Bacilosarcins A and B, novel bioactive isocoumarins with unusual heterocyclic cores from the marine-derived bacterium Bacillus subtilis

Article abstract of DOI:10.1016/j.tet.2008.04.076

Two novel isocoumarins, bacilosarcins A (1) and B (2) were isolated from a culture broth of the marine-derived bacterium Bacillus subtilis TP-B0611. The structures and absolute configurations of 1 and 2 were determined on the basis of spectroscopic analyses and chemical conversions. Compound 1 possesses an unprecedented 3-oxa-6,9-diazabicyclo[3.3.1]nonane ring system while 2 has a 2-hydroxymorpholine moiety that is rare in nature. These compounds showed growth inhibition against barnyard millet.

Full text of DOI:10.1016/j.tet.2008.04.076

Tetrahedron 64 (2008) 6420–6425
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Bacilosarcins A and B, novel bioactive isocoumarins with unusual heterocyclic
q
cores from the marine-derived bacterium Bacillus subtilis
a
a
b
c
d
Miwa Azumi , Ken-ichi Ogawa , Tsuyoshi Fujita , Michinori Takeshita , Ryuji Yoshida ,
a
a,
*
Tamotsu Furumai , Yasuhiro Igarashi
a
Biotechnology Research Center, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan
Suntory Institute for Bioorganic Research, 1-1-1 Wakayamadai, Shimamoto, Mishima, Osaka 618-8503, Japan
Department of Chemistry and Applied Chemistry, Faculty of Science and Engineering, Saga University, Honjo-1, Saga 840-8502, Japan
College of Technology, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan
b
c
d
a r t i c l e i n f o
a b s t r a c t
Article history:
Two novel isocoumarins, bacilosarcins A (1) and B (2) were isolated from a culture broth of the marine-
derived bacterium Bacillus subtilis TP-B0611. The structures and absolute configurations of 1 and 2 were
determined on the basis of spectroscopic analyses and chemical conversions. Compound 1 possesses an
unprecedented 3-oxa-6,9-diazabicyclo[3.3.1]nonane ring system while 2 has a 2-hydroxymorpholine
moiety that is rare in nature. These compounds showed growth inhibition against barnyard millet.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 6 February 2008
Received in revised form 18 April 2008
Accepted 19 April 2008
Available online 24 April 2008
Keywords:
Bacilosarcin
Bacillus subtilis
Amicoumacin
1. Introduction
exhibit antibacterial,5,6b cytotoxic,9,10 and antiulcer6a,7 activities.
The structures of 1 and 2 were assigned on the basis of NMR analysis
There is substantial interest in discovering new lead molecules
from natural products for agricultural application including plant
and chemical conversions. Compound 1 possesses an un-
precedented 3-oxa-6,9-diazabicyclo[3.3.1]nonane ring system
whereas 2 has a 2-hydroxymorpholine substructure that is rare in
nature. The new compounds showed plant growth inhibitory
activity against barnyard millet. We herein describe the isolation,
structural elucidation, absolute stereochemical assignment, and
biological activities of 1 and 2 (Fig. 1).
growth regulators. We previously isolated an
that inhibits spore germination of plant pathogenic fungi,
a-pyrone derivative
2
a
spirocyclic polyketide with auxin-like plant growth promoting
activity,3 and an indole derivative with antifungal activity from
actinomycetes. As part of our continuing studies in this field, the
marine-derived bacterium Bacillus subtilis TP-B0611 was found to
produce two new isocoumarins, bacilosarcins A (1) and B (2) as well
asthreepreviouslyreportedisocoumarins,amicoumacinsA, B, andC.
Isocoumarin-type metabolites from microorganisms are
characterized by the amino-containing substituent, which is pre-
sumably derived from leucine, at 3-position in the dihydrocoumarin
core. There have been several such isocoumarin compounds as
4
2. Results and discussion
During the course of screening marine-derived bacteria for
bioactive compounds, we assessed the strain B. subtilis TP-B0611
from intestine content of a sardine (Sardinops melanosticta) col-
lected in Toyama Bay, Japan. Upon large-scale liquid fermentation,
compounds 1 and 2 were isolated from the culture extract using
plant growth inhibition bioassay to detect bioactivity and to direct
the fractionation.
