MBJ-0110, a novel cyclopeptide isolated from the fungus Penicillium sp. f25267

Full text of DOI:10.1038/ja.2015.78

The Journal of Antibiotics (2015), 1–3
2015 Japan Antibiotics Research Association All rights reserved 0021-8820/15
www.nature.com/ja
&
NOTE
MBJ-0110, a novel cyclopeptide isolated from the
fungus Penicillium sp. f25267
1
2
1
1
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Teppei Kawahara , Masashi Itoh , Ikuko Kozone , Miho Izumikawa , Noriaki Sakata , Toshio Tsuchida and
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Kazuo Shin-ya
The Journal of Antibiotics advance online publication, 8 July 2015; doi:10.1038/ja.2015.78
We have constructed an isolated natural compound library designed to (Purif-Pack ODS-30, Shoko Scientific, Yokohama, Japan; 40–100%
1
facilitate extensive biological screenings. The library consists of over aq. MeOH with 10% stepwise increments in the MeOH concentra-
1000 isolates, including over 140 ‘JBIR compounds’ that were tion). The fractions were monitored using an ultra performance liquid
discovered in our laboratory. To enrich this library, we recently chromatography-diode array detection-evaporative light scattering-
initiated a screening program for rare microbial products using the mass spectrometry system and 1 was isolated based on peak-guided
advanced compound-identification system designated as ‘MBJ’s special fractionation. The 50% MeOH eluate (30.9 mg) was subjected to
2
,3
selection’. As a result, our program yielded novel compounds preparative reversed-phase HPLC using a Capcell Pak C18 MG II
named ‘MBJ compounds’, such as
cytotoxic hydroxamate column (20 mm inside diameter (i.d.)× 150 mm; Shiseido, Tokyo,
a
4
MBJ-0003 from Micromonospora sp. 29867; cytotoxic eremophilane Japan) with a solvent system of 20% CH CN/H O containing 0.1%
3
2
derivatives MBJ-0009 and MBJ-0010 from Nectria sp. f26111;5 formic acid (flow rate: 10 ml min ), to yield semi-purified 1 (6.9 mg,
–1
MBJ-0011, MBJ-0012 and MBJ-0013 from Apiognomonia retention time (Rt)= 15.3 min). Final purification was carried out by
2
sp. f24023; cytotoxic chaetoglobosin derivatives MBJ-0038, MBJ-0039 preparative HPLC using an X-Bridge C18 column (19 mm i.d. × 150-
3
and MBJ-0040 from Chaetomium sp. f24230; bicyclic depsipeptides
mm; Waters, Milford, MA, USA) with a solvent system of 20%
MBJ-0086 and MBJ-0087 from Sphaerisporangium sp. 33226;6 and CH CN/H O containing 0.1% formic acid (flow rate: 10 ml min ) to
–1
3
2
aziridine-containing peptide MBJ-0035 from Streptosporangium afford 1 (3.5 mg, Rt= 13.3 min).
7
sp. 32552. Further screening for novel compounds led to the
MBJ-0110 (1) was obtained as a colorless amorphous powder:
identification of MBJ-0110 (1) from the culture of Penicillium [α]24 –186 (MeOH; c 0.18); UV end; IR (attenuated total reflectance)
sp. f25267. Herein we report the fermentation, isolation, structure νmax: 3400 (hydroxy) and 1683 (carbonyl) cm . The molecular
elucidation and preliminary biological activity data.
formula of 1 was established as C H N O by high-resolution
Penicillium sp. f25267 was isolated from a soil sample collected in (HR)-ESI–MS (m/z 564.3018 [M+H] , calcd for C H N O m/z
D
−
1
2
7 41 5 8
+
2
7 42 5 8
the Shiga Prefecture, Japan. The strain was cultured in 250-ml 564.3033). Its peptidic nature was evident from the resonances
Erlenmeyer flasks, each containing 25 ml of a seed medium consisting corresponding to α-methine protons (δH 4.00–5.08) and the reso-
of 2% potato starch (Tobu Tokachi Nosan Kako Agricultural nances corresponding to the carbonyl carbons (δ 169.0–175.9) in the
C
1
13
Cooperative Assoc., Hokkaido, Japan), 1% glucose (Junsei Chemical,
H and C NMR spectra of 1, respectively. The direct connectivity
Tokyo, Japan), 2% soybean powder (SoyPro, J-Oil Mills, Tokyo, between protons and carbons was established by a HSQC spectrum;
1
3
1
Japan), 0.1% KH PO and 0.05% MgSO ·7H O (pH 7.4 before Table 1 summarizes the C and H NMR spectroscopic data for 1.
