Graphiumins, new thiodiketopiperazines from the marine-derived fungus Graphium sp. OPMF00224

Article abstract of DOI:10.1038/ja.2015.41

Eight new thiodiketopiperazines, designated as graphiumins A to H (1-8), were isolated along with bisdethiobis(methylthio)-deacetylaranotin (9) and bisdethiobis(methylthio)-deacetylapoaranotin (10) from the culture broth of the marine-derived fungus Graph

Full text of DOI:10.1038/ja.2015.41

The Journal of Antibiotics (2015), 1–8
2015 Japan Antibiotics Research Association All rights reserved 0021-8820/15
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&
ORIGINAL ARTICLE
Graphiumins, new thiodiketopiperazines from the
marine-derived fungus Graphium sp. OPMF00224
Takashi Fukuda1, Minori Shinkai1, Eri Sasaki1, Kenichiro Nagai1, Yuko Kurihara2, Akihiko Kanamoto2
and Hiroshi Tomoda1
Eight new thiodiketopiperazines, designated as graphiumins A to H (1–8), were isolated along with bisdethiobis(methylthio)-
deacetylaranotin (9) and bisdethiobis(methylthio)-deacetylapoaranotin (10) from the culture broth of the marine-derived fungus
Graphium sp. OPMF00224. The structures of the graphiumins were elucidated based on spectroscopic analyses (1D and 2D
NMR data, ROESY correlations and CD data) and chemical methods. The absolute configuration of the common (3S)-3-hydroxy-
octanoyl acid residue in 1, 3 and 4 was determined by hydrolysis, benzoyl derivatization and HPLC analysis using a chiral
column. Five graphiumins moderately inhibited yellow pigment production by methicillin-resistant Staphylococcus aureus.
The Journal of Antibiotics advance online publication, 22 April 2015; doi:10.1038/ja.2015.41
INTRODUCTION
RESULTS
Methicillin-resistant Staphylococcus aureus (MRSA) is a major noso- Collection and identification of strain OPMF00224
Strain OPMF00224 was isolated from marine sediment collected at
− 17 m on Ishigaki Island, Okinawa, Japan. The sediment was treated
comial pathogen that is resistant to many other antibiotics including
currently used β-lactams.1 Although vancomycin is widely used to
treat MRSA infections, vancomycin-resistant S. aureus has been by the direct inoculation method using kelp agar made with 130%
identified and reported.2 Therefore, it has become increasingly
artificial seawater (cut kelp 5 g, New Ocean (artificial sea water
important and necessary to discover new antibiotics effective against
MRSA infections. S. aureus strains including MRSA produce a yellow
pigment called staphyloxanthin.3–5 Several research groups recently
identified staphyloxanthin as one of the important virulence factors of
S. aureus.6–8 Staphyloxanthin is located in the cell membrane of
S. aureus and has been associated with enhancing the survival and
infectiousness of S. aureus.5,6 Liu et al.6 reported that the enzyme
dehydrosqualene synthase-deficient mutant that could not produce
staphyloxanthin failed to survive in a mouse host. This finding
indicated that compounds inhibiting the production of staphylox-
anthin could be a source of new anti-infectious agents against
MRSA. On the basis of this concept, BPH-652,7 zaragozic acid,9
7-benzyloxyindoles10 and flavones11 were reported to be inhibitors
of staphyloxanthin production. We started to search for such
inhibitors from natural sources using our established screening
system,12 leading to the discovery of tylopilusins13 and citridone A
and its derivatives.14 Continuing our screening program, new com-
pounds designated graphiumins A (1) to H (8), along with two
structurally related known bisdethiobis(methylthio)-deacetylaranotin
(9)15 and bisdethiobis(methylthio)-deacetylapoaranotin (10),15 were
isolated from the culture broth of the marine-derived fungus
Graphium sp. OPMF00224 (Figure 1). In this study, we describe the
fermentation, isolation, structure elucidation and biological activity of
these graphiumins.
powder, Japan Bio Chemical) 46.8 g, agar 20 g, tap water 1l). The
strain was cultured on Low Carbon Agar medium made with 100%
artificial seawater for identification. DNA extraction, PCR, sequencing
and Basic Local Alignment Search Tool searching followed.16,17 The
primer set ITS-5 (5′-GGAAGTAAAAGTCGTAACAAGG-3′) and
NL-4 (5′-GGTCCGTGTTTCAAGACGG-3′) was used to amplify the
region under the conditions of 30 cycles at 94 °C for 30 s, 60 °C for
30 s and 72 °C for 90 s. The sequence is available at the DNA Data
Bank of Japan/European Molecular Biology Laboratory/GenBank
databases under the accession number AB934385. On the basis of
the Basic Local Alignment Search Tool search and its microscopic
features, the strain was identified as a Graphium sp.
Fermentation
The strain was inoculated into a 500-ml Erlenmeyer flask containing
100 ml seed medium (2.0% glucose, 0.2% yeast extract, 0.05%
MgSO4·7H2O, 0.5% polypeptone, 0.1% KH2PO4 and 0.1% agar, pH
6.0). The flask was shaken on a rotary shaker at 27 °C for 3 days. The
seed culture (2.0 ml) was transferred into a 1000-ml culture box
containing 150 ml production medium (5.0% oat meal, 0.2% yeast
extract, 0.1% Na tartrate, 0.1% KH2PO4, 0.8% DAIGO authentic
seawater). Fermentation was carried out at 27 °C for 24 days under
static conditions.
