Micropeptins from the freshwater cyanobacterium Microcystis aeruginosa (NIES-100)

Article abstract of DOI:10.1021/np800631t

Micropeptins C (1), D (2), E (3), and F (4) have been isolated from the freshwater cyanobacterium Microcystis aeruginosa (NIES-100). The structures were elucidated by analyses of MS, NMR spectra, and chemical degradation. Micropeptins C, D, E, and F inhib

Full text of DOI:10.1021/np800631t

J. Nat. Prod. 2009, 72, 777–781
777
Micropeptins from the Freshwater Cyanobacterium Microcystis aeruginosa (NIES-100)
Takaya Kisugi and Tatsufumi Okino*
Graduate School of EnVironmental Earth Science, Hokkaido UniVersity, Sapporo 060-0810, Japan
ReceiVed October 9, 2008
Micropeptins C (1), D (2), E (3), and F (4) have been isolated from the freshwater cyanobacterium Microcystis aeruginosa
(NIES-100). The structures were elucidated by analyses of MS, NMR spectra, and chemical degradation. Micropeptins
C, D, E, and F inhibited chymotrypsin with IC50’s of 1.1, 1.2, 1.0, and 1.5 µg/mL, respectively.
Cyanobacteria are well-known as producers of a variety of
bioactive peptides.1 The genus Microcystis produces six classes of
peptides. The classes consist of microcystins,2 aeruginosins,3
microginins,4 anabaenopeptins,5,6 micropeptins7 (aeruginopeptins,8
cyanopeptolins,9 nostopeptins,10 and oscillapeptins11), and mi-
croviridins,12 all of which are known protease inhibitors. Micropep-
tins have a characteristic unit, 3-amino-6-hydroxy-2-piperidone
(Ahp), and a cyclic structure with six amino acid residues. This
unique unit (Ahp) is also distributed in marine cyanobacterial
depsipeptides such as largamides,13 somamides,14 and tasipeptins.15
Most of these depsipeptides show inhibitory activity against serine
1
Figure 1. H-1H COSY, TOCSY, HMBC, and NOESY correla-
proteases, and the structure-activity relationship has been ex-
plained.16 Microginin and micropeptins A and B isolated from
Microcystis aeruginosa (NIES-100) were the first examples of
cyanobacterial protease inhibitors. In the course of our screening
program for protease inhibitors, we reinvestigated the strain NIES-
100 to find chymotrypsin inhibitors. Here, we describe the isolation
and structural elucidation of novel Ahp-containing depsipeptide
inhibitors.
tions of 1.
The 80% MeOH and MeOH extract of freeze-dried cyanobac-
terial cells was partitioned between water and diethyl ether. The
aqueous layer was further extracted with n-BuOH, and the n-BuOH
layer was subjected to ODS flash column chromatography. By
bioassay-guided fractionation and LC-MS analyses, fractions eluted
with 40% and 60% MeOH were found to contain new active
compounds. These were purified with reversed-phase HPLC to yield
micropeptins C (1), D (2), E (3), and F (4) as colorless, amorphous
solids.
The molecular formula of 1 was established to be C53H69N7O14
by the ESI-TOFMS and NMR spectroscopic data. The 1H NMR in
DMSO-d6 revealed five doublet NH proton signals between δ 7.0
and 8.5 ppm and one N-Me proton signal at 2.75 ppm. The aromatic
protons between 6.56 and 7.18 ppm indicated the presence of two
p-substituted and one monosubstituted benzene ring. A broad singlet
at 6.00 ppm correlated to a carbon at 73.7 ppm in the HMBC
spectrum and was a characteristic proton of the hydroxy group in
Ahp. The structure of the Ahp unit was confirmed by analyses of
1H-1H COSY and TOCSY. In addition, the 1H-1H COSY,
TOCSY, and HMBC spectra determined the presence of Glu, Thr,
Tyr, Phe, N-Me-Tyr, and Val residues. However, the Phe residue
was suggested to be an N,N-disubstituted derivative because of no
correlation to an amide proton (Figure 1). The last unit was
determined as hexanoic acid (HA).