5
6
7
8
baciphelacin, amicoumacins, xenocoumacins, Y-05460 M-A,
PM-94128,9 and Sg17-1-4 reported to date. These compounds
possess the common chromophore, 3,4-dihydro-8-hydroxy-
isocoumarin in their structures and many of them are produced by
the genus Bacillus. These isocoumarins have been reported to
10
Compound 1 was obtained as a colorless powder. High-resolu-
þ
tion EIMS revealed an [M] at m/z 491.2272, corresponding to the
1
13
molecular formula C24
33 3 8
H N O (calcd 491.2267). H and C NMR
spectral data (Table 1) were in full agreement with the assigned
13
q
molecular formula. The C NMR and DEPT spectra of 1 displayed
signals for 24 carbons including 8 aliphatic carbons, 7 carbons
bonded to nitrogen and/or oxygen, 6 carbons in the olefinic region,
See Ref. 1.
*
Corresponding author. Tel.: þ81766567500; fax: þ81766562498.
E-mail address: yas@pu-toyama.ac.jp (Y. Igarashi).
0
040-4020/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2008.04.076

M. Azumi et al. / Tetrahedron 64 (2008) 6420–6425
6421
1
'
16'
15'
moiety, which contained two isolated spin systems. One spin sys-
tem consisting of the three aromatic protons H-5, H-6, and H-7
displayed the coupling patterns for 1,2,3-trisubstituted benzenoid
ring system. The HMBC correlations from H-5 and H-7 to C-9 and
from H-6 to C-8 and C-10, and from 8-OH to C-7 and C-8 established
the hydroxyl group at C-8 and substitutions at C-9 and C-10. The
second spin system was elucidated starting from the two secondary
HO
O
13'
3
'
1
4'
2
4
'
4'
O
7'
5
6'
NH
1
0
3
8'
NH
6
7
5'
9'
N
10'
H
12' O
11'
O
OH
9
2
8
1
OH
O
1
0
0
methyl groups (H-1 and H-2 ) that showed HMBC correlations to
0
0
0
HO
O
one another, to C-3 and to C-4 . The proton signal attached to C-4
0
O
showed a COSY correlation to a methine proton H-5 , which in turn
H
showed COSY correlations to H-3 and an XH proton (d 6.98). The
methine proton H-3 showed COSY correlation to H-4. This XH
NH
N
H
O
OH
proton was considered an NH proton on the basis of the carbon
CONH2
0
chemical shift of C-5 (
d
C
49.6) and an HMBC correlation from the
2
OH
O
0
XH proton to C-7 . The HMBC correlations from H-4 to the aromatic
carbons C-5, C-9, and C-10 established the connectivity between C-
Figure 1. Structures of bacilosarcins A (1) and B (2).
4
and C-10. The deshielding characters of H-3 and C-3 nuclei were
and 3 carbonyl carbons. These data also indicated that 1 contained
indicative of the ester linkage between C-1 and C-3 to complete the
isocoumarin-type substructure A.
10 double bond equivalents, which on the basis of the carbon
chemical shifts were assigned to 3 carbonyls, 3 C]C double bonds,
Substructure B was constructed starting from the methylene
0 0
proton (H-11 ) that showed a COSY correlation to H-10 and HMBC
and 4 rings. The IR spectrum was consistent with the presence of
ꢀ1
ꢀ1
0 0
correlations to C-9 and C-12 . This fragment was expanded by COSY
hydroxyl (3265 cm ) and carbonyl functional groups (1655 cm ).
0 0 0 0
correlations from H-10 to H-9 and from H-9 to H-8 , and a TOCSY
The UV spectrum was nearly identical to that reported for ami-
6
0
0
0
coumacins (
l
max 206, 247, 314 nm), suggesting that 1 possessed
correlation from H-8 to 8 -OH. The carbon chemical shifts of C-9
0
a chromophore similar to dihydroisocoumarin.