1Graduate School of Pharmaceutical Sciences, Kitasato University, Tokyo, Japan and 2OP BIO FACTORY Co., Ltd, Okinawa, Japan
Correspondence: Professor H Tomoda, Graduate School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641 Japan.
E-mail: tomodah@pharm.kitasato-u.ac.jp
Received 11 December 2014; revised 1 March 2015; accepted 24 March 2015

New thiodiketopiperazines from the marine-derived fungus
T Fukuda et al
2
12
14
16
18
11
13
15
17
O
O
OH
7
OH
O
O
6
O
1'
O
8
9
H
H
3'
2'
O
O
N
5' 4'
N
S
S2
S2
4
5
S
N
N
2
O
9'
8'
O
3
1
H
H
7'
6'
O
O
O
O
OH
OH
16'
14'
12'
17'
15'
13'
11'
O
18'
O
1
2
R1O
H
R1O
H
O
O
O
8
8
S
S
N
O
O
N
N
5'
N
H
O
6'
8'
8'
S
H
OR2
S
O
OR2
R2
R2
R1
R1
12'
14'
16'
12'
14'
16'
12
14
16
12
14
16
18'
18'
11'
11'
18
13'
18
13'
15'
15'
17'
17'
11
11
11
11
13
O
13
O
15
15
17
17
O
O
O
O
3
4
6
7
OH
OH
OH
OH
13
13
H
H
OH
20
OH
20
19
12
21
16
19
12
21
16
18
18
H
H
11
11
13
13
15
15
17
17
O
O
14
14
5
9
8
OH
OH
O
O
H
H
H
10
H
Figure 1 Structures of graphiumins A (1) to H (8), bisdethiobis(methylthio)-deacetylaranotin (9) and bisdethiobis(methylthio)-deacetylapoaranotin (10).
Isolation
kinds of carbonyl groups. These spectral similarities indicate that they
The culture broth (150 ml× 30) was extracted with ethanol (4.5 l) for are all structurally related.
2 h. After this extract had been evaporated to an aqueous solution, the
residue was partitioned between water and ethyl acetate to yield the Structure elucidation of graphiumins
crude extract (600 mg) after the evaporation of the ethyl acetate Graphiumin A (1): graphiumin A (1) was obtained as a pale-yellow
fraction. The crude extract was dissolved in a small volume of solid. The molecular formula for 1 was established as C34H44N2O10S2
chloroform, applied onto a silica gel column (30 g, 3.4 × 15 cm, ([M+Na]+ m/z 727.2343, calcd [M+Na]+ 727.2335) on the basis of
0.04–0.063 mm) and eluted stepwise with 100% chloroform, 50:1, high-resolution ESI-MS measurements, indicating 1 contained 14
1
25:1, 10:1, 5:1, 1:1 (v/v) of chloroform-methanol solvent and 100% degrees of unsaturation (Table 1). H and 13C NMR data (in CDCl3)
methanol (200 ml each). Graphiumins were observed in the last supported the molecular formula (Tables 2 and 3). The 13C NMR
fraction eluted with 50:1 chloroform-methanol. This fraction was spectrum showed 17 resolved signals, which were classified into one
further purified by reversed-phase C-18 HPLC (10 × 250 mm; PEGA- methyl, six methylenes, six methines, including three sp2 methines and
SIL ODS, Senshu Scientific Co., Tokyo, Japan) under the following four quaternary carbons, including two carbonyl carbons (C-1 and
conditions: solvent, a 40-min linear gradient from 60 to 80% C-11). On the basis of its molecular formula, 1 appeared to be a
aqueous acetonitrile, at a flow rate of 3.0 ml min− 1 with UV detection symmetrical compound. The 1H NMR spectrum of 1 showed one
at 210 nm. Under this condition, graphiumins A, B, C, D, E, F, methyl signal, three olefinic methine signals, two oxygenated methine
G, H, bisdethiobis(methylthio)-deacetylaranotin (9) and bisdethiobis signals and many methylene signals. The connectivity of all proton and
(methylthio)-deacetylapoaranotin (10) were eluted as peaks with carbon atoms was established by HMQC experiments (Table 2). An
retention times of 30.0, 34.0, 31.4, 16.2, 12.3, 37.7, 18.5, 13.9, 7.6 analysis of 1H-1H COSY data allowed two partial structures I and II to
and 8.5 min, respectively. These peaks were collected and concentrated be assigned (Figure 2). An analysis of HMBC spectroscopic data gave
to yield 1.0, 1.3, 4.2, 1.1, 17.1, 3.8, 3.5, 3.2, 2.5 and 1.7 mg as pale- further structural information on 1. The cross-peaks from H2-3
yellow solids, respectively.