Figure 2. NOESY correlations of the Ahp unit.
were determined to be the L-form by chiral HPLC analysis of the
hydrolysate of 1. The relative configuration of Ahp was determined
as shown in Figure 2. The absolute configuration of Ahp was
deduced by the modified method of Ishida et al.17 Pentahomoserine
was obtained from the hydrolysate followed by the reduction of 1
with NaBH4 and clarified to be the L-form by chiral HPLC analysis.
Therefore, the absolute configuration of Ahp could be assigned as
(3S,6R)-3-amino-6-hydroxy-2-piperidone.
The sequence of 1 was deduced by HMBC correlations from
R-methines and amide protons to carbonyl carbons of amino acid
residues, but the HMBC correlations from Ahp to Tyr, and from
N-Me-Tyr to Val, could not be observed. Their connections were
confirmed by the NOESY spectrum (Figure 1). Furthermore, the
absolute configurations of the amino acid residues, except for Ahp,
The molecular formula of 2 was established to be C55H73N7O14
by the ESI-TOFMS and NMR spectroscopic data. 1H and 13C NMR
spectra of 2 were similar to those of 1, but the molecular weight of
2 was 28 mass units larger than 1. The differences in 13C NMR
were an additional two methylene carbons (28.4, 28.5 ppm).
Therefore, micropeptin D (2) was suggested to be substituted by
* To whom correspondence should be addressed. Tel: +81-11-706-4519.
Fax: +81-11-706-4867. E-mail: okino@ees.hokudai.ac.jp.
10.1021/np800631t CCC: $40.75
2009 American Chemical Society and American Society of Pharmacognosy
Published on Web 03/04/2009

778 Journal of Natural Products, 2009, Vol. 72, No. 4
Notes
Table 1. NMR Spectroscopic Data (600 MHz, DMSO-d6) for Micropeptin C (1)
unit
HA
C/H no.
δC, mult.
δH (J in Hz)
HMBCa
NOESYb
1
2
3
4
5
6
1
2
172.4, qC
35.0, CH2
24.9, CH2
30.8, CH2
21.8, CH2
13.8, CH3
171.9, qC
51.7, CH
27.0, CH2
2.10, m
1.49, m
1.21, m
1.27, m
1,3,4
Glu-NH
2
1,2,4,5
2,3,5
3,4,6
4,5
0.84 t, (7.0)
Glu
4.35, m
1.69, m
1.84, m
2.20, m
1,3
2
3
3,3′,4,NH, Thr-NH
NH
Phe-2, Thr-NH
2,NH
3
3′
4
30.3, CH2
174.0, qC
2,3,5
5
NH
1
2
3
4
NH
1
2
3
3′
4
7.97, d (7.7)
2, HA-1
2,3,3′,4, HA-2
Thr
Tyr
168.8, qC
54.2, CH
72.0, CH
17.5, CH3
4.51, d (9.4)
5.37, q (6.3)
1.10, d (6.3)
7.70, brd
1, Glu-1
Glu-4, Val-1
2,3
3,4,NH, Tyr-NH
2,4,NH, Ahp-NH, Tyr-NH
2,3,NH, Tyr-NH
Glu-1
2,3,4, Glu-2,3,3′,
169.5, qC
53.3, CH
34.9, CH2
4.36, m
2.53, dd (9.7, 14.2)
3.10, brdd (3.4, 14.2)
3,3′,5,NH, Ahp-NH
2,3′,5,NH
2,5,NH
2,4,5
1,2,4,5
128.0, qC
129.5, CH
114.9, CH
155.6, qC
5,5′
6,6′
7
6.88, d (8.0)
6.56, d (8.0)
3,7
4,7
2,3,3′,6,NH
NH
2
3
8.42, d (8.6)
1
2,3,3′,5,5′, Thr-2,3,4, Ahp-NH
Ahp
168.8, qC
48.8, CH
21.6, CH2
3.60, m
1.61, m
2
4
3,3′,4,4′,NH
2,3′,NH
4
4′
5
5′
6
NH
OH
1
2.40, m
1.54, m
1.68, m
5.04, brs
7.08, d (8.8)
6.00, brs
2,3,4′,NH,OH
2,3′,5,OH, Phe-5
3′,5,OH
3′,4,4′,OH, Phe-3,3′,5, Val-NH
2,3,3′, Tyr-2,NH, Thr-3
