Cyclic depsipeptides, ichthyopeptins A and B, from Microcystis ichthyoblabe

Article abstract of DOI:10.1021/np060303s

Bioassay-guided isolation of antiviral compounds from the cultured cyanobacterium Microcystis ichthyoblabe provided two novel cyclic depsipeptides, ichthyopeptins A (1) and B (2). Their structures were determined by 1D ( ¹H and ¹³C) and 2D (COSY, TOCSY, ROESY, HMQC, and HMBC) NMR spectra, ESIMS-MS, and amino acid analysis. The fraction containing both cyclic depsipeptides exhibited antiviral activity against influenza A virus with an IC₅₀ value of 12.5 μg/mL.

Full text of DOI:10.1021/np060303s

1084
J. Nat. Prod. 2007, 70, 1084-1088
Cyclic Depsipeptides, Ichthyopeptins A and B, from Microcystis ichthyoblabe
Elmi N. Zainuddin,†, Renate Mentel,† Victor Wray,§ Rolf Jansen,§ Manfred Nimtz,§ Michael Lalk,† and Sabine Mundt*,†
Institute of Pharmacy, Friedrich-Ludwig-Jahnstrasse 17, and Institute of Medical Microbiology, Martin-Luther-Strasse 6, Ernst-Moritz-Arndt
UniVersity, D-17487 Greifswald, Germany, and Helmholtz Centre for Infection Research, Inhoffenstrasse 7, D-38124 Braunschweig, Germany
ReceiVed June 30, 2006
Bioassay-guided isolation of antiviral compounds from the cultured cyanobacterium Microcystis ichthyoblabe provided
two novel cyclic depsipeptides, ichthyopeptins A (1) and B (2). Their structures were determined by 1D (1H and 13C)
and 2D (COSY, TOCSY, ROESY, HMQC, and HMBC) NMR spectra, ESIMS-MS, and amino acid analysis. The
fraction containing both cyclic depsipeptides exhibited antiviral activity against influenza A virus with an IC50 value of
12.5 µg/mL.
One of the strategies to combat viral infectious diseases is the
search for new metabolites from natural products as antiviral agents.
Cyanobacteria have been known to be an enormous resource for
compounds with varying bioactivities including antimicrobial,
antiviral, and enzyme inhibitory effects. One of the compounds with
antiviral potential derived from cyanobacteria is cyanovirin, a
protein with 101 amino acids isolated from Nostoc ellipsosporum.1
Several compounds such as cyanopeptolins, micropeptins, oscilla-
peptins, nostopeptins, agardhipeptins, anabaenopeptins, nodulapep-
tins, microviridins, and aeruginosins are protease inhibitors. They
are widely distributed in species of cyanobacteria such as Micro-
cystis, Oscillatoria, Nostoc, Nodularia, and Anabaena, and most
compounds are cyclic depsipeptides containing the 3-amino-6-
hydroxy-2-piperidone (Ahp) unit.2
Our previous investigations of several cultured strains of cyano-
bacteria have shown antiviral effects against influenza A virus,
whereas the propagation of respiratory syncytial virus, herpes
simplex virus, and adenovirus was not affected.3-5 Here we report
the bioassay-guided fractionation of a methanolic extract of
Microcystis ichthyoblabe that has resulted in the isolation and
identification of two novel cyclic depsipeptides containing the Ahp
unit, ichthyopeptins A (1) and B (2).
Results and Discussion
As the strongest activity during screening against influenza A
virus was found for the methanolic extract of M. ichthyoblabe,6
this extract was selected for bioassay-guided isolation. Fractionation
of the methanolic extract on silica gel using an elution system from
100% EtOAc to 100% MeOH and to 100% H2O resulted in nine
fractions, M-1 to M-9. All fractions were initially tested for
cytotoxicity against MDCK cells, but no cytotoxicity was observed.