sterilization). The flasks were incubated on
(
2
4
4
2
1
1
13
a
rotary shaker The H sequences and H− C long-range couplings from α-methine
220 r.p.m.) at 25 °C for 3 days. Aliquots (0.5 ml) of the broth were protons to the corresponding amide carbonyl carbons, which were
transferred to 500-ml Erlenmeyer flasks containing 50 ml of a elucidated by double quantum-filtered COSY and constant time-
production medium of the same composition, which were then HMBC8 spectra, respectively, revealed the involvement of an
cultured on a rotary shaker (220 r.p.m.) at 25 °C for 4 days.
isoleucine (Ile), a pipecolic acid (Pip), a proline (Pro) and an aspartic
The whole culture broth (2 l) was extracted with an equal volume of acid (Asp) residue, as shown in Figure 1b. In addition to the above-
n-BuOH. After concentration in vacuo, the extract was successively mentioned amino-acid moieties, the presence of a 4-hydroxypipecolic
1
partitioned between EtOAc (350 ml × 3) and H O (300 ml). The acid (C-22 to C-27) moiety was proved based on a H sequence from
2
aqueous layer was evaporated to dryness and the residue (1.4 g) was an α-methine proton H-23 (δH 4.00) to nitrogen-bearing methylene
fractionated by reversed-phase medium-pressure liquid chromatography protons H -27 (δH 3.39 and 2.98) via aliphatic methylene protons
2
1
2
3
Japan Biological Informatics Consortium (JBIC), Koto-ku, Tokyo, Japan; Bioresource Laboratories, MicroBiopharm Japan Co., Ltd. (MBJ), Iwata, Shizuoka, Japan and National
Institute of Advanced Industrial Science and Technology (AIST), Koto-ku, Tokyo, Japan
Correspondence: Dr K Shin-ya, National Institute of Advanced Industrial Science and Technology (AIST), 2-4-7 Aomi, Koto-ku, Tokyo 135-0064, Japan.
E-mail: k-shinya@aist.go.jp
Received 7 April 2015; revised 8 June 2015; accepted 15 June 2015

New cyclopeptide from Penicillium
T Kawahara et al
2
Table 1 13C and H NMR spectroscopic data for 1
1
H -24 (δH 2.62 and 2.07), an oxymethine proton H-25 (δ 5.17,
2
H
1
13
δC 68.9) and aliphatic protons H -26 (δH 2.19, 2.02), and H– C
2
Position
δC
δH, multiplicity (J in Hz)
long-range couplings from H-23 and H-24 (δH 2.07) to an amide
carbonyl carbon C-22 (δC 169.0), and from H -27 to an α-methine
1
2
3
4
5
6
7
8
9
169.7
59.2
39.7
26.7
11.4
15.9
173.0
54.8
25.0
20.9
25.5
45.0
175.9
59.7
30.0
26.3
50.2
171.3
37.0
51.7
175.7
169.0
55.5
32.8
68.9
26.8
40.6
2
carbon C-23 (δ 55.5).
4.36, d (7.8)
1.68, ovla
C
The amino-acid sequence in 1 was determined by the HMBC
correlations from an α-methine proton H-2 (δH 4.36) to a carbonyl
carbon C-7 (δ 173.0), from an α-methine proton H-8 (δ 5.08) and
1.52, m; 1.19, m
0.95, t (7.2)
C
H
0.93, d (6.6)
ε-methylene protons H -12 (δH 3.97 and 3.00) to a carbonyl carbon
2
C-13 (δC 175.9), from an α-methine proton H-14 (δH 4.95) to a
carbonyl carbon C-18 (δC 171.3), from an α-methine proton H-20
5.08, br s
a
a
a
a
2.20, ovl ; 1.62, ovl
(
δH 4.82) to C-22 and from H-25 to an ester carbonyl carbon C-1
a
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
1.78, ovl ; 1.70, ovl
1
13
(δ 169.7). Although only H– C HMBC information suggested two
C
a
1.73, ovl ; 1.73, ovl
structural possibilities, α- and β-aspartyl amide linkages, we concluded
that the aspartyl acid moiety is linked to adjacent Pro by β-amino
3.97, br d (15.6); 3.00, ovla
bond because of the existence of a ROESY correlation between H -17
4.95, dd (4.8, 8.4)
2.47, m; 1.80, ovla
b
1
15
(δH 3.70) and H -19 (δH 3.10) and H– N HMBC correlations from
a
a
a
a
H -16 (δ 2.08, 1.96), H -15 (δH 1.80) and H -19 (δ 2.96) to a
2.08, ovl ; 1.96, ovl
2
H
b
b
H
a
3.76, ovl ; 3.70, ovl
nitrogen atom of Pro (δN 140). Therefore, the structure of 1 was
determined as shown in Figure 1b.
