JBIR-69, a new metabolite from Streptomyces sp. OG05

Full text of DOI:10.1038/ja.2009.132

The Journal of Antibiotics (2010) 63, 95–96
2010 Japan Antibiotics Research Association All rights reserved 0021-8820/10 $32.00
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&
NOTE
JBIR-69, a new metabolite from Streptomyces sp.
OG05
Takeshi Fujiwara1, Aya Nagai1, Motoki Takagi1 and Kazuo Shin-ya2
The Journal of Antibiotics (2010) 63, 95–96; doi:10.1038/ja.2009.132; published online 15 January 2010
Keywords: cytotoxicity; HL-60; Streptomyces; thioester
Actinobacteria have been extensively studied thus far, and they are to yield JBIR-69 (1, 15mg) together with 3-isopropylmalate methyl
known to produce pharmaceutically important compounds.1,2 Soil ester (6 mg).3
serves as the primary source of actinobacteria and, therefore, we
Compound 1 was obtained as a colorless oil ([a]D ꢁ11.8, 291C,
attempted to obtain novel metabolites from actinobacteria isolated c 0.28 (CHCl3), UV (MeOH) lmax (e) 233 (15 800)). The infrared (IR)
from soil samples. During our screening program for novel meta- spectrum (KBr) of 1 revealed the characteristic absorptions of esters
bolites, a new metabolite possessing a thioester named JBIR-69 (1, (vmax 1730cmꢁ1), amide (vmax 1670cmꢁ1), hydroxyl, and/or amide
Figure 1a), together with a known compound, 3-isopropylmalate NH (vmax 3400cmꢁ1) groups. Its molecular formula was established as
methyl ester, was isolated from the culture broth of Streptomyces sp. C13H21NO7S (m/z [M-H]ꢁ 334.0960, +0.2 mmu) by high-resolution
OG05. This paper describes the fermentation, isolation, structural electrospray ionization-mass spectrum.
elucidation, and, in brief, the biological activity of 1. The structure of 1 was mainly elucidated by the analyses of NMR
Streptomyces sp. OG05 was isolated from a soil sample collected in spectra, including heteronuclear single-quantum coherence, double-
Okinawa Prefecture, Japan, and was cultured on a rotary shaker quantum-filtered correlation (DQF-COSY), and constant-time hetero-
1
(180 r.p.m.) at 27 1C for 5 days in 500-ml Erlenmeyer flasks containing nuclear multiple-bond correlation (HMBC).4 The H and 13C NMR
100 ml of 2% glycerol (Nacalai Tesque, Kyoto, Japan), 1% molasses spectral data revealed by the heteronuclear multiple quantum coherence
(Dai-Nippon Meiji Sugar, Tokyo, Japan), 0.5% casein (Kanto Chemi- spectrum for 1 are listed in Table 1. The DQF-COSY and HMBC
cal, Tokyo, Japan), 0.1% polypeptone (Nihon Pharmaceutical, Tokyo, analyses revealed the two partial structures (Figure 1b). The sequence
Japan), and 0.4% CaCO3 (Kozaki Pharmaceutical, Tokyo, Japan; pH from an amide proton 2-NH (dH 6.68) to methylene protons 3-H (dH
7.2 before sterilization).
3.67 and 3.22) through an a-methine proton 2-H (dH 4.91, dC 52.2) was
The mycelium collected from the culture broth (1l) by centrifuga- observed in the DQF-COSYof 1. In the HMBC spectrum, 1H–13C long-
tion was extracted with Me2CO (200ml). After concentration in range couplings from the a-methine proton 2-H and the methylene
vacuo, the residue aqueous concentrate with pH adjusted to 2–3 protons 3-H to a carbonyl carbon C-1 (dC 171.6), and from the amide
using 2 N HCl was extracted twice with EtOAc. The organic layer was proton 2-NH and a singlet methyl proton 10-H (dH 2.12) to an amide
dried over anhydrous Na2SO4 and concentrated in vacuo. The dried carbonyl carbon C-9 (dC 172.4) were observed. Thus, the carbonyl and
residue (180mg) was subjected to normal-phase medium-pressure acetyl moieties were elucidated to be substituted at the C-2 and 2-N
liquid chromatography (Purif-Pack SI-60, size: 20, Moritex, Tokyo, positions, respectively. In a similar manner, the sequence from an
Japan) with a Hexane-EtOAc (0–30% EtOAc) linear gradient system, oxymethine proton 6-H (dH 4.46) to a methyl proton 12-H (dH 0.96)
followed by elution with a CHCl3–MeOH (0–90% MeOH) linear through methine protons 5-H (dH 2.64) and 11-H (dH 2.30), which in
gradient system; then, peak detection was carried out with UV turn spin coupled to a methyl proton 13-H (dH 1.06), was established.
absorption at 254 nm. The 10–20% MeOH elute fractions (30 mg) 1H–13C long-range couplings from 5-H and 11-H to a carbonyl carbon
were further purified by reversed-phase high-pressure liquid chromato- C-4 (dC 200.3) and from 5-H and 6-H to an ester carbonyl carbon C-7
graphy (HPLC) using a PEGASIL ODS column (Senshu Pak, 20 (dC 174.7) were recognized in the HMBC spectrum of 1. A methoxyl
i.d.ꢀ150 mm, Senshu Scientific, Tokyo, Japan) with a H2O–MeOH proton 8-H (dH 3.81) was long-range coupled to the ester carbonyl
(0–100% MeOH) linear gradient system containing 0.1% formic acid carbon C-7, which established the substitution position of the methoxyl
1Biomedicinal Information Research Center (BIRC), Japan Biological Informatics Consortium (JBIC), Koto-ku, Tokyo, Japan and 2Biomedicinal Information Research Center
(BIRC), National Institute of Advanced Industrial Science and Technology (AIST), Koto-ku, Tokyo, Japan
Correspondence: Dr M Takagi, Biomedicinal Information Research Centre (BIRC), Japan Biological Informatics Consortium (JBIC), 2-4-7 Aomi, Koto-ku, Tokyo 135-0064, Japan
or Dr K Shin-ya, Biomedicinal Information Research Center (BIRC), National Institute of Advanced Industrial Science and Technology (AIST), 2-4-7 Aomi, Koto-ku, Tokyo
135-0064, Japan.
E-mails: motoki-takagi@aist.go.jp or k-shinya@aist.go.jp
Received 7 November 2009; revised 9 December 2009; accepted 14 December 2009; published online 15 January 2010