However, antiviral activity was detected in an in vitro system using
influenza A virus/MDCK cells and showed the highest activity in
fraction M-2, which eluted with 75% EtOAc/MeOH. This fraction
was further separated on a silica gel column using a gradient system
from 100% EtOAc to 100% MeOH. Of the 11 fractions obtained,
three fractions, M2-2, M2-4, and M2-6, showed peaks in the
analytical HPLC. Fraction M2-4 was selected for further separation
and purification by preparative RP-HPLC with a gradient system
of CH3CN/H2O, resulting in two pure fractions, M2-4-P1 (tR 17.93
min, 8.2 mg) and M2-4-P2 (tR 18.40 min, 7.4 mg), which were
obtained as white, amorphous powders after lyophilization.
The combined NMR and MS data of fraction M2-4-P1 allowed
unambiguous identification of the novel cyclic depsipeptide ich-
thyopeptin A (1). The positive HRESIMS of 1 showed the [M +
H]+ ion at m/z 1043.5080, the dehydrated ion [M - H2O + H]+ at
m/z 1025.5511, and the mono-sodiated ion [M + Na]+ at m/z
1065.4900, which are compatible with a molecular formula of
ichthyopeptin A of C53H70N8O14 (calcd for [M + H]+ 1043.5090).
Amino acid analysis identified five residues (Gln or Glu, Tyr, Val,
Ile, and Thr) in equal ratios in ichthyopeptin A (1).
The assignment of the NMR spectra of 1 was accomplished using
a combination of homonuclear 2D NMR techniques. Spin systems
1
were identified from 2D H COSY and TOCSY spectra, starting
from the backbone amide protons. Sequence specific assignments
1
were determined from the cross-peaks in 2D H ROESY spectra
based on short observable distances between HN, HR, and Hâ of
amino acid i and HN of amino acid i+1, and from three-bond 13C-
1H correlations in 2D HMBC spectra. The full assignments and
chemical shift data are presented in Table 1. Using these techniques,
spin systems corresponding to Gln, Tyr, Val, Phe, Ile, and Thr were
identified in 1. These were confirmed from the long-range correla-
tions in the 2D HMBC spectrum, which indicated the phenylalanine
residue carried an N-methyl group and were compatible with amino
acid analysis of the hydrolysate. The same spectra indicated a spin
* To whom correspondence should be addressed. Phone: ++49-3834-
864869. Fax: ++49-3834-864885. E-mail: smundt@uni-greifswald.de.
† Ernst-Moritz-Arndt-University Greifswald.
§ Helmholtz Centre for Infection Research Braunschweig.
Faculty of Marine Science, Hasanuddin University Tamalanrea, KM10,
Makassar, 90245 Indonesia.
1
system that showed characteristic H and 13C chemical shifts and
10.1021/np060303s CCC: $37.00
© 2007 American Chemical Society and American Society of Pharmacognosy
Published on Web 06/28/2007

Cyclic Depsipeptides from Microcystis ichthyoblabe
Journal of Natural Products, 2007, Vol. 70, No. 7 1085
Table 1. 1H and 13C NMR Data of Ichthyopeptin A (1) in CD3CN/D2O (1:1)
unit
PAA
position
δH
δC
1H correlations to 13C intra-residue; inter-residue
sequential NOEs in 1H ROESYb
1
2
3
4
5, 9
6, 8
7
NH
1
175.7
73.5
40.0
129.3
131.6
115.9
2, 3A, 3B; Gln-2
4.19
2.70, 2.90
Gln-NHa (×2)
7.01
6.67
155.5 or 155.7
Gln
7.71
PAA-3 (×2), Thr-NHa
Thr-NHa
Thr-NHa
173.2
53.0
2, 3; Thr-2, Thr-NHa
3, 4A, 4B
2
3
4
4.27
1.81
1.94, 2.03
