Planktocyclin, a cyclooctapeptide protease inhibitor produced by the freshwater cyanobacterium Planktothrix rubescens

Article abstract of DOI:10.1021/np0700873

The freshwater cyanobacterium Planktothrix rubescens produces the cyclooctapeptide cyclo(Pro-Gly-Leu-Val-Met-Phe-Gly-Val). The chemical structure is new. This homodetic cyclic octapeptide was named planktocyclin (1). It consists solely of proteinogenic L-amino acids and is a strong inhibitor of mammalian trypsin and α-chymotrypsin and a moderately active inhibitor of human recombinant caspase-8. Mass spectrometric and 2D-NMR spectroscopic data allowed the determination of its structure. Synthetic planktocyclin was identical to the natural product.

Full text of DOI:10.1021/np0700873

J. Nat. Prod. 2007, 70, 1611–1615
1611
Planktocyclin, a Cyclooctapeptide Protease Inhibitor Produced by the Freshwater
Cyanobacterium Planktothrix rubescens
,
†
‡
‡
§
§
‡
Heike I. Baumann,* Simone Keller, Falko E. Wolter, Graeme J. Nicholson, Günther Jung, Roderich D. Süssmuth, and
Friedrich Jüttner*
,
†
Limnological Station, Institute of Plant Biology, UniVersity of Zürich, Seestrasse 187, 8802 Kilchberg, Switzerland, DiVision of Organic
Chemistry, Institute of Chemistry, Technische UniVersität Berlin, Strasse des 17. Juni 124, 10623 Berlin, Germany, and Institute of Organic
Chemistry, Eberhard-Karls-UniVersität Tübingen, Auf der Morgenstelle 18, 72076 Tübingen, Germany
ReceiVed March 1, 2007
The freshwater cyanobacterium Planktothrix rubescens produces the cyclooctapeptide cyclo(Pro-Gly-Leu-Val-Met-
Phe-Gly-Val). The chemical structure is new. This homodetic cyclic octapeptide was named planktocyclin (1). It consists
solely of proteinogenic L-amino acids and is a strong inhibitor of mammalian trypsin and R-chymotrypsin and a moderately
active inhibitor of human recombinant caspase-8. Mass spectrometric and 2D-NMR spectroscopic data allowed the
determination of its structure. Synthetic planktocyclin was identical to the natural product.
1
1
Planktonic freshwater cyanobacteria have proved to be a rich
source of new oligopeptides showing high structural diversity and
bioactivity. The two most important genera (numerically, economi-
cally, and from the standpoint of toxicology) are Microcystis and
Planktothrix. They colonize in a large number of ecotypes and
chemotypes eutrophic (Microcystis) and mesotrophic to eutrophic
chymotrypsin inhibitors are oscillatorin and the planktopeptins
12
isolated from a bloom on Lake Bled, Slovenia.
1
13
Oscillarin is an effective inhibitor of thrombin. Isolates from
other cyanobacterial genera are also very effective serine protease
inhibitors. Micropeptins are cyclodepsipeptides isolated from dif-
ferent Microcystis strains. These compounds contain the 3-amino-
6-hydroxy-2-piperidone unit, which is important for their inhibition
mechanism. The compound A90720A isolated from the terrestrial
Microchaete loktakensis is also a member of this class of inhibitor
14
(
Planktothrix) lakes throughout the world. On the basis of
morphological and cytological features, Planktothrix has been
2
separated as a new genus from Oscillatoria. In addition to some
1
4
minor species, P. agardhii and P. rubescens have been differentiated
by morphological features. However, a biochemical trait, such as
phycobilins, turned out to allow a much more reliable differentiation
with strong effects on trypsin. Chymotrypsin and elastase
inhibition were found for the cyclic nostopeptin A and nostopeptin
1
4
B, which were isolated from Nostoc minutum.
3
15
of the two species. P. agardhii possesses only phycocyanins and
Recently we described oscillapeptin J, which turned out to be
a potent inhibitor of trypsin, being active in the nanomolar range.
is therefore blue-green colored, whereas P. rubescens additionally
contains phycoerythrins, which cause a magenta red color. The strict
differentiation of these two species has important implications for
research on oligopeptide metabolites because the oligopeptidic
compounds differ essentially in both species. Unfortunately, in many
publications on bioactive oligopeptides, both species have been
confused, and only in the limited cases in which the strains came
from a culture collection can the correct name be applied.
1
6
In addition it exhibited properties of a crustacean-specific toxin.
