Aeruginoguanidines 98-A-98-C: Cytotoxic unusual peptides from the cyanobacterium Microcystis aeruginosa

Article abstract of DOI:10.1016/S0040-4020(02)00870-0

Aeruginoguanidines 98-A-98-C (1-3) were isolated from the cyanobacterium Microcystis aeruginosa (NIES-98). The structures of aeruginoguanidines 98-A-98-C (1-3) were determined by two-dimensional ¹H-¹H and ¹H-¹³C NMR correlation experiments and confirmed by mass spectral analysis. The absolute stereochemistry of 1, consisting of Hphpa trisulfate (1-(4-hydroxy-3-hydroxymethyl)phenyl-1-hydroxy-2-propylamine 4′,3′,1-tri-O-sulfate), MpArg (N^α-methyl-N^ω-prenylarginine) and MgArg ((Z)-N^α-methyl-N^ω-geranylarginine), was determined by the NMR analyses of phenylglycine methyl ester (PGME) or Boc phenylglycine (BPG) derivatives of acid hydrolysates. These compounds showed moderate cytotoxicity against the P388 murine leukemia cells.

Full text of DOI:10.1016/S0040-4020(02)00870-0

Tetrahedron 58 (2002) 7645–7652
Aeruginoguanidines 98-A–98-C: cytotoxic unusual peptides from
the cyanobacterium Microcystis aeruginosa
†
Keishi Ishida, Hisashi Matsuda, Yuji Okita and Masahiro Murakami
*
Laboratory of Marine Biochemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku,
Tokyo 113-8657, Japan
Received 27 May 2002; accepted 17 July 2002
Abstract—Aeruginoguanidines 98-A–98-C (1–3) were isolated from the cyanobacterium Microcystis aeruginosa (NIES-98). The
structures of aeruginoguanidines 98-A–98-C (1–3) were determined by two-dimensional 1H–1H and 1H–13C NMR correlation experiments
and confirmed by mass spectral analysis. The absolute stereochemistry of 1, consisting of Hphpa trisulfate (1-(4-hydroxy-3-
hydroxymethyl)phenyl-1-hydroxy-2-propylamine 40,30,1-tri-O-sulfate), MpArg (N a-methyl-N v-prenylarginine) and MgArg ((Z)-N a-
methyl-N v-geranylarginine), was determined by the NMR analyses of phenylglycine methyl ester (PGME) or Boc phenylglycine (BPG)
derivatives of acid hydrolysates. These compounds showed moderate cytotoxicity against the P388 murine leukemia cells. q 2002 Elsevier
Science Ltd. All rights reserved.
1. Introduction
2. Results and discussion
The freshwater cyanobacterium Microcystis aeruginosa has
been extensively studied from the environmental, toxico-
logical, biological, and chemical points of view because
they are responsible for water blooms that frequently
produce potent hepatotoxins.1 These hepatotoxic cyclic
peptides, microcystins, have been isolated from in a number
of strains of Microcystis and some other cyanobacteria.2
Moreover, these peptides have been found to be a highly
effective inhibitors of protein phosphatase 1 and 2A.3 On the
other hand, in the course of our screening program of new
protease inhibitors from microalgae, we have reported that
M. aeruginosa is very rich source of the protease inhibitory
peptides such as aeruginosins,4 microginins,5 micro-
peptins,6 and microviridins.7
M. aeruginosa (NIES-98) was obtained from the NIES-
collection8 and cultured in our laboratory to yield 115 g (dry
weight) from 420 L of culture. The 80% methanol extract of
freeze-dried alga was partitioned between water and diethyl
ether. The aqueous layer was further extracted with
n-butanol and n-butanol layer was subjected to ODS flash
column chromatography followed by reversed-phase HPLC
with aqueous MeCN containing 0.05% TFA to yield
aeruginoguanidines 98-A–98-C (1–3).
The molecular formula of 1 was established to be
C39H67N9O14S3 by the HRFABMS and NMR spectral
data (Table 1). The IR spectrum showed the presence of
sulfates (1260 cm21), guanidinyl groups (1620 cm21), and
amide carbonyl groups (1660 cm21).
In the course of isolation of protease inhibitors, aeruginosins
98-A–98-C4b from M. aeruginosa (NIES-98), we found that
this strain produces unusual peptides in large quantities. We
now report the isolation and structural elucidation of
aeruginoguanidines 98-A (1), 98-B (2), and 98-C (3) from
the freshwater cyanobacterium M. aeruginosa (NIES-98).
Aeruginoguanidine 98-A (1) was composed of three curious
units, Hphpa trisulfate (1-(4-hydroxy-3-hydroxymethyl)-
phenyl-1-hydroxy-2-propylamine 40,30,1-tri-O-sulfate),
MpArg (N a-methyl-N v-prenylarginine), and MgArg ((Z)-
N a-methyl-N v-geranylarginine), from the detailed analyses
of 2D NMR and FABMS spectra.
2.1. Hphpa trisulfate
The positive and negative FABMS of 1 using glycerol as a
matrix revealed desulfated ions at m/z 822 (M22SO3þH)þ,
724 (M23SO32H2OþH)þ and 900 (M2SO32H)2, which
Keywords: cyanobacterium; Microcystis aeruginosa; peptide; cytotoxicity.
*
Corresponding author. Tel.: þ81-3-5841-5298; fax: þ81-3-5841-8166;
1
supported the presence of three sulfate groups. H and 13C
e-mail: amura@mail.ecc.u-tokyo.ac.jp
Present address: Central Research Laboratory, Nippon Suisan Kaisha, Ltd
559-6 Kitano-Machi, Hachioji, 192-8557 Japan.