28.3 or 30.3
31.7
5
177.9
a
NH2
6.46, 7.14
8.09
Thr
Tyr
NHa
Gln-2, Gln-3, Gln-NHa
1
2
3
171.2
56.1
73.5
18.9
2; Tyr-NHa
Ahp-NHa
4.47
5.45
1.18
8.11
Tyr-NHa
4
NHa
Ahp-NHa, Thr-3
1
172.9
Ahp-NHa
2
4.51
56.4
3
2.64, 3.32
35.9
4
129.6
5,8
6,8
7.00
6.65
130.9
116.1
7
155.7 or 155.5
Ahp
NHa
7.75
Thr-3
Tyr-NHa
Val-4
1
2
171.5
50.3
4.53
3
4
5
1.81, 2.54
1.81
4.98
21.6
∼30.3
75.5
Val
1
2
3
4
172.2
57.7
28.2
2; N-CH3 Phe
4.29
1.93
0.45
Phe-2, Phe-3A, Phe-5,9
18.1
Ahp-2
5
1
2
3
4
5,9
6,8
7
-0.19
18.6
171.7
62.5
Phe-5,9, Phe-6,8
N-Me Phe
3A, 3B; Ile-NHa
N-Me
4.99
2.73, 3.36
Ile-NHa, Val-2
34.7
Ile-NHa (×2), Val-2, Ile-2
138.0
130.6
129.9
127.9
31.6
7.20
7.25
7.16
2.73
7.89
Val-2, Val-5
Val-5
N-Me
Ile
NHa
Phe-2, Phe-3
Phe-3A
1
2
3
4
5
6
174.8
57.5
37.5
25.7
10.6
2; Thr-3
4.36
1.74
1.03, 1.26
0.73
0.80
b
a From the CD3CN/H2O (1:1) spectrum. In all cases the NH always shows a correlation to the directly bonded carbonyl carbon. Each partner
is indicated; hence each correlation appears twice in this column.
COSY correlations of the 3-amino-6-hydroxy-2-piperidone (Ahp)
system that has been documented in other peptides from similar
cyanobacteria.5-8 Finally, the presence of a further Phe-like system
was evident in which the R-H at δH 4.19 was attached to a carbon
with a shift of δC 73.5 indicative of a 2-hydroxy-3-(4′-hydroxy-
phenyl)acetic acid (PAA) derivative. This was confirmed from mass
spectrometric data. A combination of these structural elements was
compatible with the molecular mass of 1042.
Careful inspection of the NOE correlations in the 2D ROESY
spectrum afforded the partial sequence NH-Tyr-Ahp-Val-(N-Me-
Phe)-Ile-CO (Table 1). Long-range correlations in the HMBC
spectrum indicated the sequence NH-Gln-Thr-Tyr and showed that
Ile was attached to the â-hydroxyl group of Thr to form a cyclic
lactone system. Finally the PAA was found to be the acyl residue
attached to the peptidic NH of Gln through the observation of a
correlation of HR of Gln with the CO of PAA. These data afforded
the cyclic peptide structure shown in the Figure 1. Furthermore, a
detailed analysis of the ESIMS/MS (Table 2) confirmed the
sequence assignment. At this stage it was necessary to carefully
look at the long-range correlations in both CD3CN/H2O (1:1) and
CD3CN/D2O (1:1) to establish the mode of attachment of the PAA
residue to the Gln residue. From the observation of long-range
correlations to the two carbonyl carbons at δ 173.2 and 177.9, the
mode of attachment of the PAA residue to the Gln residue was
established as shown in 1. Enantioselective GC-MS analysis
indicated the five regular amino acids (Gln, Tyr, Val, Ile, and Thr)
had the L-configuration. Appropriate reference material was not
available for the three remaining moieties, although their configura-
tions can be inferred from the absolute stereochemistries reported
in the literature for N-Me-Phe8,9 and Ahp and PAA8,10 in related
depsipeptides.
A similar approach afforded the structure of the major component
of fraction M2-4-P2, ichthyopeptin B (2). The positive ion
HRESIMS of 2 identified the [M + H]+ ion at m/z 979.5160,

1086 Journal of Natural Products, 2007, Vol. 70, No. 7
Zainuddin et al.
