Cyanopeptolin 954, a chlorine-containing chymotrypsin inhibitor of Microcystis aeruginosa NIVA Cya 43

Article abstract of DOI:10.1021/np050079r

A new depsipeptide, cyanopeptolin 954 (1), was isolated from the freshwater cyanobacterium Microcystis aeruginosa NIVA Cya 43. The structure of the compound was elucidated by chemical and spectroscopic analyses, including 2D NMR and GC-MS of the hydrolysate. The major structural differences compared to previously characterized heptadepsipeptides of Microcystis are the replacement of the basic amino acid in position 4 by L-leucine, the presence of L-phenylalanine in position 6, and the uncommon residue 3′-chloro-N-Me-L- tyrosine in position 7. Cyanopeptolin 954 inhibited chymotrypsin with an IC ₅₀ value of 45 nM. Nostopeptin BN920, formerly isolated from the cyanobacterium Nostoc, was isolated from the same strain of Microcystis, and a cis amide bond between Phe (6) and N-Me-Tyr (7) was shown. Nostopeptin BN920 inhibited chymotrypsin with an IC₅₀ value of 31 nM.

Full text of DOI:10.1021/np050079r

1324
J. Nat. Prod. 2005, 68, 1324-1327
Cyanopeptolin 954, a Chlorine-Containing Chymotrypsin Inhibitor of
Microcystis aeruginosa NIVA Cya 43
Eric von Elert,*,† Lukas Oberer,‡ Petra Merkel,† Thomas Huhn,§ and Judith F. Blom
Limnological Institute, University of Konstanz, 78457 Konstanz, Germany, Novartis Pharma AG, Preclinical Research,
CH-4002 Basel, Switzerland, Institute of Organic Chemistry, University of Konstanz, 78457 Konstanz, Germany, and
Limnological Station, Institute of Plant Biology, University of Zu¨rich, Seestrasse 187, 8802 Kilchberg, Switzerland
Received March 6, 2005
A new depsipeptide, cyanopeptolin 954 (1), was isolated from the freshwater cyanobacterium Microcystis
aeruginosa NIVA Cya 43. The structure of the compound was elucidated by chemical and spectroscopic
analyses, including 2D NMR and GC-MS of the hydrolysate. The major structural differences compared
to previously characterized heptadepsipeptides of Microcystis are the replacement of the basic amino
acid in position 4 by L-leucine, the presence of L-phenylalanine in position 6, and the uncommon residue
3′-chloro-N-Me-L-tyrosine in position 7. Cyanopeptolin 954 inhibited chymotrypsin with an IC50 value of
45 nM. Nostopeptin BN920, formerly isolated from the cyanobacterium Nostoc,1 was isolated from the
same strain of Microcystis, and a cis amide bond between Phe (6) and N-Me-Tyr (7) was shown. Nostopeptin
BN920 inhibited chymotrypsin with an IC50 value of 31 nM.
The vast majority of the toxic cyanobacterial water-
blooms worldwide are formed by cyanobacteria of the genus
Microcystis. In most cases toxicity in Microcystis is due to
the synthesis of hepatotoxins of the microcystin family.
However, the extensive search for new protease inhibitors
in recent years has revealed that only about 50% of the
Microcystis water blooms show hepatotoxicity to mammals
and other animals, but almost all contain protease inhibi-
tors.2,3 It can be hypothesized that under natural conditions
these inhibitors may act as important defense molecules
against potential grazers of Microcystis. Several groups of
inhibitors of serine proteases were isolated from toxic and
nontoxic Microcystis species. The most abundant are dep-
sipeptides that contain the modified amino acid 3-amino-
6-hydroxy-2-piperidone (Ahp). Considering Ahp as a de-
rivative of glutamate, hexa-, hepta-, and octadepsipeptides
have been found, all of which contain a six-amino-acid-
membered ring with a lactone structure between the
C-terminus and the hydroxy group of threonine. In all Ahp-
containing depsipeptides in Microcystis either isoleucine