†
NMR spectra of 1 suggested the presence of trisubstituted
benzene, and the positions of substituents (C-4, 6, 7; d
0040–4020/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(02)00870-0

7646
K. Ishida et al. / Tetrahedron 58 (2002) 7645–7652
Table 1. NMR data for aeruginoguanidine 98-A (1) in DMSO-d6
13C mult
1H J (mult, Hz)
HMBC (C, no)
Hphpa trisulfate
1
2
3
4
5
6
7
8
77.6 d
48.9 d
14.7 q
133.0 s
127.0 d
128.7 s
149.7 s
120.5 d
126.6 d
62.7 t
5.14 d 6.1
4.05 qdd 6.7, 6.9, 6.1
0.92 d 6.7
2, 3, 4, 5, 9
1, 3, 4
1, 2
7.17 d 2.0
1, 7, 9, 10
7.30 d 8.4
4, 6, 7
1, 5, 7
5, 6, 7
5, 6, 7
11
9
7.06 dd 8.4, 2.0
4.92 d 12.8
4.97 d 12.8
7.57 d 6.9
10a
10b
NH
MpArg
11
12
13a
13b
14
15a
15b
16
17
18
169.5 s
56.7 d
24.9 t
4.83 dd 9.6, 6.0
1.60 m
1.79 m
1.40 m
3.18 m
3.23 m
11, 13, 22
23.8 t
47.0 t
16
16
155.6 s
45.7 t
3.87 br
5.08 m
16, 17, 18
20, 21
118.3 d
136.8 s
25.4 q
17.6 q
30.6 q
19
20
21
22
1.69 s
1.65 s
3.01 s
7.12 br
18, 19, 21
18, 19, 20
12, 23
17vNH
MgArg
23
24
25a
25b
26a
26b
27a
27b
28
29
30
31
32a
32b
33
34
35
36
37
38
39
24–NH
27–NH
29–NH
28vNH
168.4 s
58.0 d
25.2 t
133.0, 128.7, 149.7) were determined by H–1H COSY,
HMQC, and HMBC spectra (Table 1 and Fig. 1). In the
1H–1H COSY spectrum, the connectivities from H-1 (d
5.14) to H-3 (d 0.92) and 2-NH (d 7.57), were determined.
The correlations in the HMBC spectrum from H-1 to C-4,
C-5 (d 127.0) and C-9 (d 126.6) revealed the connectivity
between C-1 and C-4 (Fig. 1). The HMBC correlations from
primary hydroxy methylene protons (H-10; d 4.92 and 4.97)
to C-5, C-6 and C-7 allowed a hydroxy methyl group to
connect with C-6 (Fig. 1). The aromatic proton (H-8; d 7.30,
d J¼8.4 Hz), which correlated with H-9 (d 7.06, dd J¼8.4,
2.0 Hz) in the 1H–1H COSY, was confirmed the corre-
lations with C-4 and C-6 by HMBC spectrum (Fig. 1). These
results suggested that this unit has a feature of Hphpa (1-(4-
hydroxy-3-hydroxymethyl)phenyl-1-hydroxy-2-propyl-
amine). Judging from the chemical shifts data, the structure
of this unit was determined to be Hphpa trisulfate whose
sulfate groups were adjacent to C-1 (d 77.6), 7 and 10 (d
62.7).
1
4.48 br
1.81 m
1.97 m
1.60 m
1.77 m
3.18 br
3.35 br
21.5 t
46.4 t
28
155.7 s
45.3 d
119.0 d
140.3 s
31.42 t
3.92 br
5.12 t 6.6
28, 30, 31
32, 38
2.05
2.08 m
2.04 m
5.10 m
25.7 t
123.8 d
131.3 s
25.5 q
17.6 q
23.0 q
31.37 q
1.64 s
1.58 s
1.72 s
2.52 s
8.75 br
7.18 br
7.18 br
7.12 br
34, 35, 37
34, 35, 36
30, 31
24
2.2. MpArg and MgArg
The 13C NMR signals at d 155.6 and 155.7 showed the
existence of two guanidino groups. The connectivity from
the former guanidinyl carbon (d 155.6) to amide carbonyl
(MpArg C-11) and N-methyl (MpArg C-22) groups was
determined by 1H–1H COSY and HMBC experiments.
Furthermore, the HMBC correlation from the broad
methylene protons (H-17; d 3.87) of the prenyl group,
which was easily determined by 1H–1H COSY and HMBC
spectra, to this guanidinyl carbon confirmed that this unit
was MpArg (N a-methyl-N v-prenylarginine). These experi-
ments also showed the connectivity from the latter
Figure 1. Key HMBC correlations (arrows) of 1.

K. Ishida et al. / Tetrahedron 58 (2002) 7645–7652
7647
guanidinyl carbon (d 155.7) to another N-methyl group
(MgArg C-39) and presence of the geranyl group. The
Z-geometry of the trisubstituted double bond was deduced
from the NOESY cross-peak; H-30 (d 5.12)/H-38 (d 1.72)
and the chemical shifts of H-38 (dH 1.72, dC 23.0).
Moreover, a correlation between the broad methylene
protons (MgArg H-29; d 3.92) of the geranyl group and
the latter guanidinyl carbon was observed in HMBC
spectrum. The connectivity between MgArg C-23 (d
168.4) and C-24 (d 58.0) was deduced by NOESY
(MpArg H-22 (d 3.01)/MgArg H-24) and the HMBC
correlations from H-22 to C-23 (d 168.4) of MgArg, though
correlations between C-23 and protons in MgArg were not
detected by HMBC spectrum.
additive reagents (PyBOP, HOBt), and Dd values (dS2dR)
were determined at 600 MHz.9 Negative Dd were found for
the protons on N-Me side of the PGME plane, whereas
positive values were found for protons on C-3 to C-10 and
C-100 side (Fig. 2). Therefore, the absolute stereochemistry
at C-2 of the MpArg unit was S configuration. The absolute
stereochemistry of MgArg was also determined to be S
configuration by the NMR analysis of (R )- and (S )-PGME
amides of MdoArg (5; N a-methyl-N v-2,6-dimethyloctyl-
arginine), obtained by the acid hydrolysis of hydrogenated
product of 1 (Fig. 3).
The assignment of the absolute configuration of the Hphpa
trisulfate unit was quite difficult for the insolubility of
aeruginoguanidines in various solvents except DMF and
DMSO.10 Sufficient sample of hydroxyamine could not be
obtained by acid hydrolysis under various conditions.