Here we describe a further effective trypsin inhibitor of novel
structure, which we named planktocyclin. The compound belongs
to the few known cyclic octapeptides consisting exclusively of
proteinogenic L-amino acids and lacks any further amino acid
modification.
P. rubescens is not ingested by Eudiaptomus, an important
crustacean grazer that lives in lakes exhibiting mass production of
4
this cyanobacterium. The ultimate reason for this food avoidance
5
,6
may be the presence of microcystins and protease inhibitors.
Protease inhibitors inhibit the digestion of the protein fraction of
the ingested food and lead to starvation of the grazers. The
efficiency of inhibitors of P. rubescens on digestive enzymes of
7
crustaceans has been shown. Trypsin and chymotrypsin were
determined to be the major digestive proteases in the gut of
8
crustaceans, and it is not surprising that effective inhibitors against
both serine proteases are found in all strains of P. rubescens so far
investigated. Therefore, these inhibitors are particularly important
as a chemical defense of P. rubescens against potential grazers.
In this context, several serine protease inhibitors have been
described for P. rubescens. Oscillapeptin G was first isolated as a
9
weak tyrosinase inhibitor. In subsequent studies, the structure was
Results and Discussion
revised and inhibitory activities were found for chymotrypsin and
elastase. Other serine protease inhibitors with properties of
Massive blooms of P. rubescens occurred in May 2002 on Lake
Hallwilersee, Switzerland. The surface film was collected and stored
at -23 °C for analysis. Several strains of P. rubescens were isolated
1
0
17
*
To whom correspondence should be addressed. (H.I.B.) Tel: +41-76-
823708. E-mail: heibaum@gmx.ch. (F.J.) Tel: +41-44-7153425. E-mail:
by picking from this lake and subsequently grown under low light
intensity. The 60% aqueous methanolic extracts of bloom samples
and of the biomass of a culture designated P. rubescens H9 were
separated by HPLC on a reversed-phase C18 column, and the
elution profiles were studied by diode array and ESIMS detection.
4
juttner@limnol.uzh.ch.
†
University of Zürich.
‡
Technische Universität Berlin.
Eberhard-Karls-Universität Tübingen.
§
1
0.1021/np0700873 CCC: $37.00

2007 American Chemical Society and American Society of Pharmacognosy
Published on Web 10/13/2007

1
612 Journal of Natural Products, 2007, Vol. 70, No. 10
Baumann et al.
Figure 1. Mass fragmentation of 1 by MS-MS and assignment of
fragments.
Figure 2. Mass fragmentation of planktocyclin sulfoxide and
assignment of fragments.
In both extracts a strong peak appeared at t ) 24.7 min exhibiting
R
Protease inhibition assays with isolated planktocyclin showed
high activity against trypsin (IC50 ) 104 nM) and R-chymotrypsin
IC50 ) 10 µM) (Figure 3A). Human caspase-8 was moderately
a quasi molecular ion at m/z 801. We named this compound
planktocyclin (1). Because the compound was sensitive to oxygen,
a modified separation procedure was applied.
(
inhibited (IC50 ) 93 µM), and mammalian cathepsin B showed no
inhibition (IC50 > 500 µM). Planktocyclin has the rare structure of
a cyclic octapeptide consisting solely of proteinogenic L-amino acids
and containing only one proline. Agardhipeptins, which were
High-resolution electrospray Fourier transform ion cyclotron mass
spectrometry (ESI-FT-ICR-MS) of planktocyclin gave a monoisotopic
+
signal at 801.43277 amu, corresponding to [C39
H
61
N
8
O
8
S] (theoreti-
cal mass 801.43276, relative mass error ∆
for planktocyclin the molecular formula of C39
deduced.
m
0.01 ppm). Therefore,
20
described previously from Planktothrix agardhii [NIES-204], are
60 8 8
H N O S was
another example of cyclic oligopeptides containing only proteino-
genic amino acids. The agardhipeptins are tryptophan-rich oli-
gopeptides, but only agardhipeptin A exhibited moderate inhibitory
activity against plasmin (IC50 ) 78 µM). Other cyclic oligopeptides
of proteinogenic amino acids have recently been isolated from
After derivatization (TFA/ethyl esters) of the hydrolysate of
planctocyclin, the constituent amino acids were identified by chiral
GC-MS and quantitatively determined by enantiomer labeling. The
following amino acids in the corresponding relative molar ratios
were detected: Pro (1.00 ref), Val (2.13), Leu (0.96), Met (0.25),
Phe (1.07), and Gly (2.06). The configuration of the amino acids
was established by comparison of their retention times (first value,
in min) with those of standards (in italics) on a Lipodex E (30% in
PS255) capillary column as follows: L-Val (15.30/D-13.45, L-15.26);
L-Leu (19.09/D-17.14, L-19.00); L-Pro (24.48/D-26.18, L-24.44);
L-Met (29.01/D-28.67, L-29.00); L-Phe (30.96/D-30.81, L-30.91).