or valine is the C-terminal amino acid, and the amino group
of threonine or the N-terminal amino acid of the side chain
is linked to a short-chain carboxylic acid. The amino acid
adjacent to the C-terminus is an N-methylated aromatic
amino acid (Phe, Tyr, ring-chlorinated Tyr4, kynurenine5)
or an N-methylated heteroaromatic amino acid (Trp5-7).
Depsipeptides of Microcystis that show these basic struc-
tures haven been labeled cyanopeptolins,8 micropeptins,9
aeruginopeptins,6 and microcystilides.10 Following the rea-
soning of Bister at al.11 that the cyanopeptolins were the
first variants to be reported with a complete stereochem-
istry, we use this trivial name for the new compound
cyanopeptolin 954.
quent separation on a reversed-phase HPLC resulted in
two compounds displaying inhibitory activity against chy-
motrypsin. Upon analysis with LC-ESIMS, the MS/MS
fragmentation pattern of one compound indicated the
presence of one chlorine and suggested it to be an as yet
unknown cyanopeptolin. The new compound, cyanopeptolin
954, exhibited a quasi molecular ion [M + Na]+ at m/z
977.5. The second compound exhibited a quasi molecular
ion [M + Na]+ at m/z 943.5, which suggested it to be
identical to the depsipeptide nostopeptin BN920 previously
reported of the cyanobacterial genus Nostoc.1 The peptide
nature of cyanopeptolin 954 and nostopeptin BN920 was
suggested by amino acid analysis of the hydrolysate that
indicated the presence of Val, Leu, Glu, Thr, and Phe in
both compounds and the presence of chloro-N-Me Tyr in
cyanopeptolin 954 and of N-Me Tyr in nostopeptin BN920.
High-resolution electrospray mass spectrometry (ESI+-
MS) of the sodium adduct of cyanopeptolin 954 led to a base
peak of m/z 977.4141, yielding the molecular formula
C46H63N8O12ClNa with a deviation of 0.5 ppm from the
calculated exact mass. Therefore, uncharged cyanopeptolin
954 has the molecular formula C46H63N8O12Cl. The isotopic
distribution of the ions indicated the presence of chlorine.
The following N-acylated amino acids could be established
from 1H and 13C 1D and homo- and heteronuclear 2D NMR
spectra of cyanopeptolin 954 (Table 1): Glu, Leu, Val,
3-amino-6-hydroxy-2-piperidone (Ahp), O-acylated Thr,
N-alkylated Phe, and N-methylated 3′-chloro-Tyr. NMR
data of Ahp in cyanopeptolin 954 were consistent with
previously published data of Ahp in other depsipeptides
(see Supporting Information). The structure of N-methyl-
ated 3′-chloro-Tyr was deduced from the 2D NMR spectrum
and confirmed by comparison of the NMR data with those
published from scyptolins A and B12 and micropeptins
Results and Discussion
1
478-A and -B.13 An additional singlet in the H spectrum
Bioassay-guided fractionation of the extract of Micro-
cystis NIVA Cya 43 by solid-phase extraction and subse-
was identified as an N-acetyl group. ROESY correlations
(Table 1) from Phe (6) to Ahp (5) confirm that the nitrogen
of Phe (6) is part of the Ahp moiety with both amino acids
forming the typical hemiaminal structure.
The amino acid sequence of cyanopeptolin 954 could be
deduced from sequential ROESY correlations (NOE) like
R-Ni, i+1, â-Ni,i+1, δ-âi,i+1, or R-Ri,i+1. The R-R NOE from
Phe (6) to N-methyl-3′-chloro-Tyr (7) indicated a cis amide
* To whom correspondence should be addressed. (E.v.E.)
Phone:
+49-7531-882935.
Fax:
+49-7531-883533.
E-mail:
eric.vonelert@uni-konstanz.de.
† Limnological Institute, University of Konstanz.
‡ Novartis Pharma.
§ Institute of Organic Chemistry, University of Konstanz.
Limnological Station, University of Zu¨rich.
10.1021/np050079r CCC: $30.25
© 2005 American Chemical Society and American Society of Pharmacognosy
Published on Web 08/11/2005