(δ 2.98, 4.10) to C-1 (δ 162.5), C-2 (δ 79.7), C-4 (δ 112.9), C-5
(δ 139.6) and C-9 (δ 63.0) from H-5 (δ 6.62) to C-3, C-4 and C-6
(δ 141.4), from H-6 (δ 6.34) to C-5 and from H-9 (δ 5.11) to C-3 and
Physicochemical properties of graphiumins
The physicochemical properties of graphiumins are summarized in C-4 supported the partial structure I. Additional cross-peaks from H-8
Table 1. All graphiumins showed very similar UV spectra with (δ 5.74) to C-11 (δ 171.5) and from H-12 (δ 2.46) to C-11 supported
absorption maxima at 222 ~ 230 nm. Absorptions at 1730~ 1738 the connectivity of the partial structures I and II. The chemical shifts
and 1656~ 1670 cm− 1 in IR spectra suggested the presence of two of C-1 and C-2 and the number of nitrogen and sulfur atoms present
The Journal of Antibiotics

New thiodiketopiperazines from the marine-derived fungus
T Fukuda et al
3
in 1 supported an aranotin type epidithiodiketopiperazine skeleton.18
Taking its structural symmetry into consideration, the planar structure
of 1 was elucidated.
The stereochemistry of the central ring part in 1 was determined by
ROESY experiments, 1H-1H coupling constant measurements and CD
data. The NOE correlations between 3-H and 5-H as well as between
8-H and 9-H and the coupling constants J7-8 (2.0 Hz) and J8-9 (8.5 Hz)
indicated that 1 had the same configuration as known 9. In addition,
the CD spectrum of 1 exhibited a typical positive Cotton effect at
270 nm for a disulfide bridge on the diketopiperazine ring, indicating
that 1 has the 2R/2′R configurations.19 Accordingly, the stereochem-
istry of the central ring of 1 is as shown in Figure 2. Then, the absolute
configuration of the hydroxylated carbon in the octanoyl side chain
was determined by chiral HPLC analysis of the benzyl derivative of the
base hydrolysate of 1.20 In the case of 1, the peak was observed with a
retention time of 29.8 min, indicating that the conformations of both
C-13 and C-13′ were elucidated as S (Figure 3). On the basis of all
data, the absolute structure of 1 was elucidated as shown in Figure 1.
Graphiumin B (2): the structure of 2 was elucidated by comparing
all spectroscopic data with those of 1. The molecular formula of 2
was established as C34H44N2O10S4 ([M+Na]+ m/z 797.1769, calcd
[M+Na]+ 797.1776) on the basis of high-resolution ESI-MS measure-
ments, indicating that 2 was 64 mass units (S2) larger than 1. The
similarity in the NMR spectra to 1 and its molecular formula indicated
that it possessed at tetrasulphide in place of a disulfide bridge.
Graphiumin C (3): the molecular formula of 3 was established as
C36H50N2O10S2 ([M+Na]+ m/z 757.2802, calcd [M+Na]+ 757.2804)
on the basis of high-resolution ESI-MS measurements, indicating that
3 was 30 mass units (C2H6) larger than 1 and contained 13 degrees of
unsaturation (Table 1). The 1H and 13C NMR data (in CDCl3)
supported the molecular formula (Tables 2 and 3). The difference
between 1 and 3 was that an additional methyl signal (C δ 14.8, H δ
2.28) in 3 was observed in the 13C and 1H NMR spectra (Tables 2 and
3). In addition, the cross-peak from S-CH3 to C-2/2′ (δ 70.5) in 3
was observed in HMBC data (Figure 4). The CD spectrum of 3
exhibited a typical negative Cotton effect at 254 nm in two S-methyl
groups on diketopiperazine ring, indicating that 3 has the 2R/2′R
configurations.19 All these data indicated that 3 was di-S-methylated 1.
Graphiumin D (4): the molecular formula of 4 was established as
C28H36N2O8S2 ([M+Na]+ m/z 615.1807, calcd [M+Na]+ 615.1810) on
the basis of high-resolution ESI-MS measurements, indicating that 4
was 142 mass units (C8H14O2) smaller than 3. The clear difference
between 3 and 4 was that 4 was not a symmetrical compound. The
13C NMR spectrum showed 28 resolved signals, which were classified
into three methyls, seven methylenes, eleven methines including six
sp2 methines and seven quaternary carbons including three carbonyl
1
carbons (C-1, C-11 and C-1′) (Table 2). An analysis of H-1H COSY
data allowed three partial structures, C-6 to C-9, C-6′ to C-9′ and
C-12 to C-18, to be assigned. In addition, the signals corresponding to
1
one side chain in 4 were missing in the H and 13C NMR spectra. A
1
broad signal assigned 8′-OH (δ 4.43) instead appeared in H NMR.
These data indicated that the structure of 4 was des-8′-O hydro-
xyoctanyl 3. The structure satisfied the molecular formula and degrees
of unsaturation.