(d
C
72.5) and C-10 (
d
C
47.9) were indicative of bonding of oxygen
Several substructures were assigned by analyses of the H, 13C,
1
and nitrogen to these carbons, respectively. Finally, HMBC corre-
1
1
0
0
0
0
0
H– H COSY, TOCSY, NOESY, HMQC, and HMBC NMR spectral data
lations from H-8 and H-9 to C-7 and 12 -NH to C-11 established
recorded in CDCl
3
(Fig. 2). The first part was the isocoumarin
this as a functionalized adipate B. Substructures A and B were
Table 1
1
H and 13C NMR data for bacilosarcins A (1) and B (2)
Position
Bacilosarcin A (1)
(mult, J in Hz)a
Bacilosarcin B (2)
b
(mult, J in Hz)a
b
HMBCc
d
H
d
C
HMBC
d
H
d
C
1
3
4
169.7
81.2
30.5
170.0
81.3
29.4
0
4.59 (ddd, 13.2, 2.7, 2.5)
3.09 (dd, 16.4, 13.2)
4.54 (ddd, 12.9, 2.5, 2.4)
3.21 (dd, 16.4, 13.2)
2.85 (dd, 16.4, 2.4)
6.50 (d, 7.3)
7.37 (dd, 8.6, 7.3)
6.97 (d, 8.6)
5
3, 10
3, 10
2.84 (dd, 16.4, 2.7)
5, 9, 10
4, 7, 9
8, 10
5, 9, 10
4, 7, 9
8, 10
5
6
7
8
9
6.71 (d, 7.3)
7.42 (dd, 8.3, 7.3)
6.89 (d, 8.3)
118.2
136.6
116.3
162.1
108.1
139.4
21.8
23.1
24.8
41.0
118.6
136.5
116.0
162.3
108.9
140.6
21.6
23.3
24.8
39.0
5, 8, 9
5, 8, 9
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
2
3
4
0.96 (d, 6.4)
0.97 (d, 6.3)
1.66 (m)
1.76 (ddd, 13.6, 10.2, 5.1)
2 3 4
0.83 (d, 6.6)
0.80 (d, 6.6)
1.72 (m)
1.85 (ddd, 13.7, 11.5, 4.4)
1.38 (ddd, 13.7, 9.8, 3.9)
4.64 (m)
2 , 3 , 4
0
0
1 , 3 , 4
1 , 3 , 4
0
0
0
0
5
1 , 3 , 5
0
1.44 (ddd, 13.6, 9.0, 4.6)
3
0
0
0
0
0
0
5
7
8
9
4.39 (dddd, 10.2, 10.0, 4.6, 2.5)
49.6
172.3
68.4
72.5
47.9
49.1
172.9
71.5
69.5
51.3
4 , 7
0
0
0
0
0
0
0
0
0
0
0
3.94 (d, 9.8)
7 , 9 , 10
4.90 (d, 8.6)
5.47 (dd, 8.6, 2.4)
4.74 (m)
3.57 (dd, 16.1, 8.0)
3.44 (dd, 16.1, 5.4)
7 , 9 , 10
0
0
4.10 (dd, 9.8, 2.4)
3.50 (dd, 6.6, 2.4)
2.62 (d, 18.3)
7 , 8 , 11
7 , 8 , 11 , 15
0
0
0
0
0
0
0
1
0
12 , 14
11 , 12 , 14
0
0
0
0
0
0
11
30.3
9 , 12
29.4
9 , 12
0
0
0
0
2.45 (dd, 18.3, 6.6)
9 , 10 , 12
9 , 10 , 12
0
1
2
171.2
21.3
69.7
98.0
23.8
174.8
14.5
52.2
95.7
25.0
0
0
0
0
0
0
0
0
0
0
1
3
1.33 (s)
14 , 15
1.43 (d, 6.8)
3.72 (q, 6.8)
14 , 15
13 , 15
1
4
1
5
0
0
0
0
1
8
6
8
6
1.31 (s)
10.69 (s)
6.98 (d, 10.0)
3.80 (br s)
6.46 (br s)
14 , 15
1.45 (s)
14 , 15
d
-OH
0
0
7, 8
ND
0
0
0
0
-NH
-OH
0
7
9.15 (d, 9.0)
ND
4 , 5 , 7
0
0
9.19 (br s)e
0
12 -NH
11 , 12
12
11 , 12
.43 (br s)e
0 0
8
0
0
0
0
15 -OH
3.80 (br s)
14 , 15 , 16
ND
a
Recorded at 400 MHz.