Table 2. MS-MS Fragmentation of Ichthyopeptin A (1)
However, the fraction containing both cyclic depsipeptides
exhibited a strong antiviral activity against influenza A virus with
an IC50 value of 12.5 µg mL-1, essentially at the same level as
amantadine, with 15 µg mL-1. To date, antiviral activities reported
for cyanobacteria are based on sulfated polysaccharides,11 indolo-
carbazoles,12 chlorine-containing alkaloids,13 and proteins such as
cyanovirin-N14. This protein, consisting of 101 amino acids, is a
fusion inhibitor of HIV with virucidal activity by multivalent
interactions with high mannose oligosaccharides comprising the
HIV glycoprotein envelope and is a potential candidate for
preventing HIV transmission.14 To our knowledge, there are no
reports on the antiviral activity of Ahp-containing cyclic peptides.
However, since the processing of virus proteins is an essential step
in the life cycle of influenza virus in order to generate infective
virus particles, our results indirectly suggest that the mode of action
of the ichthyopeptins may be based on a protease inhibition.
m/z
fragmenta
134.11
195.13
293.22
356.23
376.19
487.34
519.31
539.26
650.41
669
F
X-V(-H2O)
Y*-Q(-H2O)
X-V-F(-H2O)
Y*-Q-T(-2H2O)
X-V-F-L
Y-X-V-F(-H2O)
Y*-Q-T-Y(-2H2O)
Y*-X-V-F-L
Y*-Q-T-L-F
670
Y*-Q-T(-Y)-L
715.44
733.45
751
831.46
894.49
912.50
1025.58
1043.59
Y*-Q-T-Y-X-V(-4H2O)
Y*-Q-T-Y-X-V(-3H2O)
Y*-Q-T-Y-X-V(-2H2O)
Y*-Q-T(-Y)-L-F
Y*-Q-T-Y-X-V-F(-3H2O)
Y*-Q-T-Y-X-V-F(-2H2O)
[M + H - H2O]+
[M + H]+
Experimental Section
General Experimental Procedures. All 1D (1H and 13C) and 2D
(COSY, TOCSY, ROESY, HMQC, and HMBC) NMR spectra were
recorded at 300 K on a Bruker AVANCE DMX-600 NMR spectrometer
locked to the major deuterium resonance of the various solvent systems
used, which included CD3CN/H2O (1:1) and CD3CN/D2O (1:1).
Measurements were carried out with mixing times period of 100 ms
for TOCSY and 500 ms for ROESY. Chemical shifts were referenced
to the residual proton resonance of the acetonitrile signal at δ 1.93
(1H) and δ 1.28 (13C). Mass spectra were recorded on a Micromass
Q-Tof 2 mass spectrometer. Amino acid analysis was performed on
an Applied Biosystems ABI-420-A amino acid analyzer.
a For convenience the following abbreviations are used for the
residues: Y* ) PAA, Q ) Gln, T ) Thr, Y ) Tyr, X ) Ahp, V )
Val, F ) N-Me Phe, I ) Ile, N ) Asn, L ) Leu.
[M - H2O + H]+ at m/z 1067.4955, and [M + Na]+ at m/z
1001.4960, which are compatible with the molecular formula
C49H70N8O13. Five amino acids, Asn or Asp, Leu, Ile, Val, and
Thr, were identified from the amino acid analysis of 2 and were
confirmed from the NMR data (Table 3). The latter also indicated
the presence of spin systems belonging to N-Me-Phe, Ahp, and
PAA moieties, suggesting a further cyclic depsipeptide related to
1. The absolute stereochemistries of 2 have not been determined
but can be inferred from those found for 1. NOE and inter-residue
long-range C-H correlations (Table 3) identified two sequences,
Ile-NMePhe-Val and Asn-Thr-Leu. The absence of an amidic proton
for the Ile residue suggested this residue was attached to Ahp. As
in 1 the low-field shift of H-3 of Thr indicated acylation at this
position through binding the free carboxylic acid group of Val or
PAA. Only the former is compatible with the attachment of the
PAA moiety on Asn as shown for 2. This was confirmed from the
detailed interpretation of the MS-MS fragmentation data shown
in Table 4.