3′,4,4′,5, Phe-3′, N-Me-Tyr-Me, Val-4
29.3, CH2
73.7, CH
Phe
170.3, qC
50.2, CH
35.3, CH2
2
3
4.74, dd (3.6, 11.4)
1.78, brdd (3.6, 14.0)
2.85, brdd (11.4, 14.0)
1,3, Ahp-5
5
2,4,5
3,3′,5, N-Me-Tyr-3,3′,5, Val-NH
2,3′,5, Ahp-6
2,3,5, Ahp-6,OH
3′
4
5,5′
6,6′
7
1
2
3
3′
4
5,5′
6,6′
7
N-Me
OH
1
2
3
4
4′
NH
136.7, qC
129.4, CH
127.7, CH
126.2, qC
169.1, qC
60.8, CH
32.8, CH2
6.83, d (7.2)
7.18, dd (7.2, 7.2)
7.13, t (7.2)
3,7
4,5
5
2,3,3′,6, Ahp-5,5′,6
5
N-Me-
Tyr
4.88, brd (11.2)
2.70, brddc
3,3′,5, Phe-3, Val-NH
2,5
2,5, Phe-2
4,5
4,5
3.08, brdc
127.4, qC
130.4, CH
115.3, CH
156.2, qC
30.2, CH3
6.99, d (8.0)
6.76, d (8.0)
3,7
4,7
2,3,3′,6,OH, Phe-2,3
5
2.75, s
9.32, s
2, Phe-1
6,7
5, Val-4,4′,NH, Ahp-OH
6,6′
Val
171.9, qC
55.8, CH
30.8, CH2
17.2, CH3
19.2, CH3
4.67, dd (4.4, 9.4)
2.03, m
0.70, d (6.7)
0.84, d (6.7)
7.39, d (9.4)
1,3,4,4′, N-Me-Tyr-1
4,4′
2,4′
2,4
3,4,4′,NH, Thr-NH
2,4,4′
2,3,NH, N-Me-Tyr-Me, Ahp-OH
2,3,NH, N-Me-Tyr-Me
N-Me-Tyr-1
2,3,4,4′, Ahp-4′,6,OH, N-Me-Tyr-2,N-Me, Phe-2
a HMBC correlations are from proton(s) stated to the indicated carbon. b NOESY correlations are from proton(s) stated to the indicated proton(s).
c Signal partially obscured.
octanoic acid (OA) in place of HA in 1. The structure of 2 including
The molecular formula of 4 was established to be C52H75N7O14
by the ESI-TOFMS and NMR spectroscopic data. 1H and 13C NMR
spectra of 4 were similar to those of 3. The molecular weight of 4
was 28 mass units larger than 3. In conclusion, micropeptin F (4)
was substituted by OA in place of HA in 3, which was supported
by extensive 2D NMR analyses and chiral HPLC analysis of its
hydrolysate. The amino acid sequences of 3 and 4 were reported
by Czarnecki et al. as cyanopeptoline 993 and 1021 based on mass
spectrometry.18 However, they were not fully characterized. Due
to the lack of detailed information, we could not conclude that they
are identical with 3 and 4.
the configurations was supported by extensive NMR analyses
1
including H-1H COSY, TOCSY, HSQC, HMBC, and NOESY
spectra and chiral HPLC analysis of its hydrolysate.
The molecular formula of 3 was established to be C50H71N7O14
by the ESI-TOFMS and NMR spectroscopic data. In the 1H NMR
spectrum, the isobutyl protons at 0.40, 0.49, 0.69, 0.97, and 1.55
ppm were observed instead of aromatic protons of Phe in 1. These
1
protons were assigned as a part of Leu by analyses of the H-1H
COSY and TOCSY spectra. The sequence and absolute configu-
ration of 3 were determined by the same method for 1.

Notes
Journal of Natural Products, 2009, Vol. 72, No. 4 779
Table 2. NMR Spectroscopic Data (600 MHz, DMSO-d6) for Micropeptins D (2), E (3), and F (4)
micropeptin D (2)
δH (J in Hz)
micropeptin E (3)
δH (J in Hz)
micropeptin F (4)
δC δH (J in Hz)
unit
OA
C/H no.
δC
unit
HA
C/H no.
δC
unit
OA
C/H no.