Recorded at 100 MHz.
Recorded at 600 MHz.
b
c
d
e
ND¼not detected.
0
1
2 -NH
2
.

6422
M. Azumi et al. / Tetrahedron 64 (2008) 6420–6425
1
'
of these carbons were established by the HMBC correlations from
0
0
0
0
the two methyls (H-13 and H-16 ) to C-14 and C-15 . Attachment of
2'
0
O
O
a hydroxyl group to C-15 was indicated by the HMBC correlations
5
4
H
N
7'
0
0
0
0
from 15 -OH to C-14 , C-15 , and C-16 . Substructures C and D were
5
'
N
H
8
O
'
10'
12'
O
0
linked through two nitrogen atoms at C-14 on the basis of HMBC
correlations from H-10 to C-14 and from 12 -NH to C-14 . Finally,
an ether linkage between C-9 and C-15 that was consistent with
the deshielded character of the hemiacetal carbon C-15 (
O
7
9
HN
0 0 0 0
1
H
0
0
OH
O
A
B
0
d
C
98.0)
completed the planar structure of 1.
The relative configuration of the bicyclic ring system was elu-
COSY or TOCSY
HMBC
1
6'
1
1
D
cidated by analysis of H– H coupling constants and interpretation
of the NOESY spectrum (Fig. 3). A large diaxial coupling constant
HO 15'
1
13'
4'
0 0
9.8 Hz) between H-8 and H-9 and a small coupling constant
(
0 0
(2.4 Hz) between H-9 and H-10 indicated the axial and equatorial
O
O
9
'
0
0
NH
orientations for H-9 and H-10 , respectively. The axial orientation
N
H
0
1
0'
H
of C-11 methylene was supported by an NOESY correlation be-
tween H-8 and H-11 (d 2.45). The H -16 methyl was assigned to
O
OH
12' N
0
0
0
H 3
C
be placed in the equatorial orientation since NOE was not observed
OH
O
O
0
between the methyl and H-9 . On the basis of biogenesis consid-
eration, it seemed reasonable that 1 was derived from amicouma-
cin A (3) and a biacetyl equivalent C
two water molecules. Upon treatment in trifluoroacetic acid (60 C,
4
unit along with formation of
HO
O
ꢁ
O
3
h), 1 underwent a transformation to amicoumacins B (4) and C (5)
NH
NH
(Fig. 4). The identity of amicoumacins was confirmed by HPLC-
N
H
O
DAD-ESIMS comparisons with the compounds isolated from the
O
OH
OH
O
O
Figure 2. Partial structures and COSY, TOCSY, and key HMBC correlations of 1.
16'
NH
H
H
Me
H
1
3'
0
0
H
N
O
connected on the basis of an HMBC correlation from 6 -NH to C-7
8'
Me
NH
0
0
10'
and a NOESY correlation between 6 -NH and H-8 , providing
substructure C.
9'
OH
HO
H
H
O
Substructure D consisted the remaining four carbons including
3
two singlet methyls and two quaternary sp carbons. Connectivities
Figure 3. Relative configuration and key NOESY correlations of 1.