3-Amino-6-hydroxy-2-piperidone (Ahp)-containing cyclic dep-
sipeptides are widely distributed in cyanobacteria.7 Recently,
Yamaki et al.8 published the structure of micropeptins 88-N and
88-Y from Microcystis aeruginosa NIES-88, which have a cyclic
moiety in common with the present structure of ichthyopeptin A
(1). The difference is the nature of the chain attached to the amide
nitrogen of the Thr moiety. Both ichthyopeptins A (1) and B (2)
possess the Ahp unit, which is also found in those micropeptins.
In addition, the sequence Tyr-Ahp-Val-NMePhe-Ile-Thr found in
the micropeptins is also present in ichthyopeptin A (1). However,
there are differences between the micropeptins and ichthyopeptins,
as N-butyl-Leu or N-acetyl-Tyr in the micropeptins is substituted
by the PAA unit in the ichthyopeptins. The position of Val and Ile
in 1 is the same as in both micropeptins; in 2 the position of these
amino acids is interchanged.
Column chromatography was carried out on silica gel (Si 60, 0.040-
0.063 mm, Merck, Germany). Fractions were monitored by TLC (Si
60 GF 254 nm, Merck, Germany) with n-PrOH/EtOAc/H2O (7:2:1) or
EtOAc/MeOH/H2O (100:13.5:10) as mobile phases. Detection was done
under UV light at 254 nm or by spraying with anisaldehyde/sulfuric
acid reagent and heating. Analytical and preparative HPLC were
performed on a component system (Kontron Instruments, Italy),
consisting of pumps 422 and 422 S, auto sampler 360, and diode array
detector DAD 440. A Synergi POLAR-RP column, 4.6 × 250 mm, 4
µm, 80 Å (Phenomenex, USA), and a gradient of deionized H2O (Clear
UV plus SG, Water Preparation and Recycling GmbH, Germany) and
MeCN (gradient grade, ROTH, Germany) from 100% H2O to 100%
MeCN in 30 min was used for analytical HPLC with a flow rate of
1.0 mL min-1. Preparative HPLC was performed on an equivalent
POLAR-RP column (10 × 250 mm) with a flow rate of 3 mL min-1
.
HPLC runs were recorded using the Geminyx HPLC data system 1.91
SST version 1.6. All chemicals were used as received, and solvents
were distilled prior to use except for HPLC.
Culture Conditions. The cyanobacterium Microcystis ichthyoblabe
strain BM Mi/13 was isolated from a sample of water collected from
the Passader See, Schleswig-Holstein, Germany, and established as
laboratory culture by Dr. Barbara Meyer (Max Planck Institute for
Limnology, Plo¨n). The strain is maintained in the culture collection of
the Institute of Pharmacy, EMAU Greifswald, as a stock culture. The
cyanobacterium was cultured in a glass column containing 40 L15 of
MBL medium.16 The growth was routinely monitored by measuring
the optical density at 730 nm using a spectrometer (Uvicon 930, Kontron
Instruments, Italy). After 28 days the cells were harvested by
centrifugation at 6500 rpm in a continuous flow centrifuge (Stratos,
Heraeus Instruments, Germany), lyophilized, and kept at -20 °C until
used. The yield of lyophilized biomass was 0.2 g L-1
.
Most of the described Ahp-containing compounds exhibit serine
proteases inhibitory activity. However, the fraction containing 1
and 2 did not show any trypsin inhibitory activity. This is
compatible with the data of Yamaki et al.,8 who have shown that
the residue linked to Ahp is responsible for regulating the trypsin
inhibitory activity. If the residue is Tyr or Phe, HcAla, Leu, HTyr,
Glu, or HSer, then the compound does not possess trypsin inhibitor
activity but may be able to inhibit other serine proteases such as
chymotrypsin. Clearly the Tyr and Leu residues linked to the Ahp
unit in ichthyopeptins A (1) and B (2) fall into the non-trypsin
inhibitory category.
