 1 mL/min) as mobile phase. The acquired photodiode array (PDA) data were
7
processed by Empower PDA software and targeted derivatives were detected at 340 nm.
In the HPLC conditions, the FDAA derivatives of D- and L-standards were separated well as given
their retention times (t
R
) in Table 4. The FDAA derivatives of 1–9 were analyzed by injection of the
derivatives alone and co-injection with those of related L- and D-standards, respectively.

Mar. Drugs 2014, 12
Table 4. Retention times (t
1834
R
) for the FDAA derivatives of L- and D-standards.
FDAA derivative
L-Aspartic acid
L-Glutamic acid
L-leucinol
t
R
(min)
28.60
30.85
51.14
53.11
FDAA derivative
D-Aspartic acid
D-Glutamic acid
D-leucinol
R
t (min)
30.55
33.38
59.53
59.85
L-leucine
D-leucine
3
.7. Hydrolysis of 1–9 for Determination of the 23R Absolute Configuration in 1–9
Each 0.3 mg of 1–9 was mixed, and the mixture was suspended in 1 mL of aqueous 6 N HCl in a
sealed tube and hydrolyzed at 110 °C for 1 h. The solution was then extracted three times with 1 mL of
CHCl
3
to obtain a CHCl
3
extract. This extract was purified by silica gel PTLC (0.5 mm thickness,
2
0 mm × 20 mm; developing solvent: DM 95:5) to obtain 3R-hydroxydodecanoic acid (0.8 mg,
2
5
+
R
f
= 0.5): [α]
D
−16 (c 0.05, CHCl
3
). Positive ESI-MS m/z: 239 [M + Na] ; negative ESI-MS m/z: 215
) δ: 4.02 (1H, m), 2.57 (2H, dd, J = 16.6, 3.0 Hz), 2.47 (1H, dd,
−
1
[M − H] . H NMR (400 MHz, CDCl
3
1
3
J = 16.6, 9 Hz), 1.40–1.58 (2H, m), 1.20–1.36 (14H, m), 0.87 (3H, t, J = 6.6 Hz). C NMR (CDCl
3
)
2
5
1
13
δ: 176.8, 68.0, 40.8, 36.5, 31.9, 29.7, 29.5, 29.4, 29.3, 25.4, 22.7, 14.1. The [α]
D
and H and C NMR
data are identical with those reported for 3R-hydroxydodecanoic acid in the literature [24].
3
.8. HPLC-PDAD-UV Analysis for Detecting 1–14 in the G59 and AD-2-1 Extracts
EtOAc extracts of the control G59 strain and the mutant AD-2-1 were dissolved in MeOH to
prepare sample solutions at 10 mg/mL for HPLC analysis. Crude samples of the compounds 1–14 in
MeOH at 10 mg/mL were used as reference standards in the HPLC-PDAD-UV analysis. The MeOH
solutions of 1–6 at 10 mg/mL were mixed between 1/2, 3/4, and 5/6 in a ratio of 1:1, respectively, and
the mixed sample solutions were used, because the two compounds in these pairs were known to
be eluted as a peak with the same retention time in the given HPLC condition by pilot tests of
HPLC analyses.
The HPLC-PDAD-UV analysis was carried out on a Venusil MP C18 column (5 µm, 100 Å,
4
.6 mm × 250 mm; Agela Technologies) using the Waters HPLC equipment mentioned above. The
sample and standard solutions were filtered using 0.22 µm pore membrane filters, respectively, and
each 5 µL of the solutions was injected into the column. Then, elution was performed using
2
MeOH–H O in linear gradient (20% MeOH at initial time 0 min→100% MeOH at 60 min→100%
MeOH at 90 min; flow rate, 1 mL/min). The acquired photodiode array (PDA) data were processed
using the Empower PDA software to obtain targeted HPLC-PDAD-UV data.
In the HPLC-PDAD-UV analysis, examination of 1–14 was performed on the basis of both HPLC
retention times and UV absorption curves.
Retention times of 1–9: 56.12 min (1/2), 54.57 min (3/4), 50.62 min (5/6), 46.52 min (7), 52.77 min
(
8), and 52.57 min (9). Because 1–9 showed only end UV absorptions, they were hardly identified by
the HPLC-PDAD-UV analysis both in the mutant AD-2-1 and the control G59 extracts due to the lack
of typical UV absorptions and the baseline drift (Figure S1-A1 and A2 in the Supplementary Information).