ꢁ
Figure 4. HPLC analysis of transformation of bacilosarcins to amicoumacins. (a) Treatment of 1 with TFA at 60 C for 3 h. (b) Treatment of 2 with TFA at rt for 0.5 h. Compound 1:
bacilosarcin A, compound 2: bacilosarcin B, compound 3: amicoumacin A, compound 4: amicoumacin B, compound 5: amicoumacin C.

M. Azumi et al. / Tetrahedron 64 (2008) 6420–6425
6423
HO
O
HO
O
HO
O
O
O
O
NH
NH
NH
N
N
H
O
O
OH
OH
OH
1
CONH2
CONH2
2
OH
O
O
O
OH
O
O
O
OH
COOH
CONH2
OH NH2
OH NH2
OH NH2
4
5
3
Figure 5. Chemical correlations of bacilosarcins (1 and 2) and amicoumacins (3–5).
fermentation mixture. To further confirm the absolute configura-
tion, CD spectra of 4 derived from 1 and the authentic 4 were
compared. The CD spectrum of 4 obtained from 1 by acid-catalyzed
degradation showed positive Cotton effects at 221 nm (D3 þ0.437)
and 240 nm (D3 þ1.643) and negative Cotton effects at 259 nm (D3
Table 2
Plant growth inhibition activity of bacilosarcins A (1) and B (2) and related
compounds
a
Compound
Concentration (
mM)
2
10
50
ꢀ
2.183) and 315 nm (D3 ꢀ0.555), that was in good agreement with
Bacilosarcin A (1)
Bacilosarcin B (2)
Amicoumacin A
Amicoumacin B
Herbimycin A
17
NA
35
NA
NA
40
NA
95
14
82
7
98
13
58
those for the authentic 4 (D3219 þ1.112, D3240 þ1.779 D3260 ꢀ2.311,
D3314 ꢀ0.809). The absolute configuration of 1 was thus determined
as illustrated in Figure 1. A plausible mechanism for the conversion
is presented in Figure 5. After hydrolysis of 1 into biacetyl and 3, the
latter compound was transformed into 5 by acid-catalyzed lacto-
nization, followed by hydrolysis of lactone leading to 4.
NA
a
Growth inhibition of barnyard millet is indicated in % of control. NA¼not active.
Compound 2 was isolated as a colorless powder. FT-ICRMS
analysis of 2 showed a molecular formula of C24
[
H
35
N
3
O
8
(obsd
MþH] at m/z 494.24966; calcd [MþH] 494.24969), which dif-
fered from 1 by an extra molecule of hydrogen. Because 2 was
unstable in CDCl , NMR measurements were performed in pyri-
dine-d . The NMR data of 2 were similar to those of 1, with differ-
ences attributable to a reductive cleavage of the C–N bond between
interest, amicoumacin A (3) showed more potent activity than 1.
This may imply that 1 behaves as a prodrug of 3 in plant cells. The
activity levels shown by 1 and 3 are higher than that of herbimycin
A, a potent herbicidal compound from Streptomyces, suggesting
that they may be lead molecules for plant growth regulators.
þ
þ
11
3
5
0
0
C-14 and N-12 . This was revealed by the absence of the singlet
3. Conclusions
0
methyl signal for H-13 and the appearance of signals for a doublet
methyl (H-13 ,
0
0
d
d
H
1.43) and a quartet methine (H-14 ,
H
3.72), and
Two new isocoumarins, bacilosarcins A (1) and B (2) were iso-
lated from the culture extract of B. subtilis. The structures of baci-
losarcins are unique for their nitrogen-containing heterocyclic
cores. The 3-oxa-6,9-diazabicyclo[3.3.1]nonane ring system in 1 is
unprecedented both in natural and synthetic compounds. The
2-hydroxymorpholine substructure in 2 is rare in natural products
although it is more popular in synthetic community.1 There have
been a few compounds containing 2-hydroxymorpholine such as
was supported by the COSY correlation between these protons and
0
0
0
0
0
the HMBC correlations from H-9 to C-15 , H-10 to C-14 , H-13 to
0
0
0
0
0
C-14 and C-15 , H-16 to C14 and C-15 . The relative and absolute
configurations of 2 were determined by an NOESY experiment and
chemical correlation with 1 and amicoumacins. The coupling values
2
for JH-8
morpholine ring had a chair conformation, which was supported by
,H-9 ,H-10
of 8.6 Hz and JH-9 of 2.4 Hz indicated that the
0 0 0 0
0
0
13
14
a NOESY correlation between H-11 and the axial H-14 (Fig. 6).
convolutamine E and akashin C reported to date.