Extraction and Isolation. The lyophilized cells (5 g) of M.
ichthyoblabe were successively extracted three times with 250 mL of
n-hexane followed by MeOH under stirring for 1 h, respectively. After
separation by centrifugation at 4500 rpm at 4 °C for 10 min the
methanolic supernatants were pooled and evaporated to provide a crude
extract of about 0.5 g. The crude methanolic extract (500 mg) was
separated on Si gel [open column, 3 × 40 cm, flow rate 2 mL min-1
realized by a VAC V-500 vacuum pump (Bu¨chi, Switzerland)] using
a stepwise gradient of EtOAc/MeOH/H2O, each 250 mL [100% EtOAc
(M-1), 75% EtOAc in MeOH (M-2), 50% EtOAc in MeOH (M-3),
25% EtOAc in MeOH (M-4), 100% MeOH (M-5), 75% MeOH in H2O

Cyclic Depsipeptides from Microcystis ichthyoblabe
Journal of Natural Products, 2007, Vol. 70, No. 7 1087
Table 3. 1H and 13C NMR Data of Ichthyopeptin B (2) in CD3CN/H2O (1:1)
unit
PAA
position
δH
δC
1H correlations to 13C intra-residue; inter-residue
sequential NOEs in 1H ROESYb
1
a
2
4.20
73.8
3
2.68, 2.98
40.2
4
129.8
131.4
116.0
156.1
5, 9
6, 8
7
7.05
6.68
Asn
NH
1
8.05
173.0
50.5
37.3
2; Thr-2
2
3
4
NH2
NHa
1
2
3
4.72
2.70
Thr-NH
Asn-2
6.51, 7.24
7.95
Thr
Leu
171.6
56.5
73.7
18.7
2
4.63
5.50
1.27
8.03
Leu-NH
Leu-NH
Ahp-NH
4
NH
1
Thr-2, Thr-3
a
2
3
4
5
4.30
1.53, 1.80
1.51
0.86
0.75
53.6
39.9
25.4
23.4
21.0
6
Ahp
NH
1
7.72
Thr-3
a
2
3
4
5
1
2
3
4
4.50
1.80, 2.55
1.80
50.2
21.9
30.4
75.7
172.5
56.2
33.8
24.5
14.3
14.6
a
4.99
Ile
2; N-CH3 Phe
4.35
1.74
1.04, 0.58
0.82
-0.25
Phe-2
5
6
1
N-Me Phe
2
3
5.08
2.72, 3.38
62.6
34.7
Ile-2, Val-NH
Val-NH, Val-4
4
5,9
6,8
7
N-Me
NH
1
2
3
4
5
7.20
7.23
7.15
2.74
7.91
130.5
129.8
127.8
Val-2
Val
Phe-2, Phe-3
Phe-5,9
a
4.30
1.95
0.81
0.75
59.0
31.2
19.5
19.0
Phe-3
b
a Cannot be unambiguously assigned. Each partner is indicated; hence each correlation appears twice in this column.
(M-6), 50% MeOH in H2O (M-7), 25% MeOH in H2O (M-8), and
100% H2O (M-9)]. Fraction M-2 (yield 28 mg of yellow oil) exhibited
the highest anti-influenza activity. A portion of 60 mg of M-2 was
further separated on Si gel [open column, 1.2 × 30 cm, flow rate 0.2
mL min-1] with a step gradient from 100% EtOAc to 100% MeOH
(steps of 10%, each 30 mL), and 11 fractions were collected according
to the bands detected on TLC. All fractions were analyzed by HPLC
with a linear gradient from 2% MeCN in H2O to 100% MeCN in 25
min, flow rate 1 mL min-1. Only fraction M-2-4 [eluted with EtOAc/
MeOH (90:10) from Si gel, yield 2.9 mg of a yellowish oil] showed
two detectable peaks, which were collected by preparative HPLC (46
runs, 500 µg/injection, flow rate 3 mL min-1) with the same H2O/
MeCN gradient. The pure compounds ichthyopeptins A (1) and B (2)
eluted at tR ) 17.93 min and tR ) 18.40 min, respectively. The collected
peaks of 46 HPLC runs were combined, and after removing the solvent
under reduced pressure 8.2 mg of ichthyopeptin A (1) and 7.4 mg of
ichthyopeptin B (2) were obtained as white, amorphous solids.