In MS-MS experiments, the preferential cleavage site of the
21
marine sponges. The presence of one or more prolines in these
peptides and their reduced conformational flexibility was regarded
22,23
as essential for bioactivity of the molecules.
The seeds of higher plants are also important sources of proline-
containing bioactive cyclic peptides consisting of proteinogenic
amino acids. Cherimolacyclopeptides A and B are cyclooctapeptides
from the seeds of Annona cherimola, a small tree native to Ecuador
and Peru. Both peptides were found to be cytotoxic against KB
24
tumor cells. The seeds of Linum usitatissimum, which is cultured
18
macrocycle was, as expected, the secondary amide of Pro, leading
to a series of fragment ions originating from the linear acylium
ion (Figure 1). The major fragmentation sequence involved the loss
of C-terminal amino acids to yield ions of the B-series with m/z
worldwide, contain cyclolinopeptides with immunosuppressive
25
activities on human and mouse peripheral-blood lymphocytes.
Several cyclic peptides from the seeds of Vaccaria segetalis
26
(
Caryophyllaceae), named segetalins, have estrogen-like activity.
7
1
02.6 (B7), 645.7 (B6), 498.5 (B5), 367.4 (B4), 268.4 (B3), and
55.3 (B2). This fragmentation series corresponds to an amino acid
The seeds have been used as a Chinese drug for invigorating blood
circulation, for regulating menstrual disturbance, and to dispel
edema.
sequence of Pro-Gly-Leu-Val-Met-Phe-Gly-Val in the cyclic peptide
with a head-to-tail cyclopeptidic ring closure between Pro and Val.
In addition, the Y3 (m/z 304), Y4 (m/z 435), and Y5 (m/z 534)
fragment ions were detected in correspondence to the structure of
Many oligopeptides of cyanobacteria are synthesized by giant
27–29
multienzyme complexes, the nonribosomal peptide synthetases.
However, recently a ribosomal biosynthetic pathway was shown
for the precursor peptides of the patellamides. The patellamides
were isolated from Prochloron didemni, the cyanobacterial symbiont
1
. Fragment ions of planktocyclin sulfoxide confirmed the amino
acid sequence of 1 by MS-MS experiments (Figure 2).
The structure of cyclo(Pro-Gly-Leu-Val-Met-Phe-Gly-Val) (1)
30
of the tunicate Lissoclinum patella, and are pseudosymmetrical
1
13
was confirmed by 1D and 2D NMR spectra. H NMR and C NMR
shifts are given in Table 1. The spin systems of the eight amino
acids were identified using COSY and TOCSY experiments (Table
cyclic dimers, each having an unusually fused oxazoline-thiazole
31
unit. Structurally less complex cyclic oligopeptides such as
planktocyclins, which contain only proteinogenic amino acids, are
also strong candidates for such ribosomally coded peptide syntheses.
In summary we have shown that P. rubescens produces a novel
protease inhibitor that belongs to the seldom found structures of
all L-configured cyclooctapeptides.
13
1
). C NMR signals were assigned from HSQC and HMBC spectra.
The peptide sequence of planktocyclin was deduced from NOESY
data (sequential walk). NOE correlations between the R proton of
Val-8 and both δ and δ′ protons of Pro-1 suggest that the Val-8-
Pro-1 amide bond is in trans configuration. This finding is further
1
3
confirmed by the γ carbon C chemical shift of Pro-1 at 24.7
Experimental Section
1
9
ppm. To corroborate the structure elucidation, a reference
substance was synthesized via Fmoc peptide synthesis strategy on
a solid support. The NMR spectra of the isolated and synthetic
compound were compared and found to be fully identical (Table
General Experimental Procedures. The 2D-NMR spectra (COSY,
TOCSY, NOESY, HSQC, HMBC) were measured on an AMX 600
MHz NMR spectrometer (Bruker) equipped with a 5 mm Z-grad triple
resonance probe head and on a DRX500 NMR spectrometer (Bruker)
equipped with a 5 mm Z-grad broad band inverse probe head. Spectra
1
) and, thus, confirmed the structure of planktocyclin (1).