Treatment of 2 in trifluoroacetic acid (rt, 0.5 h) gave a mixture of 1,
3
, 4, and 5. The CD spectrum of 4 derived from 2 displayed the
4. Experimental
Cotton effect pattern (D3219 þ1.384, D3241 þ1.324 D3258 ꢀ2.675,
D3313 ꢀ0.708) similar to that for the authentic 4, thereby confirming
the absolute configuration of 2. Oxidative formation of the imine
intermediate is plausibly involved in the conversion of 2 to 1.
4.1. General
Optical rotations were measured using a JASCO DIP-3000 po-
larimeter. CD spectra were measured in MeOH on a JASCO J-720
spectropolarimeter. UV spectra were recorded on a Hitachi U-3210
spectrophotometer. IR spectra were recorded on a Perkin Elmer
Spectrum 100 spectrophotometer. NMR spectra were obtained on
H H14'
H
Me
O
CONH2
Me
H
H
N
1
8'
a JEOL JNM-LA400 or a Bruker AVANCE 600 spectrometer. The H
10'
NH
13
9
'
and C chemical shifts were referenced to the solvent signals
OH
HO
H
H
(
d
d
H
¼7.26 and
d
C
¼77.0 in CDCl
3
or
H
d ¼7.19 and
d
C
¼123.5 in pyridine-
O
5
). FT-ICRMS was measured on a Bruker Daltonics Apex-Q94e
Figure 6. Relative configuration and key NOESY correlations of 2.
spectrometer. HR-ESITOFMS were recorded on a Bruker microTOF
focus spectrometer. HR-EIMS was recorded on a JEOL GC-mate2
spectrometer. LC–ESIMS spectra were measured on an Agilent 1100
MSD spectrometer with a diode-array detector. HPLC analyses were
carried out on an Agilent HP1100 system using a Cosmosil 5C18-AR-
Bacilosarcin A (1) showed 82% inhibition at 50 mM against
growth of barnyard millet sprouts while bacilosarcin B (2) showed
very weak activity at the same concentration (Table 2). Of particular

6
424
M. Azumi et al. / Tetrahedron 64 (2008) 6420–6425
13
1
II column (Nacalai Tesque Inc., 4.6ꢂ250 mm). Silica Gel 60-C18
Nakalai Tesque 250–350 mesh) was used for ODS column chro-
matography. HPLC separation was performed using a Cosmosil
(3.23); H and C NMR (Table 1); FT-ICRMS molecular ion calcd for
þ
þ
(
24 36
C H N
3
O
8
m/z 494.24969 [MþH] , found 494.24966 [MþH] .
5
C18-AR-II column (Nacalai Tesque Inc., 20ꢂ250 mm).