Enantioselective Analysis of Amino Acids. Peptide was hydrolyzed
using 6 N HCl at 100 °C for 16 h, conditions that result in the
conversion of Gln to Glu. After drying, the resulting free amino acids
were derivatized with 4 N HCl/propan-2-ol (1h, 110 °C), and after
removal of reagents, the amino acid isopropyl esters were then acylated
by pentafluoropropionic acid anhydride in CH2Cl2 (150 °C, 12 min).
Excess reagents were again removed and the amino acid derivatives
analyzed on a Chirasilval column (50 m) connected to a GCQ ion trap
mass spectrometer. The constituent amino acids were identified by their
characteristic mass spectra, and their enantiomerity was determined by
comparison to standard D,L amino acids.
Antiviral Assay. MDCK cells, 4 × 104 in 200 µL of MEM with
5% FCS (Invitrogen GmbH, Germany), were seeded per well in 96-
well tissue culture plates (Falcon, USA) and incubated in a humidified
5% CO2 atmosphere for 24 h. Confluent monolayers were preincubated
with 100 µL of medium containing the extract or the fractions in
nontoxic concentrations (100, 50, 25, 12.5 µg mL-1) for 30 min. Cells
were infected with 30 TCID50 of influenza virus A/WSN/33/London
(H1N1) and incubated for 72 h. Antiviral effects were determined by
the dye uptake assay using neutral red.17 Cell controls without extract
and virus controls were included. Antiviral activity was calculated as
percentage of protection from virus-induced cell destruction in relation
to infected cells without test compounds and the mock-infected control.
The 50% inhibitory concentration (IC50) was determined from graphic
plotting, percent protection against extract concentrations.

1088 Journal of Natural Products, 2007, Vol. 70, No. 7
Zainuddin et al.
Table 4. MS-MS Fragmentation of Ichthyopeptin B (2)
her Ph.D. studies and O. Pulz, [Biotechnology Department of the
Institute for Cereal Processing (IGV), Bergholz-Rehbru¨cke, Germany]
for financial support. Further we thank H. Bartrow, U. Felgentra¨ger,
B. Jaschok-Kentner, and C. Kakoschke for their skillful technical
assistance.
m/z
fragmenta
134.11
209.15
279.20
362.17
370.25
475.27
483.34
493.28
622.35
640.35
683.46
701
753.44
844.49
862.50
961.57
979.58
F
X-L(-H2O)
Y*-N(-H2O)
Y*-N-T(-2H2O)
X-L-F(-H2O)
Y*-N-T-L(-2H2O)
L-X-I-F(-H2O)
X-I-F-V
L-X-I-F-V
Y*-N-T-V-F
Y*-N-T-L-X-I(-3H2O)
Y*-N-T-L-X-I(-2H2O)
Y*-N-T(-L)-V-F
Y*-N-T-L-X-I-F(-3H2O)
Y*-N-T-L-X-I-F(-2H2O)
[M + H - H2O]+
[M + H]+
References and Notes
(1) Burja, A. M.; Banaigs, B.; Abou-Mansour, E.; Burgess, J. G.; Wright,
P. C. Tetrahedron 2001, 57, 9347-9377.
(2) Radau, G. Pharmazie 2000, 55, 555-560.
(3) Wegner, U.; Mundt, S.; Kliefoth, C.; Meyer, B.; Lindequist, U.;
Mentel, R. Hyg. Med. 2000, 25, 75-78.
(4) Mundt, S.; Nowotny, A.; Mentel, R.; Lesnau, A.; Lindequist, U.