Planktocyclin from Planktothrix rubescens
Table 1. 1H and 13C NMR Data for Planktocyclin in DMSO-d
Journal of Natural Products, 2007, Vol. 70, No. 10 1613
6
natural
synthetic
a
1H
13C
1H
13C
position
Pro-1
CO
b
60.4
28.5
172.3
60.6
28.5
RCH
4.08
1.74
4.08
1.75
2.10
1.85
2.00
3.54
3.75
ꢀCH2
2
.09
1.84
.00
3.55
.72
γCH2
δCH2
24.7
47.0
b
24.9
47.2
168.5
42.5
b
2
3
Gly-2
Leu-3
CO
NH
RCH2
8.76
3.39
8.70
3.37
3.95
42.5
b
3
.97
CO
NH
7.61
4.58
1.49
1.52
0.81
0.78
7.63
4.59
1.52
1.51
0.81
0.79
RCH
50.3
40.2
23.7
21.8
22.9
b
50.2
40.0
23.5
21.6
22.7
b
ꢀCH2
γCH
δCH3
δ′CH3
CO
Figure 3. Planktocyclin concentration inhibition curves for trypsin
2
2
(
R ) 0.996; P < 0.0001) and R-chymotrypsin (R ) 0.936; P )
Val-4
Met-5
0.226). Mean values of 3 replicates and standard deviation.
NH
8.45
3.71
2.10
0.89
0.92
8.30
3.71
2.13
0.89
0.92
RCH
61.0
28.6
18.7
19.3
b
60.9
28.5
18.7
19.2
b
Cultivation and Harvest of Planktothrix rubescens. P. rubescens
H9 was isolated from Lake Hallwilersee, Switzerland (47°18′08.28′′
N; 8°12′39.86′′ E), and cultivated as a monoxenic batch culture in 300
mL Erlenmeyer flasks. The flasks were irradiated with light from
ꢀCH
γCH3
γ′CH3
CO
-
2
-1
fluorescent tubes (7 µmol m
s ). The cyanobacterium was grown at
NH
8.16
4.09
1.72
8.10
4.09
1.70
2.25
2.35
1.95
32
RCH
52.9
29.8
53.1
29.8
20 °C on a cyanobacterial mineral medium without shaking. To reduce
ꢀCH2
the buoyancy of the filaments, the suspensions were pressurized with
N at 20 bar before centrifugation. The biomass was stored frozen at
2
2
.26
γCH2
δCH3
CO
2.34
1.95
29.3
14.0
b
29.3
14.0
b
-23 °C. Additional wet biomass (∼880 g) of P. rubescens was obtained
from a bloom collected from Lake Hallwilersee in May 2002. LC-MS
and MS-MS analyses of planktocyclin derived from the bloom and the
cultured strain H9 were performed to confirm the identity of the
compound. The retention times and mass fragmentation patterns of
planktocyclin of both sources were so close to each other that they
were regarded as identical.
Phe-6
NH
8.11
4.31
2.89
8.13
4.27
2.87
3.24
RCH
54.1
36.0
54.4
35.8
ꢀCH2
3
.12
γC
δCH
εCH
137.9
128.6
127.9
126.4
b
138.1
128.9
128.1
126.6
168.2
Isolation of Planktocyclin. The thawed biomass of P. rubescens
7.17
7.23
7.19
7.16
7.23
7.19
was extracted with 60 % (v/v) MeOH/H
amount of H O already present in the biomass. After centrifugation,
separation of the supernatant, and evaporation of most of its solvents,
the resulting nearly dry residue was diluted in 30% (v/v) CH CN/H
and chromatographed on a reversed-phase C18 column (250 × 4.6 mm,
µm, 120 Å, Grom-Sil ODS-4 HE, Stagroma, Rottenburg-Hailfingen,
Germany). A linear gradient of UV-treated deionized H O (A) and
CH CN (B) was used for separation (30 to 35% B in 10 min, 35 to
0% B in 15 min, 70 to 100% B in 2 min) at 30 °C and a flow rate of
2
O, taking into account the
2
ꢁ
CH
CO
Gly-7
Val-8
3
2
O
NH
RCH2
8.18
3.46
8.08
3.48
3.76
42.5
42.9
5
3
.81
2
CO
NH
RCH
b
b
3
7.08
4.54
2.01
0.77
0.87
7.09
4.54
2.03
0.77
0.87
7
1
54.1
31.0
17.4
19.0
54.5
30.0
17.1
18.9
-
1
mL min . Planktocyclin was eluted under these conditions at
ꢀ
CH
γCH3
retention times between 24 and 25 min. Acidification of the eluent,
although giving better separations, was avoided in order to minimize
hydrolysis and oxidation reactions (sulfur-containing compound) in the
subsequent purification steps. The combined eluents containing plank-
γ′CH3
a
COSY and TOCSY experiments have been used to identify spin
b
systems of amino acids. Assignment ambiguous.