1
172.4
1
172.4
1
172.4
2
3
4
5
6
7
8
1
2
3
3′
4
5
NH
1
2
3
4
NH
1
2
3
3′
4
5,5′
6,6′
7
NH
2
3
4
4′
5
5′
6
NH
OH
1
2
3
35.1 2.11, m
25.2 1.48, m
28.5 1.23, m
28.4 1.23, m
31.2 1.21, m
22.0 1.24, m
13.9 0.83, t (6.8)
171.9
51.7 4.34, m
27.0 1.69, m
1.83, m
30.3 2.19, m
174.1
2
3
4
5
6
1
2
3
3′
4
5
NH
1
2
3
4
NH
1
2
3
3′
4
5,5′
6,6′
7
NH
2
3
4
4′
5
6
NH
OH
1
2
3
3′
4
5
5′
1
2
35.0 2.13, m
24.9 1.49, m
30.8 1.21, m
21.8 1.25, m
13.8 0.84, t (7.2)
171.9
51.7 4.36, m
27.0 1.71, m
1.85, m
30.3 2.20, m
174.1
7.99, d (7.8)
168.9
2
3
4
5
6
7
8
1
2
3
3′
4
5
NH
1
2
3
4
35.1 2.11, m
25.2 1.48, m
28.4 1.23, m
28.5 1.23, m
31.2 1.22, m
22.0 1.24, m
13.9 0.84, t (6.8)
171.9
51.7 4.36, m
27.0 1.71, m
1.85, m
30.3 2.22, m
174.1
Glu
Glu
Glu
Thr
Thr
Tyr
7.98, d (7.7)
54.3 4.55, d (9.5)
72.0 5.46, q (6.4)
17.5 1.13, d (6.4)
7.72, d (9.5)
169.9
7.99, d (7.7)
168.9
Thr
Tyr
168.8
54.2 4.50, d (9.5)
72.0 5.37, q (6.4)
17.5 1.10, d (6.4)
7.70, d (9.5)
54.3 4.55, d (9.5)
72.0 5.46, q (6.4)
17.5 1.13, d (6.4)
7.71, d (9.5)
169.9
53.6 4.45, m
35.1 2.62, dd
(10.0, 14.5)
53.5 4.45, m
NH
1
2
169.5
35.1 2.61, dd (9.8, 14.6) Tyr
3.22, dd (3.9, 14.6)
128.1
129.6 6.93, d (8.4)
115.0 6.57, d (8.4)
155.6
53.3 4.35, m
34.9 2.53, dd (9.8, 14.3)
3.10, dd (3.8, 14.3)
128.0
129.6 6.88, d (8.3)
114.9 6.56, d (8.3)
155.6
3
3′
4
3.21, dd (3.6, 14.5)
128.1
8.48, d (8.6)
5,5′
6,6′
7
NH
2
3
4
4′
5
129.6 6.93, d (8.3)
115.0 6.59, d (8.3)
155.6
Ahp
Leu
169.1
49.1 4.38, m
21.7 1.73, m
2.52, m
29.8 1.71, m
73.4 4.88, br
8.42, d (8.7)
Ahp
168.8
8.48, d (8.6)
48.8 3.61, m
21.5 1.61, m
2.40, m
29.2 1.55, m
1.68, m
73.7 5.04, brs
7.09, d (8.9)
6.00, brd (2.7)
170.3
Ahp
Leu
169.1
49.2 4.37, m
21.7 1.72, m
2.53, m
29.7 1.71, m
73.4 4.88, br
7.32, d (9.0)
7.32, d (9.1)
5.99, brd (3.0)
170.8
6
47.7 4.58, dd (3.8, 10.7)
38.4 0.40, m
1.55, m
NH
OH
1
2
3
3′
4
5
5′
1
2
3
3′
6.00, brs
Phe
170.8
50.3 4.74, dd (3.8, 11.3)
35.3 1.78, brdd
(3.8, 14.3)
23.6 0.97, m
22.1 0.49, d (6.6)
23.9 0.69, d (6.6)
169.3
47.7 4.58, dd (3.5, 10.7)
38.4 0.41, m
1.54, m
3′
2.86, brdd
(11.3, 14.3)
N-Me-
Tyr
23.6 0.97, m
22.1 0.49, d (6.5)
23.8 0.69, d (6.5)
169.3
60.8 4.90, brdda
32.8 2.66, brdda
3.07, brdda
4
136.7
3
3′
4
5,5′
6,6′
7
N-Me
OH
1
5,5′
6,6′
7
1
2
3
3′
4
129.4 6.83, d (7.3)
127.7 7.18, dd (7.3, 7.3)
126.2 7.13, t (7.3)
169.1
60.8 4.88, brd (11.3)
32.8 2.70, brdda
3.08, brda
N-Me-
Tyr
127.2
60.8 4.90, brdda
32.8 2.66, brdda
3.07, brdda
130.0 6.89, d (8.4)
115.3 6.60, d (8.4)
156.0
30.4 2.69, s
9.20, s
N-Me-
Tyr
4
127.2
5,5′
6,6′
7
130.0 6.89, d (8.2)
115.3 6.62, d (8.2)
156.0
127.5
Val
172.1
5,5′
6,6′
7
N-Me
OH
1
2
3
4
4′
130.4 6.99, d (8.3)