Pharm. Phamacol. Lett. 1997, 7, 161-163.
(5) Nowotny, A.; Mentel, R.; Wegner, U.; Mundt, S.; Lindequist, U.
Phytother. Res. 1997, 11, 93-96.
(6) Zainuddin, E. N.; Mundt, S.; Wegner, U.; Mentel, R. Med. Microbiol.
Immunol. 2002, 191, 181-182.
(7) Radau, G. Curr. Enzyme Inhib. 2005, 1, 295-307.
(8) Yamaki, H.; Sitachitta, N.; Sano, T.; Kaya, K. J. Nat. Prod. 2005,
68, 14-18.
a For convenience the following abbreviations are used for the
residues: Y* ) PAA, Q ) Gln, T ) Thr, Y ) Tyr, X ) Ahp, V )
Val, F ) N-Me Phe, I ) Ile, N ) Asn, L ) Leu.
(9) Ploutno, A.; Shoshan, M.; Carmeli, S. J. Nat. Prod. 2002, 65, 973-
978.
(10) Harada, K.; Mayumi, T.; Shimada, T.; Suzuki, M. Tetrahedron Lett.
1993, 34, 6091-6094.
Protease Inhibition Assay. Protease inhibitory effects were tested
using trypsin and BAPNA (R-N-benzoyl-DL-arginine-p-nitroanilide) as
substrate for colorimetric analysis at 405 nm.18
(11) Hayashi, T.; Hayashi, K.; Maeda, M.; Kojima, I. J. Nat. Prod. 1996,
59, 83-87.
Ichthyopeptin A (1): white, amorphous powder; UVλmax (CH3CN/
(12) Knu¨bel, G.; Larsen, L. K.; Moore, R. E.; Levine, I. A.; Patterson, G.
M. L. J. Antibiot. 1990, 43, 1236-1239.
(13) Larsen, L. K.; Moore, R. E.; Patterson, G. M. J. Nat. Prod. 1994,
57, 419-421.
(14) Tsai, C. C.; Emau, P.; Jiang, Y.; Agy, M. B.; Shattock, R. J.; Schmidt,
A.; Morton W. R.; Gustafson, K. R.; Boyd, M. R. AIDS Res. Hum.
RetroViruses 2004, 20, 11-18.
(15) Mundt, S.; Kreitlow, S.; Nowotny, A.; Effmert, U. Int. J. Hyg.
EnViron. Health 2001, 203, 327-334.
(16) Lorenzen, C. J.; Guillard, R. R. L. J. Phycol. 1972, 8, 10-14-
(17) Mothana, R. A. A.; Mentel, R.; Reiss, C.; Lindequist, U. Phytotherapy
2006, 20, 298-302.
(18) Pilgrim, H.; Haasmann, S.; Schro¨der, K. Zentralbl. Mikrobiol. 1992,
147, 400-402.
1
H2O) 205 and 276 nm; H and 13C NMR in CD3CN/D2O (1:1), see
Table 1; MS-MS fragmentation, see Table 2; HRESIMS m/z 1043.5080
[M + H]+, m/z 1025.4950 [M - H2O + H]+, and m/z 1065.4900
[M + Na]+ (calcd for C53H70N8O14, 1043.5090, 1025.4984, and
1065.4909, respectively).
Ichthyopeptin B (2): white, amorphous powder; UVλmax (CH3CN/
1
H2O) 205 and 276 nm; H and 13C NMR in CD3CN/D2O (1:1), see
Table 3; MS-MS fragmentation, see Table 4; HRESIMS m/z 979.5160
[M + H]+, m/z 961.5040 [M - H2O + H]+, and m/z 1001.4960
[M + Na]+ (calcd for C49H70N8O13, 979.5141, 961.5035, and 1001.4960,
respectively).
Acknowledgment. We thank the DAAD for a scholarship enabling
the stay of E.N.Z. at the Ernst-Moritz-Arndt University Greifswald for
NP060303S