Mar. Drugs 2014, 12
1835
Retention times of 10–14: 63.90 min (10), 59.50 min (11), 63.00 min (12), 52.12 min (13), and
0.25 min (14). All of 10–14 were detected in the mutant AD-2-1 extract but not in the parent G59
5
extract (see Figure 2 and see also Figure S1-B1,B2,C1,C2 in the Supplementary Information).
3
.9. HPLC-ESI-MS Analysis for Detecting 1–14 in the G59 and AD-2-1 Extracts
The MeOH solutions of crude 1–14 and the EtOAc extracts of G59 strain and mutant AD-2-1,
which were used in the HPLC-PDAD-UV analysis, were used also in HPLC-ESI-MS analysis for
detecting 1–14 from the EtOAc extracts.
The HPLC-ESI-MS analysis was performed on an LC-MS equipment equipped with Agilent 1100
HPLC system, AB Sciex API 3000 LC-MS/MS system and AB Sciex Analyst 1.4 software. HPLC was
carried out on the Venusil MP C18 column (5 µm, 100 Å, 4.6 mm × 250 mm; Agela Technologies) at
the conditions the same as mentioned for the HPLC-PDAD-UV analysis. The mass detector was set to
scan a range from m/z 150–1500 both in the positive and negative modes. The acquired data were
processed by Analyst 1.4 software to obtain targeted HPLC-ESI-MS data.
In the HPLC-ESI-MS analysis, both positive and negative ion peaks of 1–14 appeared with shortened
retention times than those in their HPLC-PDAD-UV analysis because of the shortened flow length from
the outlet of the HPLC column to the inlet of MS. Retention times of 1–14: 52.80 min (1/2), 51.25 min
(
3/4), 47.48 min (5/6), 43.40 min (7), 49.59 min (8), and 49.28 min (9), 62.38 min (10), 58.87 min (11),
1.83 min (12), 52.05 min (13), and 47.74 min (14). Detection of 1–14 in the control G59 and the mutant
6
+
−
AD-2-1 extracts was achieved by selective ion ([M + Na] for 1/2 and 14; [M − H] for 5/6, 7, 8, and
−
1
0–13; and [M + Cl] for 3/4 and 9) monitoring with both extracted ion chromatograms and related MS
spectra (see Figure S2A–K in the Supplementary Information).
4
. Conclusions
AD-2-1 is an antitumor fungal mutant obtained by the DES mutagenesis of originally inactive,
parent Penicillium purpurogenum G59 strain. Fourteen metabolites in the mutant AD-2-1 extract,
including seven new (1–7) and two known lipopeptides (8 and 9) as well as five known polyketides
1
0–14, were isolated by tracing newly produced metabolites in the mutant AD-2-1 extract under the
guidance of MTT assay and TLC analysis using the parent G59 extract as control. Structures of the
seven new compounds including their absolute configurations were determined by spectroscopic data
and chemical evidences, and named penicimutalides A–G (1–7), respectively. All of 1–14 inhibited
several human cancer cell lines to varying extents. Both present bioassays and HPLC-PDAD-UV and
HPLC-ESI-MS analyses demonstrated that the production of 1–14 in the mutant AD-2-1 was caused
by the activated production of silent metabolites in original fungal G59 strain by the DES mutagenesis.
Acknowledgments
This work was financially supported by the grants from the NSFC (30973631, 81172976),
NHTRDP (2013AA092901, 2007AA09Z411), NSTMP (2012ZX09301-003, 2009ZX09103-019,
2
009ZX09301-002), CAS (KSCX2-EW-G-6), COMRA (DYXM-115-02-2-09) and AMMS (2008),
China. Penicillium purpurogenum G59 was identified by Liangdong Guo, Institute of Microbiology,

Mar. Drugs 2014, 12
1836
Chinese Academy of Sciences, China. Dedicated to Professor Xin-Sheng Yao on the occasion of his
0th birthday.
8
Author Contributions
Chang-Jing Wu achieved the whole experimental work, including fermentation of fungal isolates,
isolation of newly produced metabolites, structure investigation, bioassay and chemical analysis, and
shared the task of manuscript preparation. Chang-Wei Li contributed to this work by coaching,
advising and assisting Wu’s experiments, who also shared tasks of the manuscript preparation and the
structure elucidation of new compounds. Cheng-Bin Cui was the project leader who proposed ideas for
the present work, managed and supervised the whole research work, and accomplished writing of
the paper.
Conflicts of Interest
The authors declare no conflict of interest.
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2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
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(