2
tocyclin were diluted with H O, freeze-dried, and vented with argon
after termination of lyophilization. Under these conditions very little
planktocyclin was oxidized to its sulfoxide. Preparative HPLC was
repeated twice to obtain pure planktocyclin. The colorless and fluffy
6
were recorded in and referenced to DMSO-d (2.49 ppm; 39.5 ppm).
HPLC-ESIMS experiments were performed on a 2000 Q Trap mass
spectrometer (Applied Biosystems/MDS Sciex, Darmstadt, Germany)
coupled to an Agilent 1100 HPLC system (Agilent, Waldbronn,
Germany). High-resolution FTICR-MS spectra were measured on an
APEX II ESI-FTICR mass spectrometer equipped with a 4.7 T magnet
powder was stored in 60% (v/v) CH
.5 mg per 7.0 g fresh weight. (ESI)MS-MS experiments of plank-
3 2
CN/H O. The yield was about
2
tocyclin and its sulfoxide were performed by direct MS infusion on
the ESIMS operating in the two-stage full scan type. The eluent
consisted of CH CN/H O/TFA (30:70:0.05), and the supplied voltage
(
Bruker-Daltonics, Bremen, Germany). LC-MS-MS spectra were
3
2
acquired in the positive-ion mode as enhanced product ion scans at a
scan rate of 1000 amu s- in high-resolution mode, with 5.5 kV spray
voltage, 12 V entrance potential, 350 °C TurboIonSpray source
temperature, 40 V declustering potential, 42 V collision cell entrance
potential, and 40 V collision energy with a 10 V collision energy spread.
The composition of the derivatized hydrolysate was determined by GC-
MS (Agilent MSD 5973/6890, Agilent). The mass spectra for the
determination of the molar absorption coefficient were obtained on a
GC-EIMS (Fison Instruments, GC 8000 Top, MD 800). The fluorescent
enzyme–substrates were measured on a fluorescence plate reader
for the parent ion was 30 V.
1
Amino Acid Analysis. The composition and configuration of the
amino acids of planktocyclin were determined after hydrolysis in 6 M
HCl at 110 °C for 24 h. The dry hydrolysate was derivatized to the
N-(O-)TFA/ethyl esters and analyzed by chiral GC-MS on a 20 m ×
0.25 mm Lipodex E/PS255 (30:70) capillary column. Quantitation of
3
3
amino acids was performed by the method of enantiomer labeling.
Determination of the Molar Absorption Coefficient. The molar
absorption coefficient of planktocyclin (50 µg in MeOH) was deter-
mined by quantitative analysis of L-Phe after acidic hydrolysis (6.0 M
1
3
(
SpectraMAX Gemini XS, Molecular Devices Corp., Sunnyvale, CA).
9
HCl at 110 °C for 15 h). L-U- C phenylalanine (1 µg) (Cambridge

1
614 Journal of Natural Products, 2007, Vol. 70, No. 10
Baumann et al.
[M + H]+ 801.4; ESI-FTICR-MS m/z [M + H] 801.43277 corre-
+
Isotope Laboratories, Inc., MA) was added as an internal standard before
hydrolysis. After derivatization of the dry hydrolysate with MTBSTFA
+
sponding to [C39
error ∆ 0.01 ppm.).
Synthesis of 1. Planktocyclin was synthesized by using procedures
H
61
N
8
O
8
S] (theoretical mass 801.43276, relative mass
(
(
Fluka, Switzerland) in tetrahydrofuran (THF) and trifluoroacetic acid
TFA) (50/50/0.1; v/v/v) the solution was analyzed on a GC-EIMS.
m
3
5
Analyses were conducted on a capillary column (30 m DB-1301, 0.32
for Fmoc solid-phase peptide synthesis in combination with an
36,37
mm i.d., 0.25 µm film thickness) under the following separation
alkanesulfonamide safety-catch linker.