3.10, dd (7.2, 12.0); 2.96, ovla
4.82, m
To verify the proposed structure, 1 was treated with 0.1 N NaOH
overnight at room temperature, followed by ESI–MS/MS analysis
of the alkaline hydrolysate (molecular formula: C H N O ;
2
7 43 5 9
+
HR-ESI–MS: [M+H] m/z 582.3159, C H N O 582.3139). The
2
7 44 5 9
4.00, d (7.2)
2.62, br d (13.8); 2.07, ovla
ESI–MS/MS data showed major fragment ions (m/z 185.0945,
43.0976, 324.1554, 340.1477 and 451.2165) that supported the
proposed structure (Figure 1c).
2
5.17, br s
a
a
2.19, ovl ; 2.02, ovl
1
3.39, br d (9.6); 2.98, ovla
The multiplicity and a large H spin coupling constant value of
H-23 (doublet, JH–H = 7.2 Hz) and ROESY correlations between
aOverlapped with other signals. NMR spectra were taken on a 600 NB CL NMR system (Varian,
Palo Alto, CA, USA) in CD3OD with the residual solvent peak as an internal standard (δC 49.0,
δH 3.31 p.p.m.)
.
H-23/H -27 (δH 2.98) and H-23/H -24 (δH 2.62) implied that the
ax
eq
piperidine ring is in the chair conformation and the H-23 is axially
orientated. In addition, the broad singlet signal of H-25 proved its
Figure 1 (a) Structure of 1. (b) NMR analysis of 1. COSY, bold line; 1H–13C HMBC, solid arrow; 1H–15N HMBC, dashed arrow; ROESY, bidirectional dashed
arrow. (c) ESI–MS/MS fragmentation ions of 1.
The Journal of Antibiotics

New cyclopeptide from Penicillium
T Kawahara et al
3
equatorial orientation. Taken together, the relative configurations of mesothelioma ACC-MESO-1 cell lines (IC 4100 μM), nor
5
0
C-23 and C-25 were determined as 23S* and 25S*, respectively.
antimicrobial activity against Micrococcus luteus and Bacillus subtilis.
The absolute configurations of the amino-acid residues were
The obtained structure of 1 is very rare in nature; only
determined to be L-Pro, L-Pip and L-Asp by using Marfey’s method.9 petrosifungins A and B, and JBIR-113, -114 and -115 have been
A portion of 1 (0.4 mg) was hydrolyzed in 6 N HCl at 110 °C for 12 h isolated as pipecolic acid-containing peptides of fungal origin. To the
and then dried under air flow. The resulting hydrolysate was treated best of our knowledge, there are no reports in the literature of peptide
11
12
with 0.1 M NaHCO (200 μl) and 1% N-(5-fluoro-2,4-dinitrophenyl)- compounds possessing the 4-hydroxypipecolic acid moiety.
3
L-alaninamide (L-FDAA) in Me CO (100 μl) at 40 °C for 30 min.
2
Amino-acid standards were derivatized with L-FDAA in a similar CONFLICT OF INTEREST
The authors declare no conflict of interest.
manner. The Marfey’s derivatives were analyzed using a HPLC–MS
system as follows: a Capcell Pak C18 MG II column (4.6 mm
i.d.× 150 mm) was developed with a linear gradient system of water/
ACKNOWLEDGEMENTS
This work was supported in part by a grant ‘Project focused on developing key
technologies for discovering and manufacturing drugs for next-generation
treatment and diagnosis’ from the Ministry of Economy, Trade and Industry
MeCN with 0.1% formic acid (20–50% MeCN, 15 min; flow rate,
–1
1
3
.0 ml min ). FDAA derivatives were detected by absorption at
40 nm, and assignment was secured by ion-selective monitoring.
(
METI).