115.3 6.76, d (8.3)
156.2
30.3 2.74, s
9.33, s
2
3
4
4′
NH
55.8 4.68, dd (4.7, 9.4)
30.8 2.03, m
17.3 0.71, d (6.8)
19.2 0.85, d (6.0)
7.47, d (9.4)
N-Me
OH
1
2
3
4
4′
NH
30.4 2.69, s
9.21, s
172.1
55.9 4.67, dd (4.6, 9.4)
30.8 2.03, m
17.3 0.71, d (6.8)
19.2 0.84, d (6.1)
7.48, d (9.4)
Val
Val
171.9
55.8 4.67, dd (4.5, 9..5)
30.8 2.03, m
17.2 0.70, d (6.8)
19.2 0.84, d (6.3)
7.39, d (9.5)
NH
a Signal partially obscured.
Micropeptins C, D, E, and F inhibited chymotrypsin with IC50’s
of 1.1, 1.2, 1.0, and 1.5 µg/mL, respectively. These peptides did
not inhibit trypsin and thrombin at 20 µg/mL, although micropeptins
A and B isolated from the same strain were reported as trypsin
inhibitors.7 Specificity to serine proteases of Ahp-containing
depsipeptides was related to the amino acid residue at the N-terminal
side of Ahp.15,19 When a hydrophobic residue such as Tyr or Phe
connects to Ahp, peptides are known to inhibit chymotrypsin. When
a basic residue such as Lys or Arg connects to Ahp, peptides are
trypsin inhibitors. Micropeptin T-1, which has N-Me-L-Trp instead
of N-Me-L-Tyr in micropeptin C, inhibited chymotrypsin with an
IC50 of 3.0 µg/mL, similar to that of micropeptin C.20 Micropeptin
B, containing L-Lys instead of L-Tyr as in micropeptin C, inhibited
trypsin with an IC50 of 0.25 µg/mL. Therefore, our results were in

780 Journal of Natural Products, 2009, Vol. 72, No. 4
Notes
Determination of the Absolute Configurations of the Amino
Acids. Each compound was dissolved in 6 M HCl (500 µL). The
reaction mixture was then placed in a sealed glass tube at 110 °C for
22 h. After evaporation in Vacuo, the residue was dissolved in H2O
(100 µL), and chiral HPLC analyses were carried out using a
SUMICHIRAL OA-5000 column (Sumitomo Chemical Industry, 4.6
× 150 mm, eluent: MeOH-H2O (5:95) containing 2.0 mM CuSO4;
flow rate: 1.0 mL/min; UV detection: 254 nm; oven temperature: 40
°C). Retention times (min) of authentic amino acids were as follows:
L-Thr (4.0), D-Thr (4.5), L-allo-Thr (5.4), D-allo-Thr (5.8), L-Val (9.4),
D-Val (15.7), L-Tyr (24.2), D-Tyr (39.8), N-Me-L-Tyr (28.8), N-Me-D-
Tyr (31.3), L-Glu (37.1), D-Glu (43.9), L-Leu (27.5), D-Leu (45.7), L-Phe
(79.3), and D-Phe (113.5). Retention times of the hydrolysate of 1 were
as follows: L-Thr (4.0), L-Val (9.4), L-Tyr (24.2), N-Me-L-Tyr (28.8),
L-Glu (37.1), and L-Phe (79.3). Retention times of the hydrolysate of
3 were as follows: L-Thr (4.0), L-Val (9.4), L-Tyr (24.3), L-Leu (27.6),
N-Me-L-Tyr (28.8), and L-Glu (37.1). Retention times of hydrolysates
of 2 and 4 were identical to those of 1 and 3, respectively.