Hydrazinolysis of 1 was also failure. Finally, small sample
of Hphpa (6; 0.8 mg) obtained by acid hydrolysis of 1
(50 mg) using 3 M HCl (1008C for 3 h) was used for the
microanalysis of the absolute structure determination. The
absolute configuration at C-2 of Hphpa was achieved by the
Boc-phenylglycine (BPG) method.11 The (R)- and (S)-BPG
amides of Hphpa were prepared by treatment with (R)- and
(S)-BPG in DMF using the coupling and additive reagents
(PyBOP, HOBt), and Dd values (dS2dR), which were
analyzed with 600 MHz NMR, indicated that the absolute
configuration at C-2 of Hphpa was R-configuration (Fig. 4).
The absolute configuration at C-1 of Hphpa was determined
by the comparison with four isomers ((1R,2R) and (1S,2S)
norephedrine were prepared from (1S,2R ) and (1R,2S)
norephedrine, respectively) of norephedrine (Table 2). The
similarity of the coupling constants and optical rotations
between (1R,2R ) norephedrine and Hphpa indicated that the
configurations at C-1 and C-2 of Hphpa were R.
The sequence of 1 was deduced by HMBC (Hphpa trisulfate
H-2/MpArg C-11) and NOESY correlation (MpArg
H-22/MgArg H-24).
The absolute stereochemistry of the MpArg unit was
determined as follows. The (R )- and (S)-PGME (phenyl-
glycine methyl ester) amides of MiArg (4; N a-methyl-N v-
isoamylarginine), obtained by the acid hydrolysis of a
hydrogenated product of 1, were prepared by treatment with
(R)- and (S)-PGME in DMF using the coupling and
Figure 4. Dd (dS2dR) values in ppm obtained at 600 MHz for (R )- and (S)-
BPG amides of Hphpa.
Figure 2. Dd (dS2dR) values in ppm obtained at 600 MHz for (R)- and (S)-
PGME amides of MiArg.
The fragment patterns of the positive and negative FABMS
of aeruginoguanidine 98-B (2) using glycerol as a matrix
revealed the presence of three sulfate groups. The molecular
formula of 2 was established to be C34H59N9O14S3 by the
1
HRFABMS and NMR spectral data (Table 3). The H and
13C NMR spectra of 2 were similar to those of 1, but two
methyl protons of the prenyl group were not detected in 1H
1
NMR spectrum. The H–1H COSY, HMQC and HMBC
data showed the presence of Hphpa trisulfate, MgArg and
N-Me Arg, which lacked a prenyl group. The sequence of 2
was deduced by HMBC (Hphpa trisulfate H-2/N-MeArg
C-11) and NOESY correlations (N-MeArg H-17/MgArg
1
H-19). The correspondence of H and 13C NMR spectra
at C-1, C-2, C-12, C-18 (C-24 in 1) between 1 and 2
indicated that the absolute stereochemistry of 2 was
1R,2R,12S,18S.
Figure 3. Dd (dS2dR) values in ppm obtained at 600 MHz for (R)- and (S)-
PGME amides of MdoArg.

7648
K. Ishida et al. / Tetrahedron 58 (2002) 7645–7652
Table 2. Physical and chemical data for Hphpa and norephedrine
[a]2D1 (EtOH)
1H NMR (DMSO-d6, 300 K)
H-1
H-2
Hphpa from 1
231.38 (c 0.0667)
þ35.08 (c 1.0)
235.38 (c 1.0)
229.88 (c 0.5)
þ31.28 (c 1.0)
4.27 (dd, J¼9.9, 3.4 Hz)
4.90 (br)
4.90 (br)
3.16 (m)
3.38 (br)
3.38 (br)
3.24 (m)
3.24 (m)
(1S,2R) Norephedrine
(1R,2S) Norephedrine
(1R,2R ) Norephedrine
(1S,2S) Norephedrine
4.39 (dd, J¼9.0, 3.4 Hz)
4.39 (dd, J¼9.0, 3.4 Hz)
Table 3. NMR data for aeruginoguanidines 98-B (2) and 98-C (3) in DMSO-d6
Aeruginoguanidine 98-B (2)
Aeruginoguanidine 98-C (3)
13C
1H J (mult, Hz)
13C
1H J (mult, Hz)
Hphpa trisulfate
1
2
3
4
5
6
7
8
Hphpa trisulfate
1
2
3
4
5
6
7
8
77.5 d
48.7 d
14.6 q
132.9 s
127.0 d
128.9 s
149.7 s
120.5 d
126.8 d
62.9 t
5.16 d 5.8
4.07 qdd 7.1, 6.9, 5.8
0.92 d 6.9
77.5 d
48.9 d
14.5 q
132.8 s
127.1 d
128.9 s
149.8 s
120.5 d
126.6 d
62.9 t
5.16 d 6.4
4.06 qdd 6.8, 6.7, 6.4
0.92 d 6.8
7.15 d 2.3
7.17 s
7.30 d 8.6
7.30 d 8.4
7.06 d 8.4
4.92 d 12.8
4.97 d 12.8
7.57 d 6.7
9
7.06 dd 8.6, 2.3
4.92 d 12.8
4.97 d 12.8
7.54 d 7.1
9
10a
10b
2–NH
10a
10b
2–NH
N-Me Arg
11
12
13a
13b
14a
14b
15
16
17
MpArg
11
12
13a
13b
14
15a
15b
16
17
18
169.5 s
56.5 d
25.0 t
169.6 s
56.7 d
21.7 t
4.86 dd 10.2, 5.4
1.66 m
1.80 m
1.32 m
1.40 m
3.10 m
4.84 dd 9.5, 5.9
1.62 br
1.77 br
1.40 m
3.18 m
3.22 m
25.4 t
23.8 t
46.9 t
40.2 t
158.6 s
30.6 q
155.6 s
45.7 t
118.3 d
2.99 s
7.43 t 5.9
3.87 br
5.08 d 6.4
15–NH
MgArg
18
19
20a
20b
21b
22a
22b
23
24
25
26
27a
27b
28
29
30
20
21
22
16vNH
MdoArg
23
25.4 q
17.8 q
30.6 q
1.69 s
1.65 s
3.00 s
7.16 br
169.4 s
58.0 d
25.1 t
4.48 br
1.81 m
1.97 m
1.79 m
3.20 br
3.35 br
168.4 s
58.0 d
25.1 t
46.4 t
24
4.47 br
1.82 br
1.97 br
1.63 br
1.78 br
3.23 m
3.37 br
25a
25b
26a
26b
27a
27b
28
29
30
31
32
155.7 s
45.3 d
119.0 d
140.3 s
31.42 t
3.92 brd 6.5