Graphiumin E (5): the molecular formula of 5 was established as
C31H38N2O9S2 ([M+Na]+ m/z 669.1904, calcd [M+Na]+ 669.1916) on
the basis of high-resolution ESI-MS measurements. The NMR spectra
of 5 were similar to those of 4 (Tables 2 and 3). The clear difference
between 4 and 5 lay in the side chain signals in the NMR data. The 13
C
NMR spectrum showed 31 resolved signals, which were classified into
six methyls, two methylenes, 14 methines including seven sp2 methines
The Journal of Antibiotics

New thiodiketopiperazines from the marine-derived fungus
T Fukuda et al
4
Table 2 13C NMR spectroscopic data for 1 to 8 in CDCl3 (100 MHz for 1 and 3 and 150 MHz for 2, 4, 5, 6, 7 and 8)a,b
1
2
3
4
5
6
7
8
Position
δC
δC
δC
δC
δC
δC
δC
δC
1
162.5, s
79.7, s
166.0, s
75.6, s
164.8, s
70.5, s
166.6, s
70.3, s
166.7, s
70.4, s
165.2, s
70.4, s
167.3, s
69.7, s
167.3, s
69.8, s
2
3
34.1, t
41.5, t
40.6, t
40.1, t
40.3, t
40.9, t
39.6, t
39.8, t
4
112.9, s
139.6, d
141.4, d
105.1, d
69.8, d
63.0, d
107.8, s
138.9, d
139.9, d
105.7, d
71.0, d
60.9, d
109.1, s
137.8, d
139.9, d
105.3, d
71.7, d
60.5, d
109.2, s
138.2, d
139.9, d
105.3, d
71.8, d
60.6, d
109.7, s
137.9, d
139.8, d
105.6, d
72.4, d
60.3, d
109.4, s
137.9, d
139.9, d
105.3, d
71.8, d
60.6, d
109.2, s
138.1, d
139.9, d
105.5, d
72.0, d
60.4, d
109.8, s
138.0, d
139.8, d
105.7, d
72.6, d
60.2, d
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
1'
171.5, s
42.0, t
67.5, d
36.5, t
25.3, t
31.7, t
22.6, t
14.0, q
172.0, s
42.5, t
67.7, d
36.8, t
25.2, t
31.7, t
22.6, t
14.1, q
171.6, s
42.1, t
67.4, d
36.6, t
25.2, t
31.7, t
22.6, t
14.0, q
171.5, s
42.2, t
67.4, d
36.6, t
25.2, t
31.7, t
22.6, t
14.0, q
174.6, s
42.6, d
72.2, d
41.1, d
205.8, s
137.4, s
138.5, d
15.1, q
12.2, q
13.6, q
11.0, q
164.4, s
69.3, s
171.5, s
42.1, t
67.6, d
37.4, t
25.3, t
31.8, t
22.5, t
14.0, q
171.4, s
42.2, t
67.4, d
36.6, t
25.2, t
31.7, t
22.6, t
14.0, q
174.6, s
42.6, d
72.3, d
41.2, d
206.0, s
137.9, s
138.6, d
15.1, q
12.4, q
13.5, q
11.1, q
164.4, s
73.2, s
162.5, s
79.7, s
166.0, s
75.6, s
164.8, s
70.5, s
164.6, s
69.4, s
165.0, s
74.1, s
164.7, s
73.3, s
2'
3'
34.1, t
41.5, t
40.6, t
40.0, t
40.1, t
40.1, t
38.6, t
38.7, t
4'
112.9, s
139.6, d
141.4, d
105.1, d
69.8, d
63.0, d
107.8, s
138.9, d
139.9, d
105.7, d
71.0, d
60.9, d
109.1, s
137.8, d
139.9, d
105.3, d
71.7, d
60.5, d
107.7, s
138.0, d
137.7, d
110.8, d
72.6, d
64.1, d
107.7, s
137.9, d
137.6, d
110.8, d
72.6, d
64.1, d
133.4, s
120.0, d
125.3, d
127.7, d
75.6, d
64.5, d
131.4, s
120.1, d
122.9, d
130.5, d
74.3, d
69.0, d
131.5, s
120.0, d
122.9, d
130.5, d
74.3, d
69.0, d
5'
6'
7'
8'
9'
10'
11'
12'
13'
14'
15'
16'
17'
18'
2-SMe
2'-SMe
171.5, s
42.0, t
67.5, d
36.5, t
25.3, t
31.7, t
22.6, t
14.0, q
172.0, s
42.5, t
67.7, d
36.8, t
25.2, t
31.7, t
22.6, t
14.1, q
171.6, s
42.1, t
67.4, d
36.6, t
25.2, t
31.7, t
22.6, t
14.0, q
14.8, q
14.8, q
171.8, s
43.0, t
67.4, d
37.5, t
25.2, t
31.8, t
22.5, t
14.0, q
14.5, q
14.8, q
14.7, q
14.9, q
14.8, q
14.8, q
14.9, q
14.7, q
14.9, q
14.8, q
aAssignments made by interpretation of HSQC and HMBC NMR data.
bChemical shifts are shown with reference to CDCl3 as δ 77.0.