4.5. Acid-catalyzed degradation of bacilosarcins
A (1) and B (2)
4
.2. Microorganism
A solution of 1 (50
m
g) in trifluoroacetic acid (400
m
L) was heated
ꢁ
B. subtilis TP-B0611 was isolated from the intestine of a sardine
at 60 C for 3 h. The solution was concentrated in vacuo, diluted
with MeOH, and subjected to the HPLC analysis [Cosmosil 5C18-AR-
II (4.6ꢂ250 mm), a linear gradient of 25–75% MeCN in 0.15%
(
S. melanosticta) collected in Toyama Bay, Japan. The intestine
content was streaked on the isolation medium consisting of glucose
.5%, soluble starch 0.5%, meat extract 0.1%, yeast extract (Difco
laboratories) 0.1%, NZ-case (Humco Scheffield Chemical Co.) 0.2%,
0
2 4
KH PO solution (pH 3.5) over 32 min, 0.7 mL/min, 254 nm de-
tection]. The retention times of 1, 2, amicoumacins A (3), B (4), and
C (5) were 20.6, 15.5, 15.2, 15.4, 15.9 min, respectively. Additionally,
LC–MS analysis was conducted for the identification of the degra-
dation products [Cadenza CD-C18 (4.6ꢂ75 mm, Imtact), a linear
gradient of 2.5–97.5% MeCN in 0.1% HCOOH over 30 min, 0.8 mL/
NaCl 0.2%, CaCO
3
0.1%, agar 1.5%, 0.005% amphotericin B and 0.02%
ꢁ
benomyl. After cultivation at 32 C for 1 week, the strain was iso-
lated and preserved. The isolated strain was identified as B. subtilis
on the basis of 16S rRNA analysis, which indicated 99.87% identity
ꢁ
to B. subtilis subsp. subtilis. The strain was kept at ꢀ78 C as a stock
min]. Compound g) was treated with trifluoroacetic
2
(50
m
culture in 20% glycerol.
acid (20 L) in MeOH (400 mL) at room temperature for 0.5 h. The
m
reaction mixture was analyzed in the same manner as described
4
.3. Fermentation
for 1.
Strain TP-B0611 grown on a slant culture was inoculated into
00-mL K-1 flasks each containing 100 mL of the seed medium
4
.6. Determination of absolute configuration of bacilosarcins
5
A (1) and B (2)
consisting of soluble starch 1%, glucose 0.5%, NZ-case 0.3%, yeast
extract 0.2%, triptone (Difco Laboratories) 0.5%, K HPO 0.1%,
2
4
As described above, bacilosarcin A (1, 1 mg) was treated in tri-
fluoroacetic acid to yield the mixture of amicoumacins. The mixture
was purified by preparative C-18 reversed-phase HPLC [Waters
XTerra RP18 (4.6ꢂ250 mm), a linear gradient of 25–50% MeCN in
MgSO
4
$7H
2
O 0.05%, and CaCO
3
0.3% (pH 7.0). The flasks were cul-
ꢁ
tivated on a rotary shaker (200 rpm) at 30 C for 3 days. Seed cul-
ture (3 mL) was transferred into 500-mL K-1 flasks each containing
100 mL of the production medium consisting of glucose 0.5%,
2 4
0.15% KH PO solution (pH 3.5) over 30 min, 0.7 mL/min, 254 nm
glycerol 2%, soluble starch 2%, Pharmamedia (Traders Protein) 1.5%,
yeast extract 0.3%, and Diaion HP-20 (Mitsubishi Chemical Co.) 1%.
The pH of the medium was adjusted to 7.0 before sterilization. The
detection]. The fraction containing amicoumacin B (4) was lyoph-
ilized and the remaining solid was extracted with MeOH to give 4
(116
mg). Similarly, degradation of bacilosarcin B (2, 1 mg) and HPLC
inoculated flasks were cultured on a rotary shaker (200 rpm) at
purification yielded 4 (13
mg). The yields were calculated on the
ꢁ
3
0 C for 5 days.
basis of the optical density at 315 nm. The CD spectra of 4 derived
from 1 and 2 were similar to that of 4 isolated from the fermen-
tation mixture.