5.12 td 6.5,1.8
24.9 t
46.7 t
2.05 m
2.07 m
2.04 m
5.09 m
155.7 s
45.6 t
118.9 d
140.5 s
27.8 t
25.9 t
3.92 br
5.12 t 6.1
123.8 d
131.3 s
25.5 q
17.6 q
23.0 q
31.38 q
31
32
33
34
19–NH
23vNH
1.64 d 0.7
1.57 d 1.2
1.72 d 1.1
2.52 br
8.76 br
7.17 br
2.06 m
1.45 m
1.52 m
3.85 m
33a
33b
34
33.1 t
73.3 d
148.2 s
109.9 t
35
36a
36b
37
38
39
4.74 br
4.88 br
1.64 br
1.70 s
2.52 s
8.76 br
7.18 br
17.8 q
23.0 q
31.4 q
24–NH
28vNH

K. Ishida et al. / Tetrahedron 58 (2002) 7645–7652
7649
The fragment pattern of the positive FABMS of aerugino-
guanidine 98-C (3) using glycerol as a matrix revealed the
presence of three sulfate groups. The molecular formula of 3
was established to be C39H67N9O15S3 by the HRFABMS
at dH 2.49 and dC 39.5 were used as internal references for
1H and 13C NMR spectra, respectively. FAB mass spectra,
containing high resolution FABMS, were recorded by
employing a JEOL JMS SX-102 mass spectrometer.
1
and NMR spectral data (Table 3). The H and 13C NMR
spectra of 3 resembled those of 1. The 1H–1H COSY,
HMQC and HMBC data showed the presence of Hphpa
4.2. Culture conditions
1
trisulfate and MpArg. The H and 13C NMR spectrum of 3
Culture conditions were the same as previously described.4b
4.3. Extraction and isolation
lacked methine and methyl signals (dH 5.10, dC 123.8 and
dH 1.64, dC 25.5) of geranyl group in 1, but revealed the
presence of an oxymethine and a terminal methylene signals
(dH 3.85, dC 73.3; dH 4.74, 4.88, dC 109.9). Therefore, 3 was
suggested to be substituted MhdoArg ((Z)-N a-methyl-N v-
6-hydroxy-3,7-dimethyl-2,7-octadienylarginine), which
was deduced by 1H–1H COSY, HMQC and HMBC spectra,
in place of MgArg of 1. The sequence of 3 was deduced by
HMBC (Hphpa trisulfate H-2/N-MeArg C-1) and NOESY
correlations (MpArg H-22/MhdoArg H-24).
Freeze-dried alga (M. aeruginosa (NIES-98); 115.4 g from
420 L of culture) was extracted with 80% MeOH (2 L£3)
and MeOH (1.8 L£1). Combined 80% MeOH and MeOH
extracts were concentrated to an aqueous suspension which
was then extracted with ether. The aqueous layer was
extracted with n-BuOH. The n-BuOH layer was evaporated
under reduced pressure to green dry solid (11.4 g), which
was subjected to flash chromatography on ODS (YMC-ODS
1
The correspondence of H and 13C NMR spectra at C-1,
AM120A, 10£7 cm) with aqueous MeOH (20, 30, 40, 50,
˚
C-2, C-12, C-24 between 1 and 3 indicated that the
absolute stereochemistry of 3 was 1R,2R,12S,24S.
Studies on the absolute configuration at C-34 is now in
progress.12
60, 100%) followed by CH2Cl2.
The 40% (377 mg) and 50% MeOH (535 mg) fractions were
combined, concentrated and subjected to flash chromato-
˚
graphy on ODS (YMC-ODS AM120A, 5£8 cm) with
2.3. Biological activities
aqueous MeOH (20, 30, 40, 50, 60, 100%) followed by
CH2Cl2. The 40% MeOH (113 mg) fraction was subjected
to reversed-phase HPLC (Cosmosil 5C18-MS, 10£250 mm;
20–50% MeCN containing 0.05% TFA; UV-detection at
210 nm; flow rate 2.0 mL/min) to yield 2 (9.6 mg). The 50%
MeOH fraction (249 mg) was subjected to reversed-phase
Aeruginoguanidines 98-A–98-C showed moderate cyto-
toxicity against P388 leukemia cells with IC50 values of 26,
27 and 50 mg/mL, respectively.
˚
HPLC (YMC-Pack AM-324 120A, 10£300 mm; 20–44%
MeCN containing 0.05% TFA; UV-detection at 210 nm;
flow rate 2.0 mL/min) to yield 1 (38.6 mg) and 2 (41.3 mg).
The fraction (19.1 mg) containing 3 was subjected to
3. Discussion
Aeruginoguanidines 98-A–98-C (1–3) are novel unusual
peptides consisting of Hphpa trisulfate and two N-Me Arg
derivatives. Since all these units are very unique structures,
their biosynthesis pathway is interesting.
˚
reversed-phase HPLC (YMC-Pack AM-324 120A,
10£300 mm; 23–28% MeCN containing 0.05% TFA;
UV-detection at 210 nm; flow rate 2.0 mL/min) to yield 3
(12.9 mg). The MeOH fraction (230 mg) was subjected to
˚
reversed-phase HPLC (YMC-Pack AM-324 120A,
Aeruginoguanidine 98-A is also produced by the other
strains (NIES-91, 99, 101, 299) besides M. aeruginosa
(NIES-98).13 Among these strains, M. aeruginosa (NIES-
299) produced aeruginoguanidine 98-A up to about 1 g per
100 g of dried algal cells. To our knowledge, a secondary
metabolite which is produced over 1% of dried algal cells in
cyanobacteria has never been known. Aeruginoguanidines
are composed of very hydrophylic part and hydrophobic
part. Moreover, aeruginoguanidines are insoluble in H2O
and many organic solvents at a concentration of 1 mg/mL.