The retention times of the standard FDAA derivatives were as follows:
L-Asp, 7.9 min; D-Asp, 8.2 min; L-Pip, 13.6 min; D-Pip, 12.8 min; L-Pro,
1
1
0.1 min; D-Pro, 10.7 min; L-Ile, 14.7 min; D-Ile, 16.8 min; L-allo-Ile,
4.7 min; and D-allo-Ile, 16.7 min. The retention times of the FDAA
1
2
Kawahara, T., Nagai, A., Takagi, M. & Shin-ya, K. JBIR-137 and JBIR-138, new
secondary metabolites from Aspergillus sp. fA75. J. Antibiot. 65, 535–538 (2012).
Kawahara, T. et al. Three eremophilane derivatives, MBJ-0011, MBJ-0012 and
MBJ-0013, from an endophytic fungus Apiognomonia sp. f24023. J. Antibiot. 66,
299–302 (2013).
derivatives of 1 were as follows: Asp, 7.9 min; Pip, 13.6 min; Pro,
10.1 min; and Ile, 14.7 min.
The absolute configuration of the Ile residue in 1 was established by
3
Kawahara, T. et al. New chaetoglobosin derivatives, MBJ-0038, MBJ-0039 and
MBJ-0040, isolated from the fungus Chaetomium sp. f24230. J. Antibiot. 66,
HPLC comparison of the 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl
isothiocyanate (GITC) derivative of hydrolysate of 1 with standard
7
27–730 (2013).
1
0
samples. Triethylamine (50 μl) and a GITC solution (250 μl,
4
5
6
7
8
Kawahara, T. et al. New hydroxamate metabolite, MBJ-0003, from Micromonospora
sp. 29867. J. Antibiot. 67, 261–263 (2014).
Kawahara, T. et al. Cytotoxic sesquiterpenoids MBJ-0009 and MBJ-0010 from a
saprobic fungus Nectria sp. f26111. J. Antibiot. 66, 567–569 (2013).
Kawahara, T. et al. MBJ-0086 and MBJ-0087, new bicyclic depsipeptides from
Sphaerisporangium sp. 33226. J. Antibiot. 68, 67–70 (2015).
Kawahara, T. et al. MBJ-0034 and MBJ-0035, new aziridine-containing peptides from
Streptosporangium sp. 32552. J. Antibiot. 67, 577–580 (2014).
Furihata, K. & Seto, H. Constant time HMBC (CT-HMBC), a new HMBC technique
useful for improving separation of cross peaks. Tetrahedron Lett. 39, 7337–7340
(1998).
–1
prepared at 3.9 mg ml in CH CN) were added to the acid hydro-
3
lysate of 1 or an authentic amino-acid standard. The reaction mixture
was kept at room temperature for 30 min and the reaction was then
quenched by adding 40 μl of MeCN–5% AcOH in H O (1:1). Analysis
2
of the GITC derivatives was performed on a Capcell Pak ADME
column (4.6 mm i.d. × 150 mm; Shiseido) employing an isocratic
–1
elution of 40% CH CN containing 0.1% formic acid (1.0 ml min ).
3
9
1
Marfey, P. Determination of D-amino acids. II. Use of
a
bifunctional reagent,
GITC derivatives were detected by absorption at 248 nm, and assigned
by ion-selective monitoring. The retention times of the GITC
derivatives were as follows: L-Ile, 11.6 min and L-allo-Ile, 11.3 min.
The retention time (11.6 min) of the GITC derivative of 1 implied that
the Ile residue in 1 is L-Ile.
1
,5- difluoro-2,4-dinitrobenzene. Carlsberg Res. Commun. 49, 591–596 (1984).
0 Nimura, N., Ogura, H.
& Kinoshita, T. Reversed-phase liquid chromatographic
resolution of amino acid enantiomers by derivatization with 2,3,4,5-tetra-O-acetyl-β-D-
glucopyranosyl isothiocyanate. J. Chromatogr. 202, 375–379 (1980).
1 Bringmann, G., Lang, G., Steffens, S. & Schaumann, K. Petrosifungins A and B, novel
cyclodepsipeptides from a sponge-derived strain of Penicillium brevicompactum. J. Nat.
Prod. 67, 311–315 (2004).
2 Kawahara, T., Takagi, M. & Shin-ya, K. Three new depsipeptides, JBIR-113, JBIR-114
and JBIR-115, isolated from a marine sponge-derived Penicillium sp. fS36. J. Antibiot.
65, 147–150 (2012).
1
1
We evaluated the cytotoxic and antimicrobial activities of 1, but it
showed neither cytotoxicity to human ovarian adenocarcinoma
SKOV-3 cell lines (IC 4100 μM) or human malignant pleural
5
0
The Journal of Antibiotics