Synthesis of DL-Pentahomoserines. Each of Boc-D- and Boc-L-
Glu(OBzl) (200 mg) was dissolved in anhydrous THF (1.0 mL), and
then LiBH4 (31.5 mg) was added to the solution with stirring at room
temperature under argon for a further 16 h. EtOAc (1.0 mL) was added,
and the solution was stirred at room temperature for 3 h. After
evaporation, the reaction mixture was subjected to Si gel (silica gel
60, 70-230 mesh, Merck Ltd., 15 × 235 mm) column chromatography
with CHCl3 to CHCl3-MeOH (1:1). The fraction containing Boc-
pentahomoserine was evaporated and dissolved in HCOOH (1.0 mL).
After stirred at room temperature for 4 h, the reaction mixture was
evaporated and purified using RP-HPLC (Develosil C30 UG-5, 10.0
× 250 mm; H2O; 3.0 mL/min: UV detection 210 nm) to obtain D- and
L-pentahomoserine.
Reduction of Micropeptin C (1) to F (4). Each of 1 to 4 (300 µg)
was dissolved in anhydrous MeOH (1.0 mL), and then an excess amount
of NaBH4 was added to the solution with stirring at room temperature.
After stirring for 3 h, H2O was added and evaporated. The reaction
mixture was passed through a disposable ODS column (YMC Dispo
SPE C18; H2O-MeOH) and evaporated. The MeOH extract was
dissolved in 6 N HCl (500 µL) and placed in a sealed glass tube at
110 °C for 16 h. After evaporation in Vacuo, the residue was dissolved
in H2O (300 µL), and chiral HPLC analyses were carried out using a
SUMICHIRAL OA-5000 column (Sumitomo Chemical Industry, 4.6
× 150 mm, eluent: H2O containing 2.0 mM CuSO4; flow rate: 1.0 mL/
min; UV detection: 254 nm; oven temperature: 40 °C). Retention times
(min) of standard amino acids were as follows: L-pentahomoserine (5.4),
D-pentahomoserine (8.2). Retention times of the reductive hydrolysates
of 1 to 4: L-pentahomoserine (5.4).
Protease Inhibition Assay. All the enzymes and substrates were
purchased from Sigma Chemical Co. The enzyme (R-chymotrypsin type
II) was dissolved in 50 mM Tris-HCl (pH 7.6) to prepare a 15 U/mL
solution. A 1 mg/mL solution of N-succinyl-L-phenylalanyl-p-nitroa-
nilide in the buffer was used for the substrate solution. A 30 µL buffer
solution, 50 µL enzyme solution, and 20 µL of test solution were added
to each microtiter plate well and preincubated at 37 °C for 5 min. Then,
100 µL of substrate solution was added to start the reaction. The
absorbance of the well was immediately measured at 405 nm. The
developed color was measured after incubation at 37 °C for 30 min.
Thrombin inhibitory activity was determined by the modified method
of Svendsen et al.21 The following stock solutions were prepared for
Tris-HCl buffer: (I) a mixture of equal volumes of 0.1 M imidazole-
HCl and 0.1 M Tris-HCl; (II) a mixture of equal volumes of 0.1 M
imidazole and 0.1 M Tris, both in 0.1 M NaCl. These two stock
solutions were then mixed to adjust to pH 8.2 and diluted with an equal
volume of 0.2 M NaCl. Thrombin was dissolved in Tris-imidazole
buffer to prepare a 1.3 U/mL solution. A 0.25 mg/mL solution of
N-benzoyl-Phe-Val-Arg-p-nitroanilide in buffer was used for the
substrate solution. A 90 µL enzyme solution and 20 µL test solution
were added to each mictotiter plate well and preincubated at 37 °C for
5 min. Then 90 µL of substrate solution was added to start the reaction.
The absorbance of the well was immediately measured at 405 nm. The
developed color was measured after incubation at 37 °C for 30 min.