and nine quaternary carbons including four carbonyl carbons (C-1,
Graphiumin F (6): the molecular formula for 6 was established as
C-11, C-15 and C-1′) (Table 2). An analysis of H-1H COSY data C36H50N2O9S2 ([M+Na]+ m/z 741.2845, calcd [M+Na]+ 741.2855) on
allowed partial structure III to be assigned (Figure 4). An analysis of the basis of high-resolution ESI-MS measurements indicating that 6
HMBC data gave further structural information on the side chain. The contained 13 degrees of unsaturation. The 13C NMR spectrum showed
cross-peaks from H-12 (δ 2.55) to C-11 (δ 174.6), from H-13 (δ 4.09) 36 resolved signals, which were classified into four methyls, 12
to C-11, from H-14 (δ 3.37) to C-15 (δ 205.8), from H-17 (δ 6.78) to methylenes, 12 methines including six sp2 methines and eight
C-15, from H-18 (δ 1.88) to C-16 (δ 137.4) and C-17 (δ 138.5), from quaternary carbons including two carbonyl carbons (C-1 and C-11)
1
1
H-19 (δ 1.24) to C-11, C-12 (δ 42.6) and C-13 (δ 72.2), from H-20 (Tables 2 and 3). The H NMR spectrum of 6 showed four methyl
(δ 1.20) to C-13, C-14 (δ 41.1) and C-15 and from H-21 (δ 1.75) to signals, six olefinic methine signals, four oxygenated methine signals
C-15, C-16 and C-17 supported partial structure III (Figure 4). In and many methylene signals. The NMR spectra of 6 were similar to
addition, the cross-peaks from H-8 (δ 5.72) to C-11 supported that those of 3 (Tables 2 and 3). The difference between 3 and 6 was that
partial structure III was linked to the central ring system at C-8. Taken 5′-H (δ 5.96) and 6′-H (δ 5.97) in 6 were shifted to the upper field in
1
1
together, the structure of 5 was elucidated as shown in Figure 1.
the H NMR spectrum. An analysis of H-1H COSY data allowed the
The Journal of Antibiotics

New thiodiketopiperazines from the marine-derived fungus
T Fukuda et al
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The Journal of Antibiotics

New thiodiketopiperazines from the marine-derived fungus
T Fukuda et al
6
12
18
12
14
11
O
O
8.5
13
II
OH
H
2.0
OH
O
O
H
8.0
O
6
8
8
6
H
H
N
7
8
O
9
9
O
5
N
O
5
9
H
6
H
S
N
5
S
H
3
1
3
O
I
O
Key 1H-1H coupling with J
Key NOE
1H-1H COSY
Key HMBC
Figure 2 1H-1H COSY, Key HMBC, NOE correlations and 1H-1H coupling of 1.
connectivity from C-5′ (δ 120.0) to C-9′ (δ 64.5) in the partial
structure IV to be assigned (Figure 4). These results indicated that 6
was an apoaranotin type compound.18 In addition, all data for the ring
parts in 6 were similar to those of known bisdethiobis(methylthio)-
deacetylapoaranotin (10).15 The cross-peaks from 8-H (δ 5.88) to
C-11 (δ 171.5) and from 8′-H (δ 6.22) to C-11′ (δ 171.8) in HMBC
data supported the same acyl side chains was attached at both C-8 and
C-8′. Taken together, the structure of 6 was elucidated as shown in
Figure 1.
Graphiumin G (7): the molecular formula of 7 was established as
C28H36N2O7S2 ([M+Na]+ m/z 599.1856, calcd [M+Na]+ 599.1861) on
the basis of high-resolution ESI-MS measurements, which was 142
mass units (C8H14O2) smaller than 6. This suggested that 7 was
missing one side chain from 6. In addition, the cross-peaks from H-8
(δ 5.88) to C-11 (δ 171.4) in HMBC supported that one acyl side
chain was attached to C-8. Thus, the structure of 7 was elucidated as
des-8′-O hydroxyoctanyl 6.
Graphiumin H (8): the structure of 8 was elucidated by a
comparison of all spectroscopic data with those of 5 and 6. The
molecular formula of 8 was established as C31H38N2O8S2 ([M+Na]+
m/z 653.1964, calcd [M+Na]+ 653.1967) on the basis of high-
resolution ESI-MS measurements, which was 16 mass units (O)
smaller than 5. In addition, 8 was an apoaranotin type from the
chemical shifts of H-5′ (δ 5.93) and H-6′ (δ 5.87) with the same side
chain as 5 (Tables 2 and 3). Taking all NMR data into consideration,
the structure of 8 was elucidated as shown in Figure 1.
Figure 3 Separation of (S)benzyl-3-hydroxy-octanoate and (R)benzyl-3-
hydroxy-octanoate by HPLC. (a) Derivatives prepared from authentic ( )-3-
hydroxyoctanoic acid. (b) Derivative prepared from graphiumin A.
The absolute configurations of 3 and 4 were determined in the same
way as 1. In addition, taking the biogenetic mechanism, ROESY
experiments and the value of optical rotation into consideration, 2, 6
12
18
19
12
20
14
21
11
O
18
O
S
16
3'
OH
O
13
11
15
17
6
III
5'
OH
O
O
N
9'
8'
6
O
5
7
6'
8
9
N
O
O
IV
N
OH
O
11'
1
3
5
S
O
18'
O
S
O
1H-1H COSY
Key HMBC
1H-1H COSY
Key HMBC
1H-1H COSY
Key HMBC
3
5
6
Figure 4 1H-1H COSY and Key HMBC correlations of 3, 5 and 6.
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New thiodiketopiperazines from the marine-derived fungus
T Fukuda et al
7
O
Sn
O
O
Sn
O
EXPERIMENTAL PROCEDURE
General experimental procedures
O
NH
N
Optical rotations were recorded with a DIP-1000 digital polarimeter (JASCO,
Tokyo, Japan). Melting points were measured with a micro melting apparatus
(Yanaco, Kyoto, Japan). ESI-MS spectrometry was conducted on a JMS-
T1000LP spectrometer (JEOL, Tokyo, Japan). UV and IR spectra were
measured with a U-2800 spectrophotometer (HITACHI, Tokyo, Japan) and
FT/IR-460 plus spectrometer (JASCO), respectively. The various NMR spectra
were measured with an UNITY 400 or an INOVA 600 spectrometer (Agilent
Technologies Inc, Santa Clara, CA, USA). Reversed-phase HPLC separations
were performed using a Senshu pak preparative C18 PEGASIL ODS column
(250 × 10 mm) at a flow rate of 3 ml min− 1 and CHIRALCEL OJ-H column
(150 × 4.6 mm) at a flow rate of 1.0 ml min− 1 using the SHIMAZU LS20AT
pump and SHIMAZU LS20AS UV detector.