Therefore, we are interested in how these peptides are kept
in the cyanobacterial cells.
10£300 mm; 32% MeCN containing 0.05% TFA; UV-
detection at 210 nm; flow rate 2.0 mL/min) to yield 1
(109.3 mg).
4.3.1. Aeruginoguanidine 98-A (1). [a]2D8¼þ4.98 (c 1.0,
DMF); UV (60% EtOH) lmax 268 nm (1 1010); IR (KBr)
1660, 1620, 1260, 1050, 1010 cm21; HRFABMS m/z
980.3946 [M2H]2 (C39H66N9O14S3, D 25.5 mmu).
4.3.2. Aeruginoguanidine 98-B (2). [a]2D8¼þ3.78 (c 1.0,
DMF); UV (60% EtOH) lmax 269 nm (1 1420); HRFABMS
m/z 912.3276 [M2H]2 (C34H58N9O14S3, D þ1.1 mmu),
HMBC correlations: H-1/C-3, C-4, C-5 and C-9, H-2/C-1
and C-11, H-3/C-1 and C-2, H-5/C-1, C-7, C-9 and C-10,
H-8/C-4, C-6 and C-7, H-9/C-1, C-4 and C-7, H-10a/C-5,
C-6 and C-7, H-10b/C-5, C-6 and C-7, 2-NH/C-11, H-12/
C-11, C-13, C-14 and C-18, H-15/C-13, C-14 and C-16,
H-17/C-11 and C-18, H-22a/C-23, H-24/C-23, C-25 and
C-26, H-25/C-27 and C-33, H-27a/C-25, C-26, C-28, C-29
and C-33, H-27b/C-25, C-26, C-28, C-29 and C-33, H-28/
C-27, C-29 and C-30, H-29/C-28, C-31 and C-32, H-31/
C-29, C-30, C-32, H-32/C-29, C-30 and C-31, H-33/C-25
and C-27, H-34/C-19.
4. Experimental
4.1. General experimental procedures
UV spectrum was recorded on a Hitachi 330 spectro-
photometer. Optical rotation was measured on a JASCO
DIP-1000 polarimeter. IR spectrum was measured on a
JASCO FT/IR-5300 spectrometer. 1H and 13C NMR spectra
were obtained with either Bruker AM600 or JEOL A600 in
DMSO-d6 at 27.08C. The resonances of residual DMSO-d6

7650
K. Ishida et al. / Tetrahedron 58 (2002) 7645–7652
4.3.3. Aeruginoguanidine 98-C (3). [a]2D8¼21.48 (c 0.5,
DMF); UV (50% MeOH) lmax 265 nm (1 1030);
HRFABMS m/z 996.3851 [M2H]2 (C39H66N9O15S3, D
þ1.1 mmu), HMBC correlations: H-1/C-2, C-3, C-4 and
C-5, H-2/C-1, C-3 and C-4, H-3/C-1 and C-2, H-5/C-1, C-7,
C-9 and C-10, H-8/C-4, C-6 and C-7, H-9/C-1, C-5 and C-7,
H-10a/C-5, C-6 and C-7, H-10b/C-5, C-6 and C-7, 2-NH/
C-11, H-12/C-11, C-14 and C-22, H-15a/C-14, C-16 and
C-17, H-17/C-15, C-16, C-18 and C-19, H-18/C-20 and
C-21, H-20/C-18, C-19 and C-21, H-21/C-18, C-19 and
C-20, H-22/C-12 and C-23, H-29/C-27, C-30 and C-31,
H-30/C-32 and C-38, H-32/C-31, H-34/C-32, C-33, C-35,
C-36 and C-37, H-36a/C-34 and C-37, H-36b/C-34 and
C-37, H-37/C-34, C-35 and C-36, H-38/C-30, C-31 and
C-32, H-39/C-24.
room temperature for 1.5 h. After the solvent was removed
by lyophilization, the reaction mixture was subjected to
reversed-phase HPLC (Cosmosil 5C18-MS, 10£250 mm;
10–70% MeCN containing 0.05% TFA; UV-detection
210 nm; flow rate 2.0 mL/min) to yield N a-methyl-N v-
isoamylarginine (S)-PGME amide (2.3 mg): HRFABMS
1
m/z 406.2780 [MþH]þ (C21H36N5O3, D 23.8 mmu); H
NMR specrum (CD3OD; 300 K), N a-methyl-N v-isoamyl-
arginine d 0.980 (d, J¼6.8 Hz, 6H, H-10 and 100), 1.552 (m,
2H, H-8), 1.670 (m, 1H, H-9), 1.788 (m, 1H, H-4), 1.920 (m,
2H, H-3 and H-4), 1.970 (m, 1H, H-3), 2.630 (s, 3H, N-Me),
3.381 (m, 2H, H-5), 3.420 (m, 2H, H-7), 3.862 (dd, J¼7.3,
4.3 Hz, 1H, H-2), (S)-PGME d 3.710 (s, 3H, CO2Me), 5.520
(s, 1H, H-2), 7.370–7.426 (m, 5H, phenyl). N a-methyl-N v-
isoamylarginine (R)-PGME amide was also derivatized as
described above. N a-methyl-N v-isoamylarginine (R )-
PGME amide (0.9 mg): HRFABMS m/z 406.2818
4.3.4. MiArg (N a-methyl-N v-isoamylarginine) and
MdoArg (N a-methyl-N v-2,6-dimethyloctylarginine). 1
(100 mg) was dissolved in DMF (3.0 mL) and EtOH
(5.0 mL) and then 30 mg of palladium black was added to
a solution, and the mixture was stirred at room temperature
for 4 days under hydrogen. The reaction mixture was filtered
and concentrated. This crude residue was dissolved in DMF
(3.0 mL) and 6 M HCl (5.0 mL), and heated at 1108C for 2.5
days. After the solvent was removed by evaporation, the
reaction mixture was subjected to reversed-phase HPLC
(Cosmosil 5C18-MS, 10£250 mm; 0–80% MeCN contain-
ing 0.05% TFA; UV detection at 210 nm; flow rate
2.0 mL/min) to yield MdoArg (33.4 mg) and a fraction
containing MiArg (32.8 mg). The fraction containing
MiArg was further subjected to reversed-phase HPLC
(Cosmosil 5C18-MS, 10£250 mm; 13% MeCN containing
0.05% TFA; UV detection at 210 nm; flow rate 2.0 mL/min)
to yield MiArg (16.9 mg). N a-methyl-N v-isoamylarginine:
[a ]2D3¼þ18.68 (c 0.1, MeOH); HRFABMS m/z 259.2159
1
[MþH]þ (C21H36N5O3, D 0.0 mmu); H NMR spectrum
(CD3OD; 300 K), N a-methyl-N v-isoamylarginine d 0.915
(d, J¼6.8 Hz, 3H, H-10), 0.920 (d, J¼6.4 Hz, 3H, H-100),
1.420 (m, 2H, H-7), 1.445 (m, 1H, H-4), 1.556 (m, 1H, H-9),
1.576 (m, 1H, H-4), 1.839 (m, 2H, H-3), 2.720 (s, 3H,
N-Me), 3.190 (m, 2H, H-7), 3.220 (ddd, J¼9.8, 6.0, 3.8 Hz,
2H, H-5), 3.890 (t J¼6.8 Hz, 1H, H-2), (R )-PGME d 3.720
(s, 3h, CO2Me), 5.560 (s, 1H, H-2), 7.360–7.420 (m, 5H,
phenyl).