JBIR-69 from Streptomyces sp. OG05
T Fujiwara et al
96
O
OMTPA
13
12
9
5 : + 0.044
11: + 0.201
12: + 0.105
13: + 0.096
11
5
8
7
6
5
11
12,13
10
O
CHCH(CH3)2
NH
MeOOC
C
8: - 0.010
8
R
6
4
HO
3
S
1
2
7
H
O
O
O
OH
7
CO
4
CO
2J5H-6C < 2 Hz
O
O
11
H
H
13
12
O
9
11
5
3J6H-4C = 8.8 Hz
3J6H-11C < 2Hz
4
6R
10
S
NH
O
6
7
O
5S
O
: DQF-COSY
8
S
O
OH
C
11
7
O
2
1
: HMBC
3
6
4
O
Figure 2 (a) Absolute configuration at C-6 revealed by modified Mosher’s
method. (b) Absolute configuration at C-5 established by J-based analysis.
Figure 1 (a) Structure of 1. (b) Key correlations in the DQF-COSY (bold line)
and HMBC (arrow) spectra of 1.
values obtained by subtracting the (R)-MTPA ester values from
(S)-MTPA ester values (dD¼ d(S)-MTPAꢁd(R)-MTPA) are summar-
ized in Figure 2a. From these values, the absolute configuration at C-6
was concluded to be 6R. A small coupling constant (o2 Hz) between
5-H and C-6 revealed that the oxygen atom and 5-H were in an
anti relationship. A large coupling constant between 6-H and
C-4 (3JH6ꢁC4 ¼ 8.8 Hz) and a small one between 6-H and C-11
(3JH6ꢁC11o2 Hz) indicated that they were in anti and gauche loca-
tions, respectively, as shown in Figure 2b. These results revealed the
absolute configuration at C-5 to be 5S. Thus, the absolute structure of
1 was established, as shown in Figure 1a.
The cytotoxic activity of 1 against human acute myelogenous
leukemia HL-60 cells was tested by the WST-8 [2-(2-methoxy-4-
nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium,
monosodium salt] colorimetric assay (Cell Counting Kit, Dojindo,
Kumamoto, Japan). It was found that 1 exhibited a weak cytotoxic
effect against HL-60 cells for 48h with an IC50 value of 210 mM. We
also attempted to investigate the antimicrobial activitiy of 1. However,
1 did not exhibit antimicrobial activity against Micrococcus luteus,
Escherichia coli, and Schizosaccharomyces pombe.
Table 1 1H and 13C NMR spectral data for 1 and 3-isopropylmalate
methyl ester
1
3-Isopropylmalate methyl ester
No.
13C
1H (J in Hz)
13C
1H (J in Hz)
1
2
3
171.6
52.2
29.7
4.91 (1H, m)
3.22 (1H, dd, 14.5, 4.4)
3.67 (1H, dd, 14.5, 3.9)
4
5
200.3
63.9
70.1
174.7
52.9
172.4
22.5
27.5
20.9
19.9
177.7
55.8
2.64 (1H, dd, 10.1, 2.9)
4.46 (1H, d, 3.1)
2.67 (1H, dd, 9.9, 2.8)
4.50 (1H, d, 2.9)
6
69.7
7
174.0
52.2
8
3.81 (3H, s)
3.82 (3H, s)
9
10
11
12
13
NH
2.12 (3H, s)
2.30 (1H, m)
26.9
20.9
19.8
2.27 (1H, m)
0.96 (3H, d, 6.6)
1.06 (3H, d, 6.6)
6.68 (1H, d, 6.6)
1.06 (3H, d, 6.4)
1.03 (3H, d, 6.4)
ACKNOWLEDGEMENTS
This work was supported in part by the New Energy and Industrial Technology
Development Organization of Japan (NEDO) and a Grant-in-Aid for Scientific
Research (20380070 to KS) from The Japan Society for the Promotion of
Science (JSPS).
13C (125MHz) and 1H (500 MHz) NMR spectra were obtained using an NMR System 500 NB
CL (Varian, Palo Alto, CA, USA) in CDCl3, and the solvent peak was used as an internal
standard (dC 77.0, dH 7.24).
functional group. By taking into consideration the molecular formula of
1 and the long-range coupling from 3-H to C-4, together with the 13
C
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The Journal of Antibiotics