N
N
O
Type B
O
Type A
O
O
N
N
Sn
Sn
N
N
O
O
Type C
Type D
Figure 5 Structural classification of TDKPs.
and 7 were deduced to have the same absolute configurations.
However, the absolute configurations for 5 and 8 are still unknown.
The effects of all graphiumins 1 to 10 on yellow pigment
production by MRSA were tested using the paper disk method.12
Compounds 1, 4, 5, 7, 8, 9, 10 and citridone A14 (positive compound)
showed a white zone (10, 14, 10, 12, 11, 11, 11 and 20 mm,
respectively) with no inhibition zone at 50 μg 8 mm− 1 paper disk,
suggesting that they selectively inhibited the production of the yellow
pigment without affecting the growth of MRSA.
Hydrolysis and benzoylation of graphiumins A, B and C
A total of 0.1N NaOH (1 ml) was added to a solution of a sample (0.5 mg) in a
1:3 mixture of water (250 μl) and methanol (750 μl) at room temperature. After
stirring for 4 h, the resulting mixture was evaporated to give a colorless solid,
which was used for the next reaction without further purification. Cesium
carbonate (1 mg, 5 mmol) was added to a solution of the residue in water
(100 μl) and the mixture was stirred at room temperature for 30 min. After
removal of the solvent, the cesium salt was dissolved in dimethylformamide
(100 μl) and benzyl bromide (10 μl) was then added. Stirring was continued at
room temperature for 20 h. The resulting mixture was poured into a 1:1
mixture of water and ethyl acetate. The phases were separated and the aqueous
phase was extracted with ethyl acetate. The combined organic phases were dried
over Na2SO4, filtered and concentrated under reduced pressure. The residue
was dissolved in MeOH (40 μl) for chiral HPLC analysis.
DISCUSSION
A number of thiodiketopiperazines have been reported. On the basis
of the core ring system consisting of carbon, oxygen and nitrogen
atoms, they are classified into the four types as summarized in
Figure 5. Type A has the three-ring system, to which gliotoxin21 and
phomazines A and B22 belong. Type B has the symmetrical five-ring
system including two oxepane rings, to which aranotin17 and its
derivatives15 belong. Type C has the five-ring system including one
oxepane ring, to which apoaranotin17 and its derivatives15 belong.
Type D has the symmetrical five-ring system, to which a number of
compounds such as epicoccins,23 epicorazines,24 rostratins,25
haematocins,26 scabrosin esters,27 brocazines,28 exserohilones,29 pho-
mazine C22 and SCH64874s30 belong. Graphiumins A (1) to E (5)
belong to type B, and graphiumins F (6) to H (8) belong to type C.
Regarding the side chain extending from the central ring, all of the
eight graphiumins (1 to 8) have long acyl chains. Graphiumins E (5)
and H (8) carry the same side chain of 3-hydroxy-2,4,6-trimethyl-5-
oxooct-6-enoyl acid residue.31,32 Before graphiumins discovery, only
SCH64874s,30 isolated from the culture broth of an unidentified
fungus, had been reported to have two acyl side chains, whose planar
structure is the same as that of 5 and 8. Interestingly, the other
graphiumins have the distinct 3-hydroxy-octanoyl acid residue(s) as
the common side chain. Furthermore, 1, 2, 3 and 6 were found to be
the second thiodiketopiperazines with the two acyl side chains. The
biosynthesis of graphiumins remains to be investigated.
Structurally related thiodiketopiperazines were reported to possess
various biological activities; aranotin with antiviral activity against
polio and parainfluenza virus,18,33 epicoccin A with antimicrobial
activity,23 epicoccin G with anti-HIV activity34 and phomazines,22
rostratins,25 scabrosin esters27 and brocazines28 with cytotoxic activity.
Accordingly, the graphiumins were evaluated in our in-house assays;
antimicrobial assay,35 mammalian cell cycle assay,36 lipid metabolism
assay37 and bone morphogenetic protein-induced alkaline phosphatase
expression assay.38 However, they showed no activity in these assays
even at 25 μg ml− 1 (data not shown). Thus, compounds 1, 4, 5, 7 and
8 selectively inhibited yellow pigment production by MRSA. The
mechanism of action remains to be defined.