4.3.6. N a-Methyl-N v-2,6-dimethyloctylarginine (S,R )-
PGME amides. A solution of N a-methyl-N v-dimethyl-
octylarginine (3.0 mg) in DMF (0.5 mL) was added with
(S )-PGME (2.2 mg), PyBOP (7.0 mg), HOBt (1.8 mg) and
N-methylmorphorine (3.0 mL) at 08C, and the mixture was
stirred at room temperature for 1.5 h. After the solvent was
removed by lyophilization, the reaction mixture was
subjected to reversed-phase HPLC (Cosmosil 5C18-MS,
10£250 mm; 20–100% MeCN containing 0.05% TFA;
UV-detection 210 nm; flow rate 2.0 mL/min) to yield
N a-methyl-N v-dimethyloctylarginine (S)-PGME amide
1
[MþH]þ C12H27N4O2, D þ2.5 mmu); H NMR spectrum
(DMSO-d6; 300 K), d 0.87 (m, 6H, H-10 and 100), 1.37 (m,
2H, H-8), 1.55 (m, 2H, H-4 and H-9), 1.58 (m, 1H, H-3),
1.70 (m, 1H, H-4), 1.77 (m, 1H, H-3), 2.45 (s, 3H, N-Me),
3.12 (m, 1H, H-5), 3.23 (m, 2H, H-7), 3.35 (m, 1H, H-2),
3.40 (m, 1H, H-5), 13C NMR spectrum (DMSO-d6; 300 K),
22.2 (C-10 and C-100), 23.0 (C-4), 25.4 (C-9), 25.6 (C-3),
32.0 (N-Me), 35.5 (C-8), 46.8 (C-7), 47.6 (C-5), 59.5 (C-2),
N a-methyl-N v-2,6-dimethyloctylarginine: [a]2D3¼þ18.68
(c 0.1, MeOH); HRFABMS m/z 329.2874 [MþH]þ
(2.0 mg):
(C26H46N5O3,
HRFABMS
m/z
476.3591
[MþH]þ
D
21.0 mmu); 1H NMR spectrum
(CD3OD; 300 K), N a-methyl-N v-dimethyloctylarginine d
0.877 (m, 6H, H-14 and 140), 0.965 (d, J¼6.4 Hz, 3H, H-90),
1.174 (m, 1H, H-10), 1.175 (m, 1H, H-12), 1.280 (m, 1H,
H-11), 1.380 (m, 1H, H-10), 1.480 (m, 1H, H-8), 1.520 (m,
1H, H-9), 1.540 (m, 1H, H-13), 1.680 (m, 1H, H-8), 1.800
(m, 1H, H-4), 1.924 (m, 1H, H-4), 1.948 (m, 2H, H-3), 2.630
(s, 3H, N-Me), 3.398 (m, 2H, H-5), 3.428 (m, 2H, H-7),
3.870 (dd, J¼7.7 Hz, 4.3, 1H, H-2), (S)-PGME d 3.720 (s,
3H, CO2Me), 5.515 (s, 1H, H-2), 7.380–7.440 (m, 5H,
phenyl). N a-methyl-N v-dimethyloctylarginine (R )-PGME
amide was also derivatized as described above. N a-methyl-
N v-dimethyloctylarginine (R )-PGME amide (4.7 mg):
HRFABMS m/z 476.3591 [MþH]þ (C26H46N5O3, D
21.0 mmu); 1H NMR spectrum (CD3OD; 300 K), N a-
methyl-N v-dimethyloctylarginine d 0.879 (d, J¼6.4 Hz,
3H, H-14), 0.881 (d, J¼6.8 Hz, 3H, H-140), 0.901 (d,
J¼6.4 Hz, 3H, H-90), 1.120 (m, 1H, H-10), 1.172 (m, 2H,
H-12), 1.272 (m, 1H, H-11), 1.310 (m, 1H, H-10), 1.340 (m,
1H, H-8), 1.368 (m, 1H, H-11), 1.425 (m, 1H, H-9), 1.450
(m, 1H, H-4), 1.530 (m, 1H, H-13), 1.545 (m, 1H, H-8),
1.590 (m, 1H, H-4), 1.850 (m, 2H, H-3), 2.720 (s, 3H,
N-Me), 3.205 (m, 2H, H-7), 3.220 (m, 2H, H-5), 3.900 (dd,
1
(C17H37N4O2, D 24.3 mmu); H NMR spectrum (DMSO-
d6; 300 K), d 0.84 (m, 6H, H-14 and 140), 0.87 (m, 3H, H-90),
1.07 (m, 1H, H-10), 1.12 (m, 2H, H-12), 1.20 (m, 1H, H-11),
1.26 (m, 1H, H-10), 1.28 (m, 1H, H-11), 1.30 (m, 1H, H-7),
1.41 (m, 1H, H-9), 1.51 (m, 2H, H-8 and H-13), 1.56 (m, 1H,
H-4), 1.65 (m, 1H, H-3), 1.69 (m, 1H, H-4), 1.79 (m, 1H,
H-3), 2.52 (s, 3H, N-Me), 3.20 (m, 1H, H-5), 3.25 (m, 2H,
H-7), 3.36 (m, 1H, H-5), 3.60 (m, 1H, H-2), 13C NMR
spectrum (DMSO-d6; 300 K), 19.1 (C-90), 22.5 (C-4), 22.6
(C-14 and C-140), 24.0 (C-11), 25.5 (C-3), 27.5 (C-13), 30.0
(C-9), 31.2 (N-Me), 34.0 (C-8), 36.5 (C-10), 38.8 (C-12),
46.5 (C-7), 47.5 (C-5), 61.0 (C-2).