To prepare the safety catch
-
1
conditions: 1 min at 120 °C, 120 to 250 °C at a rate of 10 °C min
.
handle, 3-carboxypropanesulfonamide was coupled to aminomethyl
polystyrene resin (100 mg, loading 0.9 mmol/g). Attachment of Fmoc-
L-Met-OH as the first amino acid residue was carried out according to
The integrals of the fragment ions at m/z 234 (30%), m/z 308 (15%),
1
2
m/z 336 (11%), and m/z 242, m/z 316 and m/z 345 of L-U- C
9
1
3
36
phenylalanine and L-U- C
9
phenylalanine, respectively, were applied
an optimized procedure described by Backes and Ellman. The peptide
to determine the amount of unlabeled amino acids. This value was
correlated to the UV absorption of planktocyclin at 273 nm in MeOH
measured before hydrolysis.
chain elongation was performed according to the following protocol:
(i) Fmoc removal: 20% piperidine/DMF (v/v), 2 × 5 min, (ii) wash
procedure: 5 × DMF, 5 × DCM, 5 × Et
2
O, (iii) coupling procedure:
3
4
Protease Inhibition Assays. Fluorogenic substrates were used
to determine the inhibition of proteases (trypsin, R-chymotrypsin,
cathepsin B, and caspase-8) by planktocyclin. The measurements were
performed in black 96-well microtiter plates. The reaction was
monitored in 2 min intervals for up to 40 min. The IC50 values agreed
independent of whether they were calculated from the kinetics or end
point measurements (which were actually used). The IC50 values were
calculated using the nonlinear regression sigmoid four-parameter
5 equiv of Fmoc-aa-OH, 5 equiv of HOBt, 5 equiv of DIC, 4 h, RT,
(iv) wash procedure as (ii). The last residue was coupled as Boc-L-
Phe-OH. For cleavage–cyclization: the Boc-protected peptidyl resin was
shaken with anhydrous N-methylpyrrolidinone (3 mL) and treated with
iodoacetonitrile (20 equiv) and N-ethyldiisopropylamine (5 equiv) for
24 h and was shielded from light. Subsequently the protecting Boc
group was removed by treating the resin with a mixture of TFA/phenol/
2
triisopropylsilane/H O (88:5:5:2) for 2 h. The peptide was liberated
b
formula: y ) d + a/(1 + (x/c) ) and the program Sigma Plot 8.0. (a )
under cyclization from the handle by shaking the peptidyl resin in THF/
DIPEA (4:1) for 16 h. The cleavage solution was lyophilized and
purified by reversed-phase HPLC, yielding 3.1 mg of planktocyclin as
a colorless solid.
maximum value, b ) slope of the curve, c ) slope of the curve at the
point of 50%, d ) minimum value, x ) the amount of compound that
inhibits the proteases by 50%).
For trypsin-inhibition experiments, dimethylated trypsin from porcine
pancreas (proteomics grade, Sigma) was used. The assay consisted of
Acknowledgment. This research was supported by the National
Science Foundation, Bern, Switzerland (F.J.) and by Schering AG/
Germany (R.D.S.).
1
0 µL of trypsin (134 mU), 140 µL of incubation buffer (50 mM Tris/
1
HCl, 150 mM NaCl, 1 mM CaCl
pH 8.0), and 30 µL of planktocyclin solution (12 different concentra-
tions between 4.0 nM and 400 µM in 60% (v/v) CH CN/H O). The
, 0.1 mg mL- bovine serum albumin,
2
Supporting Information Available: 1H NMR, 13C NMR, and
HMBC spectra of natural and synthetic planktocyclin. Inhibition curves
of cathepsin B and caspase-8 assayed with 1. This material is available
free of charge via the Internet at http://pubs.acs.org.
3
2
mixture was preincubated for 5 min at 37 °C in a 200 µL well, and the
reaction was started by addition of 20 µL of the substrate solution (50
µM Boc-Gln-Ala-Arg-7-amido-4-methylcoumarin in incubation buffer).
The fluorescence change (λex 380 nm, λem 440 nm) was monitored for
2
0 min at 37 °C.
References and Notes
Bovine pancreas R-chymotrypsin (Sigma) was used to determine
(
1) Welker, M.; von Döhren, H. FEMS Microbiol. ReV. 2006, 30, 530–
63.
the inhibition efficiency of planktocyclin. The substrate glutaryl-Gly-
Gly-Phe-7-amido-4-methylcoumarin (Bachem, Bubendorf, Switzerland)
was dissolved in 5% (v/v) DMSO, and a 300 µM solution in incubation
buffer was prepared and measured fluorometrically at λex 380 nm, λem
5
(2) Anagnostidis, K.; Komárek, J. Arch. Hydrobiol. 1988, Suppl. 80, 327–
472.
(3) Suda, S.; Watanabe, M. M.; Otsuka, S.; Mahakahant, A.; Yongmanitchai,
W.; Nopartnaraporn, N.; Liu, Y.; Day, J. G. Int. J. Syst. EVol.
Microbiol. 2002, 52, 1577–1595.