Trypsin inhibitory activity was determined by the method of
Yamaguchi et al.22 except that the enzyme solution was 300 U/mL.
a good agreement with the reported specificity to serine proteases
of Ahp-containing depsipeptides.
Experimental Section
General Experimental Procedures. IR spectra were recorded on a
Jasco FT/IR-460 Plus infrared spectrometer. Optical rotations were
measured on a Horiba SEPA-300 high sensitive polarimeter. NMR
spectra were obtained with a JEOL JMN-ECA-600 spectrometer in
DMSO-d6. The resonances of residual DMSO-d6 at δH 2.49 and δC
39.5 ppm were used as internal references for 1H and 13C NMR spectra,
respectively. High-resolution mass spectra were obtained with a Bruker
Daltonics microTOF mass spectrometer. LC-MS analyses were carried
out under the following conditions: YMC Pack Pro C18 column and
Develosil ODS HG-5 column applying a MeCN/0.01% HCOOH (in
H2O) gradient with a flow rate of 0.2 mL/min.
Biological Materials. Microcystis aeruginosa (NIES-100) was
obtained from the NIES collection (Microbial Culture Collection, the
National Institute for Environmental Studies, Japan) and cultured in 5
L bottles containing MA medium with aeration at 25 °C for 2 weeks
under a 12 h/12 h light-dark cycle. Cells were harvested by
centrifugation, lyophilized, and kept in a freezer at -20 °C until
extraction.
Isolation Procedure. The freeze-dried cells (64.5 g) were extracted
with 80% MeOH and MeOH. The combined extract (12.5 g) was
partitioned with diethyl ether and water. The aqueous layer was further
extracted with n-BuOH, and the n-BuOH layer was subjected to ODS
(YMC-GEL, ODS-A, 22 × 295 mm) flash column chromatography
with aqueous MeOH followed by CHCl3 to obtain fractions 1-6.
Fraction 2 (4:6 MeOH-H2O) was subjected to reversed-phase HPLC
(Develosil ODS HG-5, 10.0 × 250 mm) with a gradient of aqueous
MeCN (30-45%) containing 0.05% TFA to yield crude peptide
fractions I and II. Fraction I was subjected to reversed-phase HPLC
(Develosil CN-UG-5, 10.0 × 250 mm) with 33% MeCN containing
0.05% TFA to yield 3.4 mg of micropeptin E (3). Fraction II was
subjected to reversed-phase HPLC (Inertsil ODS-3, 10.0 × 250 mm)
with 45% MeCN containing 0.05% TFA to yield 2.4 mg of micropeptin
C (1).
Fraction 3 (6:4 MeOH-H2O) was subjected to reversed-phase HPLC
(Develosil ODS HG-5, 10.0 × 250 mm) with a gradient of aqueous
MeCN (35-55%) containing 0.05% TFA. Final purification was
achieved by reversed-phase HPLC (Inertsil ODS-3, 10.0 × 250 mm)
with 50% MeCN containing 0.05% TFA to yield 5.4 mg of micropeptin
F (4) and 3.1 mg of D (2).
Micropeptin C (1): colorless, amorphous solid; [R]25D -14 (c 0.05,
MeOH); IR νmax 3367, 3299, 2958, 2929, 2894, 2852, 1734, 1641 cm-1
;
1H NMR and 13C NMR data, see Table 1; HRMS (ESI-TOF) m/z
1026.4786 [M - H]- (calcd for C53H68N7O14, 1026.4830).
Micropeptin D (2): colorless, amorphous solid; [R]25D -6.5 (c 0.2,
MeOH); IR νmax 3369, 3298, 2958, 2929, 2891, 2852, 1732, 1641 cm-1
;
1H NMR and 13C NMR data, see Table 2; HRMS (ESI-TOF) m/z
1054.5161 [M - H]- (calcd for C55H72N7O14, 1054.5143).
Micropeptin E (3): colorless, amorphous solid; [R]25D -46 (c 0.1,
MeOH); IR νmax 3363, 3290, 2958, 2927, 2894, 2852, 1734, 1641 cm-1
;
1H NMR and 13C NMR data, see Table 2; HRMS (ESI-TOF) m/z
992.4993 [M - H]- (calcd for C50H70N7O14, 992.4981).