4.3.5. N a-Methyl-N v-isoamylarginine (S,R )-PGME
amides. A solution of N a-methyl-N v-isoamylarginine
(3.0 mg) in DMF (0.5 mL) was added with (S )-PGME
(2.2 mg), PyBOP (7.0 mg), HOBt (1.8 mg) and N-methyl-
morphorine (3.0 mL) at 08C, and the mixture was stirred at

K. Ishida et al. / Tetrahedron 58 (2002) 7645–7652
7651
J¼7.7, 5.6 Hz, 1H, H-2), (R)-PGME d 3.720 (s, 3H,
J¼6.8 Hz, 1H), 7.33–7.40 (m, 4H), 7.92–8.14 (br, 2H),
(1R,2S ) norephedrine: [a ]2D1¼235.38 (c 1.0, EtOH); 1H
NMR spectrum was in correspondence with (1S,2R )
norephedrine.
CO2Me), 5.560 (s, 1H, H-2), 7.370–7.450 (m, 5H, phenyl).
4.3.7. Hphpa (1-(4-hydroxy-3-hydroxymethyl)phenyl-1-
hydroxy-2-propylamine). 1 (50 mg) was dissolved in 3 M
HCl (5.0 mL), and heated at 1008C for 3 h. After the solvent
was removed by lyophilization, the reaction mixture was
subjected to reversed-phase HPLC (Cosmosil 5C18-MS,
10£250 mm; 5% MeCN containing 0.05% TFA; UV
detection at 210 nm; flow rate 2.0 mL/min) to yield Hphpa
(0.8 mg); [a ]2D1¼231.38 (c 0.0667, EtOH); HRFABMS m/z
(1S,2R ) Norephedrine (100 mg) was dissolved in pyridine
(0.5 mL) and acetic anhydride (0.5 mL), and the mixture
was stirred at room temperature for 16 h. After the solvent
was removed by evaporation and lyophilization, the reaction
mixture was dissolved in EtOAc, washed with H2O,
saturated aqueous NaCl, and dried over MgSO4 to yield
the crude diacetate. This crude material was dissolved in
MeOH (0.75 mL) and 4N NaOH (0.25 mL), and the mixture
was stirred at room temperature for 30 min. The reaction
mixture was concentrated, neutralized with 1 M HCl,
dissolved in EtOAc, washed with H2O, saturated aqueous
NaCl, and dried over MgSO4 to yield the crude acetylamide.
1
198.1147 [MþH]þ (C10H16NO3, D þ1.6 mmu); H NMR
spectrum (DMSO-d6, 300 K), d 0.90 (d, J¼6.8 Hz, 3H,
H-3), 3.16 (m, 1H, H-2), 4.27 (dd, J¼9.9, 3.4 Hz, 1H, H-1),
4.47 (br, 2H, H-70), 4.99 (t J¼4.7 Hz, 1H, 70-OH), 5.99 (d,
J¼3.4 Hz, 1H, 1-OH), 6.74 (d, J¼8.1 Hz, 1H, H-50), 7.01
(brd, 1H, H-60), 7.27 (br, 1H, H-20), 7.78-7.88 (br, 2H, NH2),
9.41 (s, 1H, 40-OH); 13C NMR spectrum (DMSO-d6,
300 K), d 15.1 (C-3), 52.4 (C-2), 58.0 (C-70), 74.6 (C-1),
114.1 (C-50), 125.8 (C-20 and C-60).
To this crude acetylamide, 300 mL of thionyl chloride was
added at 08C. The mixture was stirred at 08C for 15 min
under argon, and the solvent was removed by lyophilization.
This reaction mixture was dissolved in 6 M HCl (2.0 mL),
and heated at 1008C for 3 h under argon. After the solvent
was removed by lyophilization, the reaction mixture was
subjected to reversed-phase HPLC (Cosmosil 5C18-MS,
10£250 mm; 6% EtOH containing 0.05% TFA; UV-
detection 210 nm; flow rate 2.0 mL/min) to yield (1R,2R )
norephedrine (86 mg; 86%): [a ]2D1¼229.88 (c 0.5, EtOH);
1H NMR (DMSO-d6, 600 MHz), d 0.93 (d, J¼6.4 Hz, 3H),
3.24 (m, 1H), 4.39 (dd, J¼9.0, 3.4 Hz, 1H), 6.20 (d,
J¼3.4 Hz, 1H), 7.31–7.40 (m, 5H), 7.78–7.90 (br, 2H).
4.3.8. (R,S )-BPG-Hphpa Amides. A solution of Hphpa
(0.2 mg) in DMF (0.4 mL) was added with (S )-BPG
(2.0 mg), PyBOP (4.0 mg), HOBt (2.0 mg) and N-methyl-
morphorine (10 mL) at 08C, and the mixture was stirred at
room temperature for 12 h. After the solvent was removed
by lyophilization, the reaction mixture was subjected to
reversed-phase HPLC (Cosmosil 5C18-MS, 10£250 mm;
10–90% MeCN containing 0.05% TFA; UV-detection 210
nm; flow rate 2.0 mL/min) followed by reversed-phase
HPLC (Cosmosil 5C18-MS, 10£250 mm; 35% MeCN
containing 0.05% TFA; UV-detection 210 nm; flow rate
2.0 mL/min) to yield (S)-BPG-Hphpa amide (0.1 mg):
HRFABMS m/z 431.2207 [MþH]þ (C23H31N2O6, D
1
(1S,2S) Norephedrine ([a]2D1¼þ31.28 (c 1.0, EtOH); H
NMR spectrum was correspondence with (1S,2S ) nor-
ephedrine) was obtained from (1R,2S ) norephedrine
(100 mg) in 82% yield.
