1
08
Vol. 59, No. 1
1
3
ꢀ
2
ꢂR.
The anti-microbial activities of compounds 1—5 were
tested for the growth inhibition of 8 microbes with the paper
C-NMR data (see Table 1); (ꢀ)FAB-MS m/z: 213 [MꢀH] ; (ꢀ)HR-FAB-
ꢀ
MS m/z: 213.1244 [MꢀH] (Calcd for C H N O , 213.1239,
ꢀ
D
10 17
2
3
0.5 mmu).
Determination of Amino Acids Configuration A sample of 1 or 2
14)
disk method. The growth inhibition was studied in a con-
(
100 mg) was hydrolyzed in 6 N HCl (100 ml) at 110 °C for 12 h. After con-
centration of 125 mg/disk. As a result, compound 1 exhibited centration to dryness, the residue was dissolved in 25 ml of H O and 20 mg
2
a
of N -(5-fluoro-2,4-dinitrophenyl)-L-leucinamide (FDLA) in acetone (50 ml)
weak inhibition activity against Aspergillus niger, and com-
pounds 4 and 5 exhibited moderate inhibition activity against
Aspergillus niger. Compounds 2 and 3 did not show anti-mi-
crobial activity.
and 10 ml of 1 M NaHCO aq. were added. The mixture was heated at 37 °C
3
for 1 h. and 10 ml of 1 N HCl was added. The solution was diluted ten times
with CH CN followed by analyses by HPLC. The conditions for HPLC
3
analyses and the retention times for standard and hydrolysate FDLA deriva-
tives are provided in Table 2.
Experimental
Antibiotic Activity Assay Activities of the Compounds 1—5 were
tested by the paper disk method against Aspergillus niger, Penicillium crus-
tosum, Schizophyllum commune, Trichophyton concentricum, Saccha-
romyces cerevisae, Bacillus subtilis subsp. subtilis, Serratia marcescens
subsp. marcescens, Staphylococcus aureus subsp. aureus with 125 mg/disk.
As a result, compound 1 slightly showed an inhibition circle against As-
pergillus niger, and compounds 4 and 5 exhibited a weak inhibition circle
against Aspergillus niger.
General IR spectra were obtained with JASCO FT/IR-410 spectropho-
tometers. Optical rotations were measured with a JASCO DIP-370 digital
polarimeter. H- and C-NMR, H– H COSY, NOESY, HSQC and HMBC
spectra were recorded with a Unity plus 500 spectrometer (Varian Inc.,
U.S.A.) operating at 500 MHz for H, and 125 MHz for C, respectively.
FAB-MS were recorded on a JMS DX-303 spectrometer (JEOL Ltd., Japan),
and m-nitrobenzyl alcohol or Magic bullet used as a matrix. Preparative
HPLC was performed on a Develosil C-30-UG-5 (250ꢄ4.6 mm i.d., No-
mura Chemical Co., Aichi, Japan), at a flow rate of 1.0 ml/min, equipped
with a JASCO RID-300 detector and a JASCO BIP-I HPLC pump.
Bacterial Material and Fermentation The marine Bacillus sp. (strain
number p-0707-517) was isolated from the digestive tract of sea urchin, An-
thocidaris crassispina, collected in the Nagasaki Shitsu coast of Japan in
1
13
1
1
1
13
Acknowledgements We are grateful to Mr. M. Inada and Mr. N. Yama-
guchi of the Scientific Support Section of Joint Research Center, Nagasaki
University, for the H-NMR, C-NMR, and MR measurements.
1
13
References
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1
2
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shaking at 120 rpm for 21 d at 24 °C . The culture broth (30 l) was sonicated
followed by filtration.
2
4
4
4
)
)
Extraction and Isolation The filtered broth was extracted with EtOAc
(10 lꢄ3). EtOAc extract concentrated under reduced pressure to dryness.
5
Byun H. G., Zhang H., Mochizuki M., Adachi K., Shizuri Y., Lee W.
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The dried residue (15.2 g) was subjected to Sephadex LH-20 (CHCl3–
MeOHꢃ5 : 5) to yield 4 fractions (fractions 1—4). The third fraction was
chromatographed on silica gel using a n-hexane– acetone (from 8 : 2 to 5 : 5)
followed by CHCl –MeOH–H O (from 95 : 5 : 0 to 5 : 5 : 1) to yield 8 frac-
tions. Fraction 4 of the eight fractions (145.3 mg) was subjected to reversed
phase HPLC (5% MeOH–H O) to give 3 (t ꢃ21.2 min, 1.3 mg). Fraction 6
of the eight fractions (612.7 mg) was chromatographed on ODS using 20%
MeOH–H O as the eluent to give followed by a subject on a reversed phase
HPLC (40% MeOH–H O) to give the mixture of 1 and 2, 4 (t ꢃ4.1 min,
6
7
)
)
Rhee K. H., Int. J. Antimicrob. Agents, 24, 423—427 (2004).
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3
2
8
)
)
2
R
9
Arunrattiyakorn P., Nitoda T., Kanzaki H., Peptides, 27, 633—639
(2006).
2
2
R
10) Marfey P., Carlsberg Res. Commun., 49, 591—596 (1984).
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37.0 mg), and 5 (t ꢃ4.9 min, 9.9 mg). The mixture of 1 and 2 was subjected
R
to reversed phase HPLC (5% MeOH–H O) to give 1 (t ꢃ19.6 min, 1.3 mg)
and 2 (t ꢃ21.2 min, 4.0 mg).
2
R
R
3
0
Bacillusamide A (1): White amorphous powder; [a] ꢁ64.0° (cꢃ0.04,
D
ꢁ
1 1
Py); IR n
(dry film) 3470, 2962, 1701, 1630, 1447, 1133 cm , H- and
ꢀ
max
13
C-NMR data (see Table 1); (ꢀ)FAB-MS m/z: 212[MꢀH] , 197
1
3) Tsukamoto S., Umaoka H., Yoshikawa K., Ikeda T. Hirota H., J. Nat.
ꢀ
ꢀ
[MꢁCH ] ; (ꢀ)HR-FAB-MS m/z: 212.1420 [MꢀH]
(Calcd for
3
Prod., 73, 1438—1440 (2010).
C H N O , 212.1399, D ꢀ2.1 mmu).
1
0
18
3
2
14) Ericsson H., Hogman C., Wickman K., Scand. J. Clin. Lab. Invest., 6,
3
0
Basillusamide B (2): Light yellow amorphous powder; [a] ꢀ44.0°
D
23—36 (1954).
ꢁ1 1
(
cꢃ0.04, MeOH) ; IR nmax (dry film) 3224, 2974, 1644, 1442 cm ; H- and