(
(
4
40 nm. Ten different concentrations of planktocyclin (60 nM to 400
CN/H O. The assay consisted of
µM) were dissolved in 60% (v/v) CH
3
2
4) Kurmayer, R.; Jüttner, F. J. Plankton Res. 1999, 21, 659–683.
5) Blom, F. J.; Robinson, J. A.; Jüttner, F. Toxicon 2001, 39, 1923–
1
40 µL of incubation buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM
CaCl , pH 8.0), 30 µL of planktocyclin solution, and 10 µL of
2
1
932.
-
1
R-chymotrypsin (1.02 U mL incubation buffer), which was pre-
incubated for 5 min at 37 °C. The reaction was started by adding 20
µL of substrate solution (300 µM) and continued for 20 min at 37 °C.
The substrate for cathepsin B (from bovine spleen, g10 U mg
protein, Sigma) was N-benzyloxycarbonyl-alanine-arginine-arginine-
-methoxy-ꢀ-naphthylamide (Z-Ala-Arg-Arg-4MꢀNA acetate salt),
(
(
6) Sano, T.; Takagi, H.; Kaya, K. Phytochemistry 2004, 65, 2159–2162.
7) Baumann, H. I.; Jüttner, F. Verh. Int. Ver. Limnol. 2006, 29, 1849–
1853.
-
1
(8) von Elert, E.; Agrawal, M. K.; Gebauer, C.; Jaensch, H.; Bauer, U.;
Zitt, A. Comp. Biochem. Physiol. B 2004, 137, 287–296.
(
9) Sano, T.; Kaya, K. J. Nat. Prod. 1996, 59, 90–92.
4
(
10) Fujii, K.; Sivonen, K.; Naganawa, E.; Harada, K. I. Tetrahedron 2000,
obtained from Bachem, Bubendorf, Switzerland. The substrate was
dissolved in 5% (v/v) DMSO, and a 6.4 mM stock solution was
5
6, 725–733.
(
(
11) Sano, T.; Kaya, K. Tetrahedron Lett. 1996, 38, 6873–6876.
12) Grach-Pogrebinsky, O.; Sedmak, B.; Carmeli, S. Tetrahedron 2003,
prepared in phosphate incubation buffer (12 mM NaH
2 4
PO , 88 mM
KH PO , 1.33 mM Na EDTA, 2.7 mM dithiothreitol, 0.03% Brij 35,
2
4
2
5
9, 8329–8336.
pH 5.8). The assay containing 70 µL of phosphate incubation buffer,
(
13) Hanessian, S.; Tremblay, M.; Petersen, J. F. W. J. Am. Chem. Soc.
2004, 126, 6064–6071. The authors synthesized oscillarin and revised
its structure. The data given for the cyanobacterium from which this
compound was isolated needs to be revised. The given strain number
B 283 does not exist in the SAG culture collection, but the reversely
written number, B 382, belongs to the cyanobacterial strain Oscillatoria
agardhii. If this latter strain was the source, its current name would
be P. rubescens, and it would be identical to the well-studied strains
NIVA CYA 18, CCAP 1459/22, NIES 610, and PCC 7821. It is a
freshwater rather than marine cyanobacterium.
a 50 µL solution of cathepsin B (3.8 mU mL- incubation buffer), and
1
3
0 µL of the planktocyclin solution (eight different concentrations
between 6.0 µM and 1 mM in 60% (v/v) CH CN/H O) was pre-
3
2
incubated for 5 min at 37 °C. To start the reaction, 50 µL of the substrate
solution (0.64 mM) was added and the increase of fluorescence (λex
3
45 nm, λem 425 nm) was measured for 30 min at 37 °C.
The assay of human caspase-8 (recomb. E. coli) (according to the
manual provided with the assay kit No. CASP8F-1KT, Sigma) was
based on the hydrolysis of the peptide substrate acetyl-Ile-Glu-Thr-
Asp-7-amido-4-methylcoumarin and monitored fluorometrically (λex 360
nm, λem 440 nm). The assay consisted of 70 µL of caspase buffer pH
(14) Radau, G. Pharmazie 2000, 55, 555–560.
(15) Blom, J. F.; Bister, B.; Bischoff, D.; Nicholson, G.; Jung, G.; Süssmuth,
R. D.; Jüttner, F. J. Nat. Prod. 2003, 66, 431–434.
(
16) Blom, J. F.; Baumann, H. I.; Codd, G. A.; Jüttner, F. Arch. Hydrobiol.
7
.4 (20 mM Hepes, 2 mM EDTA, 0.1% CHAPS, 5 mM DTT, and 5%
sucrose), 15 µL of 10 different concentrations of planktocyclin (2 µM
to 1 mM in 60% (v/v) CH CN/H O), and 5 µL of caspase-8 (10 µg
2
006, 167, 547–559.