1
þ2.5 mmu); H NMR spectrum (DMSO-d6, 300 K), (S)-
BPG d 1.382 (s, 9H, tert-butyl), 5.130 (d, J¼7.3 Hz, 1H,
H-2), 7.224 (m, 1H, phenyl), 7.241 (d, J¼7.3 Hz, 1H, NH),
7.275 (t J¼6.8 Hz, 2H, phenyl), 7.315 (m, 2H, phenyl),
Hphpa d 0.800 (d, J¼6.4 Hz, 3H, H-3), 3.847 (m, 1H, H-2),
4.415 (d, J¼5.1 Hz, 1H, H-1), 4.448 (s, 2H, H-70), 6.660 (d,
J¼8.1 Hz, 1H, H-50), 6.922 (brd, J¼8.1 Hz, 1H, H-60), 7.234
(s, 1H, H-20), 7.708 (d, J¼8.6 Hz, 1H, NH), 9.160 (s, 1H,
40-OH). (R )-BPG-Hphpa amide was also derivatized as
described above. (R )-BPG-Hphpa amide (0.1 mg):
HRFABMS m/z 431.2207 [MþH]þ (C23H31N2O6, D
4.4. Assays for cytotoxic activity
P388 murine leukemia cells were cultured at 378C in a
humidified atmosphere of 5% CO2 in 95% air, with RPMI-
1640 media containing 10% fetal bovine serum,
500 units/mL penicillin, and 50 mg/mL streptomycin. The
cells (2.5£104 cells/mL) were incubated with samples
(ranging in concentration from 25 to 0.01 mg/mL) in 0.2 mL
of cultured medium at 378C for 3 days. After incubation, the
cells were treated with tetrazolium salt (MTT) for 4 h, the
plate was centrifuged at 3000g for 5 min, and the resulting
supernatant was removed by suction. The precipitates were
dissolved in dimethyl sulfoxide, and absorbance was
measured by a microtiter plate reader at a wavelength of
570 nm (reference wavelength at 630 nm). Cytotoxic
activity was evaluated based on a comparison of the
survival ratio of the cells with the control value.
1
þ2.5 mmu); H NMR spectrum (DMSO-d6, 300 K), (R)-
BPG d 1.376 (s, 9H, tert-butyl), 5.141 (d, J¼9.0 Hz, 1H,
H-2), 7.224 (m, 1H, phenyl), 7.241 (d, J¼7.3 Hz, 1H, NH),
7.275 (t J¼6.8 Hz, 2H, phenyl), 7.315 (m, 2H, phenyl),
Hphpa d 0.921 (d, J¼6.8 Hz, 3H, H-3), 3.870 (m, 1H, H-2),
4.338 (m, 1H, H-1), 4.402 (brd, J¼5.6 Hz, 2H, H-70), 4.846
(t J¼5.6 Hz, 1H,0 70-OH), 5.188 (br, 01H, 1-OH), 6.506 (m,
2H, H-50 and H-6 ), 7.115 (s, 1H, H-2 ), 7.672 (d, J¼8.1 Hz,
1H, NH), 9.092 (s, 1H, 40-OH).
4.3.9. Norephedrine. (1S,2R ) and (1R,2S) norephedrines
were purchased from Wako Pure Chemical Industries, and
subjected to reversed-phase HPLC (Cosmosil 5C18-MS,
10£250 mm; 8% EtOH containing 0.05% TFA; UV-
detection 210 nm; flow rate 2.0 mL/min) to yield (1S,2R )
norephedrine: [a ]D21¼þ35.08 (c 1.0, EtOH); 1H NMR
(DMSO-d6, 600 MHz), d 0.92 (d, J¼6.8 Hz, 3H), 3.38 (br,
1H), 4.90 (br, 1H), 6.01 (d, J¼1.7 Hz, 1H), 7.29 (t,
Acknowledgments
We indebted the Cell Resource Center for Biomedical
Research, Tohoku University for providing the murine
leukemia P388 cells. We thank for Mr H. Nakagawa, the
University of Tokyo, for valuable advise on the cytotoxic
assays. This work was partly supported by a Grant-in-Aids
for ‘Research for the Future’ Program from the Japan

7652
K. Ishida et al. / Tetrahedron 58 (2002) 7645–7652
5. (a) Okino, T.; Matsuda, H.; Murakami, M.; Yamaguchi, K.
Tetrahedron Lett. 1993, 34, 501–504. (b) Ishida, K.; Matsuda,
H.; Murakami, M.; Yamaguchi, K. Tetrahedron 1997, 53,
10281–10288. (c) Ishida, K.; Matsuda, H.; Murakami, M.
Tetrahedron 1998, 54, 13475–13484. (d) Ishida, K.; Kato, T.;
Murakami, M.; Watanabe, M.; Watanabe, M. F. Tetrahedron
2000, 56, 8643–8656.
Society for the Promotion of Science. K. I. is financially
supported by Research Fellowships of the Japan Society for
the Promotion of Science for Young Scientists.
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Murakami, M. Tetrahedron 1999, 55, 10971–10988.
´
11. Seco, J. M.; Quin˜oa, E.; Riguera, R. J. Org. Chem. 1999, 64,
4669–4675.
12. Although we tried the esterification of 3 with MTPA by the
improved Mosher method, this experiment resulted only in
recovered the starting material.
13. Aeruginoguanidine 98-A was obtained 202.7 mg, 189.9 mg,
99.8 mg, and 898.7 mg/100 g of dried alagal cells from strains
NIES-91, 99, 101, and 299 of M. aeriuginosa, respectively.