4
.4. Extraction and isolation
A 5-day old fermentation broth of strain TP-B0611 (100 mLꢂ50
4
.6.1. Compound 4 isolated from the fermentation mixture
flasks) was extracted with 1-butanol (50 mL per flask) on a rotary
shaker (200 rpm) for additional 1 h. The mixture was centrifuged
at 6000 rpm for 10 min and the organic layer was separated from
the aqueous layer. Evaporation of the solvent provided approxi-
mately 4.3 g of extract from 5 L of culture. The crude extract was
defatted by partitioning with MeOH and n-hexane several times and
the MeOH layer was concentrated in vacuo to give 3.8 g of brown
viscous oil. The oily material was repeatedly subjected to reversed-
phase ODS column chromatography with a step gradient of MeCN–
2
3
6
22
[
a
]
D
ꢀ80.8 (c 0.36, MeOH) {lit. [
a
]
D
ꢀ78.2 (c 0.08, MeOH)}. The
1
13
H and C NMR data were identical to those reported in the liter-
15
ature. CD (MeOH) D3219 þ1.112, D3240 þ1.779 D3260 ꢀ2.311, D3314
ꢀ
0.809; HR-ESITOFMS molecular ion calcd for C20 m/z
27 2 8
H N O
ꢀ
ꢀ
4
23.1773 [MꢀH] , found 423.1780 [MꢀH] .
4
.6.2. Compound 4 obtained from bacilosarcin A (1)
CD (MeOH) D3221 þ0.437, D3240 þ1.643, D3259 ꢀ2.183, D3315
ꢀ
0.555; HR-ESITOFMS molecular ion calcd for C20 m/z
27 2 8
H N O
0
4
2 4
.15% KH PO solution (pH 3.5) (20:80, 25:75, 30:70, 35:65, 40:60,
ꢀ
ꢀ
4
23.1773 [MꢀH] , found 423.1765 [MꢀH] .
5:55 and 50:50 v/v). Fractions 2–4 showed the potent seed
germination inhibitory activity and by HPLC contained bacilosarcins
and amicoumacins. Evaporation of combined fractions 2–4,
followed by extraction with 1-butanol, provided 307 mg of brown
powders. Final purification was achieved by preparative reversed-
4
.6.3. Compound 4 obtained from bacilosarcin A (2)
CD (MeOH) D3219 þ1.384, D3241 þ1.324, D3258 ꢀ2.675, D3313
ꢀ
0.708; HR-ESITOFMS molecular ion calcd for C20 m/z
27 2 8
H N O
ꢀ
ꢀ
4
23.1773 [MꢀH] , found 423.1772 [MꢀH] .
phase HPLC with a linear gradient of MeCN–0.15% KH
50:50–75:25 over 30 min) to give 1 (9.3 mg) and 2 (6.3 mg), along
with amicoumacins A (3, 10.1 mg), B (4, 1.5 mg), and C (5, 4.8 mg).
2 4
PO (pH 3.5)
(
4.7. Bioassay
4
.4.1. Bacilosarcin A (1)
Barnyard millet seeds (Echinochloa frumentacea L.) were in-
cubated for 18 h on a wet paper towel at 25 C in the dark. Ger-
2
5
ꢁ
Colorless powder; [
a
]
D
ꢀ76.9 (c 0.48, CHCl
max (log ) 206 (4.61), 247 (3.90), 314
3.76); H and C NMR (Table 1); HR-EIMS molecular ion calcd for
3
); IR (film) 3265,
ꢀ1
1
(
655 cm ; UV (MeOH)
l
3
minated seeds were selected and incubated in a solution (2 mL) of
test compounds in 0.5% aqueous MeOH containing 0.1% Tween 20
1
13
þ
þ
ꢁ
C
24 33
H N
3
O
8
m/z 491.2267 [M] , found 491.2272 [M] .
in glass containers (50 mm cube) for 5 days at 25 C in the dark.
Each experiment was carried out triplicate. The sprouts were
measured and the inhibition was indicated by percent (%) to the
control (0.5% aqueous MeOH with 0.1% Tween 20). The data were
statistically analyzed.
4
.4.2. Bacilosarcin B (2)
2
5
Colorless powder; [
a
]
D
ꢀ22.9 (c 0.78, MeOH); IR (film) 3188,
max (log ) 206 (4.07), 247 (3.34), 315
ꢀ1
1653 cm ; UV (MeOH)
l
3

M. Azumi et al. / Tetrahedron 64 (2008) 6420–6425
6425
Acknowledgements
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AuthorsthanktoDr. YoshitakaIkedaatMitsubishiTanabe Pharma
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