(
(
(
(
17) Booker, M. J.; Walsby, A. E. Br. Phycol. J. 1979, 14, 141–150.
18) Eckart, K. Mass Spectrom. ReV. 1994, 13, 23–55.
19) Douglas, D. E.; Bovey, F. A. J. Org. Chem. 1973, 38, 2378–2383.
20) Shin, H. J.; Matsuda, H.; Murakami, M.; Yamaguchi, K. Tetrahedron
3
2
-1
mL ). After preincubation for 5 min at 25 °C, the reaction was started
by addition of 10 µL of substrate solution (150 µM in caspase buffer).
The measurements were continued for 40 min.
1
996, 52, 13129–13136.
Planktocyclin (1): amorphous, colorless powder; UV (MeOH) λmax
(21) (a) Randazzo, A.; Dal Piaz, F.; Orrù, S.; Debitus, C.; Roussakis, C.;
1
13
2
73 nm (ε 1040); H NMR, C NMR data, see Table 1; ESMS m/z
Pucci, P.; Gomez-Paloma, L. Eur. J. Org. Chem. 2001, 57, 6249–

Planktocyclin from Planktothrix rubescens
Journal of Natural Products, 2007, Vol. 70, No. 10 1615
6
255. (b) Napolitano, A.; Bruno, I.; Rovero, P.; Lucas, R.; Peris, M. P.;
(30) Ireland, C. M.; Durso, A. R.; Newman, R. A.; Hacker, M. P. J. Org.
Chem. 1982, 47, 1807–1811.
(31) Schmidt, E. W.; Nelson, J. T.; Rasko, D. A.; Sudek, S.; Eisen, J. A.;
Haygood, M. G.; Ravel, J. Proc. Natl. Acad. Sci. U.S.A. 2005, 102,
Gomez-Paloma, L.; Riccio, R., Tetrahedron 2001, 57, 6249–6255. (c)
Tabudravu, J.; Morris, L. A.; Kettenes-van den Bosch, J. J.; Jaspars,
M. Tetrahedron Lett. 2001, 42, 9273–9276.
7
315–7320.
32) Jüttner, F.; Leonhardt, J.; Möhren, S. J. Gen. Microbiol. 1983, 129,
07–412.
33) Frank, H.; Nicholson, G. J.; Bayer, E. J. Chromatogr. 1978, 167, 187–
96.
(
(
(
(
(
22) Napolitano, A.; Bruno, I.; Rovero, P.; Lucas, R.; Peris, M. P.; Gomez-
Paloma, L.; Riccio, R. Tetrahedron 2001, 57, 6249–6255.
23) Vanhoof, G.; Goossens, F.; Demeester, I.; Hendriks, D.; Scharpe, S.
FASEB J. 1995, 9, 736–744.
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Tetrahedron 2004, 60, 405–414.
25) Morita, H.; Shishido, A.; Matsumoto, T.; Itokawa, H.; Takeya, K.
Tetrahedron 1999, 55, 967–976.
26) Morita, H.; Yun, Y. S.; Takeya, K.; Itokawa, H.; Shirota, O.
Phytochemistry 1996, 42, 439–441.
27) Neilan, B. A.; Dittmann, E.; Rouhiainen, L.; Bass, R. A.; Schaub, V.;
(
(
4
1
(34) (a) The substrate for the trypsin assay was introduced by Kawabata,
S. I.; Miura, T.; Morita, T.; Kato, H.; Fujikawa, K.; Iwanaga, S.;
Takada, K.; Kimura, T.; Sakakibara, S. Eur. J. Biochem. 1988, 172,
17–25. (b) The assay for cathepsin B was adopted from: Greenspan,
P. D., et al. J. Med. Chem. 2001, 44, 4524–4534.
(
35) Wiesmüller, K.-H.; Feiertag, S.; Fleckenstein, B.; Kienle, S.; Stoll,
D.; Herrmann, M.; Jung, G. In Combinatorial Peptide and Nonpeptide
Libraries; Jung, G., Ed.; VCH Verlagsgesellschaft: Weinheim, 1996;
pp 203–246.
(
(
(
Sivonen, K.; Borner, T. J. Bacteriol. 1999, 181, 4089–4097.
28) Stachelhaus, T.; Schneider, A.; Marahiel, M. A. Science 1995, 269,
(
(
36) Backes, B. J.; Ellman, J. A. J. Org. Chem. 1999, 64, 2322–2330.
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6
9–72.
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–14.
3
NP0700873