Analysis of stereochemistry of the benzyl-3-hydroxy-octanoate
by HPLC
To determine the side chain stereochemistries, aliquots (20 μl) of the
methanolic solutions of the benzyl derivatives of the base hydrolysates were
analyzed using a SHIMAZU LS20AT pump under the following conditions:
column, CHIRALCEL OJ-H column (150 × 4.6 mm); flow rate, 1.0 ml min− 1
;
detection, UV at 254 nm. Using 35% aqueous CH3CN in 0.05% trifluoroacetic
acid as a solvent for HPLC, benzyl (3S)-3-hydroxy-octanoate and benzyl (3R)-
3-hydroxy-octanoate, which were synthesized from authentic ( )-3-hydro-
xyloctanoic acid (SIGMA-ALDRICH, St Louis, MO, USA) in the same manner,
were eluted as peaks with retention times of 29.2 and 31.4 min, respectively
(Figure 3). Under the same conditions, all products from graphiumins A, B and
C were eluted as a peak with retention time of 29.2 min (Figure 3).
Assay for inhibition of yellow pigment production in MRSA using
paper disks
MRSA K-24 strain, a clinical isolate, was used as a yellow pigment-producing
strain. MRSA was cultured in Mueller–Hinton broth at 37 °C for 20 h and
adjusted to 1 ×108 colony forming units/ml. The inoculum (100 μl) was spread
on TYB agar (1.7% tryptone, 1% yeast extract, 0.5% NaCl, 0.25% K2HPO4,
15% agar and 1.5% monoacetate) (25 ml) on a plate (100 × 140 mm). Paper
disks (8 mm i.d.) containing a 50 μg sample were placed on the plate and
incubated at 37 °C for 72 h. Inhibition of the production of yellow pigments by
a sample is expressed as the diameter (mm) of the white zone on the plate.
ACKNOWLEDGEMENTS
We wish to thank Ms Noriko Sato (School of Pharmaceutical Sciences, Kitasato
University) for measuring the NMR spectra. This work was supported by
Takeda Science Foundation (TF) and JSPS KAKENHI Grant Number
25870704 (TF).
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New thiodiketopiperazines from the marine-derived fungus
T Fukuda et al
8
20 Nagai, K. et al. Synthesis and biological evaluation of a beauveriolide analogue library.
J. Comb. Chem. 8, 103–109 (2006).
1
Centers for Disease Control and Prevention (CDC). Staphylococcus aureus with reduced
susceptibility to vancomycin–United States. MMWR Morb. Mortal. Wkly. Rep. 46,
765–766 (1997).
Hiramatsu, K. et al. Methicillin-resistant Staphylococcus aureus clinical strain with
reduced vancomycin susceptibility. J. Antimicrob. Chemother. 40, 135–136 (1997).
Marshall, J. H. & Wilmoth, G. J. Pigments of Staphylococcus aureus, a series of
triterpenoid carotenoids. J. Bacteriol. 147, 900–913 (1981).
21 Brian, P. W.
& Hemming, H. G. Gliotoxin, a fungistatic metabolic product of
Trichoderma viride. Ann. Appl. Biol. 32, 214–220 (1945).
22 Kong, F., Wang, Y., Liu, P., Dong, T. & Zhu, W. Thiodiketopiperazines from the
marine-derived fungus Phoma sp. OUCMDZ-1847. J. Nat. Prod. 77, 132–137
(2014).
23 Wang, J. M. et al. Thiodiketopiperazines produced by the endophytic fungus Epicoccum
nigrum. J. Nat. Prod. 73, 1240–1249 (2010).
24 Deffieux, G., Baute, M. A., Baute, R. & Filleau, M. J. New antibiotics from the fungus
Epicoccum nigrum. II. Epicorazine A: structure elucidation and absolute configuration.
J. Antibiot. (Tokyo) 31, 1102–1105 (1978).
25 Tan, R. X., Jensen, P. R., Williams, P. G. & Fenical, W. Isolation and structure
assignments of rostratins A-D, cytotoxic disulfides produced by the marine-derived
fungus Exserohilum rostratum. J. Nat. Prod. 67, 1374–1382 (2004).
26 Suzuki, Y. et al. Haematocin, a new antifungal diketopiperazine produced by Nectria
haematococca Berk. et Br. (880701a-1) causing nectria blight disease on
ornamental plants. J. Antibiot. (Tokyo) 53, 45–49 (2000).
27 Chai, C. L., Elix, J. A., Huleatt, P. B. & Waring, P. Scabrosin esters and derivatives:
chemical derivatization studies and biological evaluation. Bioorg. Med. Chem. 12,
5991–5995 (2004).
28 Meng, L. H., Li, X. M., Lv, C. T., Huang, C. G. & Wang, B. G. Brocazines A-F, cytotoxic
bisthiodiketopiperazine derivatives from Penicillium brocae MA-231, an endophytic
fungus derived from the marine mangrove plant Avicennia marina. J. Nat. Prod. 77,
1921–1927 (2014).
29 Gross, U., Nieger, M. & Brase, S. A unified strategy targeting the thiodiketopiperazine
mycotoxins exserohilone, gliotoxin, the epicoccins, the epicorazines, rostratin A and
aranotin. Chem. Eur. J 16, 11624–11631 (2010).
30 Hegde, V. R., Dai, P., Patel, M., Das, P. R. & Puar, M. S. Novel thiodiketopiperazine
fungal metabolites as epidermal growth factor receptor antagonists. Tet. Lett. 38,
911–914 (1997).
2
3
4
Marshall, J. H.
& Wilmoth, G. J. Proposed pathway of triterpenoid carotenoid
biosynthesis in Staphylococcus aureus: evidence from a study of mutants. J. Bacteriol.
147, 914–919 (1981).