Micropeptin F (4): colorless, amorphous solid; [R]25D -39.5 (c 0.05,
MeOH); IR νmax 3365, 3292, 2958, 2929, 2894, 2852, 1732, 1641 cm-1
;
1H NMR and 13C NMR data, see Table 2; HRMS (ESI-TOF) m/z
1020.5287 [M - H]- (calcd for C52H74N7O14, 1020.5294).

Notes
Acknowledgment. We thank Y. Kumaki, High-Resolution NMR
Laboratory, Graduate School of Science, Hokkaido University, for
performing the NMR measurements.
Journal of Natural Products, 2009, Vol. 72, No. 4 781
(10) Okino, T.; Qi, S.; Matsuda, H.; Murakami, M.; Yamaguchi, K. J. Nat.
Prod. 1997, 60, 158–161.
(11) Itou, Y.; Ishida, K.; Shin, H. J.; Murakami, M. Tetrahedron 1999, 55,
6871–6882.
(12) Ishitsuka, M. O.; Kusumi, T.; Kakisawa, H. J. Am. Chem. Soc. 1990,
Supporting Information Available: 1D and 2D NMR spectra for
micropeptins C (1), D (2), E (3), and F (4). This material is available
free of charge via the Internet at http://pubs.acs.org.
112, 8180–8182.
(13) Plaza, A.; Bewley, C. A. J. Org. Chem. 2006, 71, 6898–6907.
(14) Nogle, L. M.; Williamson, R. T.; Gerwick, W. H. J. Nat. Prod. 2001,
64, 716–719.
(15) Williams, P. G.; Yoshida, W. Y.; Moore, R. E.; Paul, V. J. J. Nat.
Prod. 2003, 66, 620–624.
References and Notes
(1) Burja, A. M.; Banaigs, B.; Abou-Mansour, E.; Burgess, J. G.; Wright,
P. C. Tetrahedron 2001, 57, 9347–9377.
(2) Namikoshi, M.; Rinehart, K. L.; Sakai, R. J. Org. Chem. 1992, 57,
866–872.
(3) Murakami, M.; Ishida, K.; Okino, T.; Okita, Y.; Matsuda, H.;
Yamaguchi, K. Tetrahedron Lett. 1995, 36, 2785–2788.
(4) Okino, T.; Matsuda, H.; Murakami, M.; Yamaguchi, K. Tetrahedron
Lett. 1993, 34, 501–504.
(5) Harada, K.; Fujii, K.; Shimada, T.; Suzuki, M. Tetrahedron Lett. 1995,
36, 1511–1514.
(16) Linington, R. G.; Edwards, D. J.; Shuman, C. F.; McPhail, K. L.;
Matainaho, T.; Gerwick, W. H. J. Nat. Prod. 2008, 71, 22–27.
(17) Ishida, K.; Matsuda, H.; Murakami, M. Tetrahedron 1998, 54, 5545–
5556.
(18) Czarnecki, O.; Henning, M.; Lippert, I.; Welker, M. EnViron.
Microbiol. 2006, 8, 77–87.
(19) Yamaki, H.; Sitachitta, N.; Sano, T.; Kaya, K. J. Nat. Prod. 2005, 68,
14–18.
(20) Kodani, S.; Suzuki, S.; Ishida, K.; Murakami, M. FEMS Microbiol.
Lett. 1999, 178, 343–348.
(21) Blomba¨ck, M.; Blomba¨ck, B.; Olsson, P.; Svendsen, L. Thromb. Res.
1974, 5, 621–632.
(22) Shin, H. J.; Murakami, M.; Matsuda, H.; Yamaguchi, K. Tetrahedron
1996, 52, 8159–8168.
(6) Fastner, J.; Erhard, M.; Do¨hren, H. Appl. EnViron. Microbiol. 2001,
67, 5069–5076.
(7) Okino, T.; Murakami, M.; Haraguchi, R.; Munekata, H.; Matsuda,
H.; Yamaguchi, K. Tetrahedron Lett. 1993, 34, 8131–8134.
(8) Harada, K.; Mayumi, T.; Shimada, T.; Suzuki, M. Tetrahedron Lett.
1993, 34, 6091–6094.
(9) Jakobi, C.; Oberer, L.; Quiquerez, C.; Ko¨nig, W. A.; Weckesser, J.
FEMS microbiol. Lett. 1995, 129, 129–134.
NP800631T