Clauditz, A., Resch, A., Wieland, K. P., Peschel, A. & Goötz, F. Staphyloxanthin plays a
role in the fitness of Staphylococcus aureus and its ability to cope with oxidative stress.
Infect. Immun. 74, 4950–4953 (2006).
Liu, G. Y. et al. Staphylococcus aureus golden pigment impairs neutrophil killing
and promotes virulence through its antioxidant activity. J. Exp. Med. 202,
209–215 (2005)
Liu, C. I. et al. A cholesterol biosynthesis inhibitor blocks Staphylococcus aureus
virulence. Science 319, 1391–1394 (2008).
Song, Y. et al. Inhibition of staphyloxanthin virulence factor biosynthesis in Staphylo-
coccus aureus: in vitro, in vivo, and crystallographic results. J. Med. Chem. 52,
3869–3880 (2009).
Liu, C. I., Jeng, W. Y., Chang, W. J., Ko, T. P. & Wang, A. H. Binding modes of zaragozic
acid A to human squalene synthase and staphylococcal dehydrosqualene synthase. J.
Biol. Chem. 287, 18750–18757 (2012).
5
6
7
8
9
10 Lee, J. H. et al. Indole and 7-benzyloxyindole attenuate the virulence of Staphylococcus
aureus. Appl. Microbiol. Biotechnol. 97, 4543–4552 (2013).
11 Lee, J. H., Park, J. H., Cho, M. H. & Lee, J. Flavone reduces the production of virulence
factors, staphyloxanthin and α-hemolysin, in Staphylococcus aureus. Curr. Microbiol.
65, 726–732 (2012).
12 Sakai, K. et al. Method of search for microbial inhibitors of staphyloxanthin production
by MRSA. Biol. Pharm. Bull. 35, 48–53 (2012).
13 Fukuda, T., Nagai, K. & Tomoda, H. ( )-Tylopilusins, diphenolic metabolites from the
31 Chinworrungsee, M., Kittakoop, P., Saenboonrueng, J., Kongsaeree, P.
& Thebt-
aranonth, Y. Bioactive compounds from the seed fungus Menisporopsis theobromae
BCC 3975. J. Nat. Prod. 69, 1404–1410 (2006).
32 Kawashima, A., Yoshimura, Y., Mizutani, T., Hanada, K. & Omura, S. Jpn. Kokai Tokkyo
Koho Patent No. JP2062880 (19900302) (1990).
33 Trown, P. W. et al. LL-S88-alpha, an antiviral substance produced by Aspergillus
terreus. Antimicrob. Agents Chemother. 8, 225–228 (1968).
fruiting bodies of Tylopilus eximius. J. Nat. Prod. 75, 2228–2231 (2012).
14 Fukuda, T., Shimoyama, T., Nagamitsu, T.
&
Tomoda, H. Synthesis and
biological activity of citridone
A and its derivatives. J. Antibiot. (Tokyo) 67,
445–450 (2014).
15 Guo, C. J. et al. Biosynthetic pathway for the epipolythiodioxopiperazine acetylaration in
Aspergillus terreus revealed by genome-based deletion analysis. J. Am. Chem. Soc.
135, 7205–7213 (2013).
34 Zhang, Y., Liu, S.
& Liu, X. Epicoccins A-D, epipolythiodioxopiperazines from a
Cordyceps-colonizing isolate of Epicoccum nigrum. J. Nat. Prod. 70,
1522–1525 (2007).
16 Kobayashi, K. et al. Bafilomycin L, a new inhibitor of cholesteryl ester synthesis in
mammalian cells, produced by marine-derived Streptomyces sp. OPMA00072. J.
Antibiot. (Tokyo) 68, 126–132 (2015).
35 Iwatsuki, M. et al. Lariatins, novel anti-mycobacterial peptides with
structure, produced by Rhodococcus jostii K01-B0171. J. Antibiot. 60,
357–363 (2007).
a lasso
17 Kurihara, Y. et al. Entomopathogenic fungi isolated from suspended-soil-inhabiting
arthropods in East Kalimantan, Indonesia. Mycoscience 49, 241–249 (2008).
18 Neuss, N. et al. Aranotin and related metabolites from Arachniotus aureus (Eidam)
Schroeter. IV. Fermentation, isolation, structure elucidation, biosynthesis, and antiviral
properties. Antimicrob. Agents Chemother. 8, 213–219 (1968).
19 Wang, J. M. et al. Study on absolute configurations of α/α’ chiral carbons of
thiodiketopiperazines by experimental and calculated circular dichroism spectra.
Tetrahedron 69, 1195–1201 (2013).
36 Hagimori, K., Fukuda, T., Hasegawa, Y., Omura, S. & Tomoda, H. Fungal malformins
inhibit bleomycin-induced G2 checkpoint in Jurkat cells. Biol. Pharm. Bull. 30,
1379–1383 (2007).
37 Ohshiro, T. & Tomoda, H. Isoform-specific inhibitors of ACATs: recent advances and
promising developments. Future Med. Chem. 3, 2039–2061 (2011).
38 Fukuda, T. et al. Trichocyalides A and B, new inhibitors of alkaline phosphatase activity
in bone morphogenetic protein-stimulated myoblasts, produced by Trichoderma sp.
FKI-5513. J. Antibiot. (Tokyo) 65, 565–569 (2012).
The Journal of Antibiotics