Cl-Containing Chymotrypsin Inhibitor of M. aeruginosa
Journal of Natural Products, 2005, Vol. 68, No. 9 1325
Table 1. NMR Data of Cyanopeptolin 954 (1) in DMSO-d6
selected
ROESY
selected
amino
acid
Ac1
HMBC
position
1
δC
169.6
δH
correlations correlations
Gln-NH,
Ac-2
2
1
22.8
172.9
1.85
Gln-NH
Gln2
Thr-NH,
Gln-2
2
52.4
31.9
174.2
4.40
2.08/2.11
Thr-NH
3
4
Gln-3
NH
NH2
1
8.05
7.21/6.72
Thr3
169.6
Leu-NH,
Thr-2
2
3
55.1
72.2
4.55
5.39
Leu-NH
Figure 1. Chemical structure of cyanopeptolin 954 (1, R ) Cl) and
nostopeptin BN920 (2, R ) H). Double-headed arrows designate
important ROESY correlations, and the dotted arrow indicates an
HMBC correlation. mTyr, N-methyl-Tyr; cmTyr, 3′-chloro-N-methyl-
Tyr.
Leu-NH,
Ahp-NH
4
NH
1
18.1
1.17
7.97
Leu4
Ahp5
170.6
50.6
39.1
24.5
Ahp-NH
Ahp-NH
2
4.22
1.68/1.30
1.45
NOE of the Gln-NH to the acetyl group (Table 1). Thus
the gross structure was determined as 1.
3
4
5
23.7/21.2 0.84/0.73
The absolute configuration of cyanopeptolin 954 was
determined by spectral and chemical methods. The stereo-
chemistry of usual amino acids (Thr, Leu, Phe, and Val)
was determined by chiral GC-MS amino acid analysis of
the hydrolysate of 1, which allowed us to assign the
L-configurations to all these amino acids. Upon acid hy-
drolysis, Gln was converted to Glu and determined to be
the L-enantiomer, indicating the L-configuration of Gln.
NH
1
8.35
Ahp-NH
Ahp-2
169.3
2
48.9
22.0
29.7
74.1
3.64
3
2.40/1.55
1.70/1.58
5.07
4
5
Phe-3,
Phe-2′/6′
NH
OH
1
7.07
6.03
Val-NH
cmTyr-
NCH3
Phe6
170.7
17
Oxidation of cyanopeptolin 954 with CrO3 followed by
acid-catalyzed hydrolysis led to the formation of L-Glu as
the major degradation product of Ahp, indicating the
l-configuration of the R-position of Ahp. The absolute
stereochemistry of (3S,6R)-L-Ahp was deduced from NOEs
and corresponded to the configuration observed in other
cyanopeptolines18 (see Supporting Information). The ster-
eochemistry of chloro-N-Me-Tyr was investigated by HPLC
analysis with Marfey’s reagent19 and was determined to
be the L-enantiomer. The inner ring current effect of the
aromatic ring from N-Me-Phe or N-Me-Tyr induced a
highfield shift to the â-, γ-, and δ-protons of either Leu or
Thr in those cyanopeptolins.8,12,14 This effect was also
observed in cyanopeptolin 954, where H-â′ of Phe-6 had a
normal shift of 2.89 ppm but H-â′′ was shifted upfield to
1.83 ppm and the aromatic protons H 2′/6′ of Phe-6 were
shifted upfield to 6.80 ppm. A strong characteristic NOE
between Ahp-δ-OH (5) and Val-NH (8) was detected in
cyanopeptolin 954 and nostopeptin BN920, as well as in
cyanopeptolins A-D,8 cyanopeptolin S,16 scyptolins A and
B,12 and hofmannolin.14 This confirms the assumption of
identical configurations of the amino acids in this part of
the molecule. Therefore the amino acid N-methyl-(3′-
chloro)-Tyr is in the L-configuration.
2
3
1′
50.7
35.8
136.9
129.7
128.2
126.6
169.6
4.72
2.89/1.83
cmTyr-2
2′/6′
3′/5′
4′
6.80
7.17
7.13
cmTyr7
1
Val-NH,
cmTyr-2
2
3
61.0
32.9
4.90
3.11/2.75
Val-NH
1′
129.5
130.9
120.0
152.3
117.0
129.6
30.7
2′
7.16
3′
4′
5′
6.99
6.96
2.77
6′
NCH3
OH
1
Val-NH
Thr-3
10.1
Val8
172.3
56.1
31.2
2
4.74
2.08
3
4
19.7/17.6 0.87/0.73
7.42
NH
bond between these two amino acids, as reported for
cyanopeptolins A-D,8 scyptolins A and B,12 hofmannolin,14
and A90720A.15 In the cyanopeptolins with cis amide bonds
published before, the two amino acids that form the cis
amide bond are N-Me-Phe and either Leu (cyanopeptolins
A-D8) or Thr (scyptolins A and B12), or the cis amide bonds
are formed by N-Me-Tyr and either Leu (A90720A15) or 4′-
O-Me-Tyr in hofmannolin14 instead of N-methyl-(3′-chloro)-
Tyr and Phe in the new compound. In cyanopeptolin 954
all other amide bonds were trans, and the sequential NOEs
are listed in Table 1. The lactone ring closure between
Thr-3 and Val-8 was verified by HMBC correlation from
the â-proton of Thr-3 to the quaternary carbonyl C atom
of Val-8. A common feature of many cyanopeptolins is the
moiety located N-terminally from Thr-2. This moiety was
composed of Gln and acetic acid. The acetylation of the
R-NH2 group of Gln was confirmed by the strong sequential
The molecular formula of nostopeptin BN920 was de-
duced from the base peak of ESI+MS (m/z 943.4537)
yielding the molecular formula C46H64N8O12Na with a
deviation of 0.2 ppm from the calculated exact mass.
Therefore, uncharged nostopeptin BN920 has the molec-
ular formula C46H64N8O12. With the exception of N-methyl-
ated 3′-chloro-Tyr the same amino acids as in 1 were found
1
in the H and 13C 2D NMR spectra of nostopeptin BN920
(data not shown). Instead of N-methylated 3′-chloro-Tyr,
N-methylated Tyr was detected. The amino acid sequence
of nostopeptin BN920 was deduced from sequential NOEs
and suggested this peptide to be identical to nostopeptin
BN920.1 However, this peptide has not been reported in
Microcystis before, and sequential NOEs unequivocally
revealed a cis amide bond between Phe (6) and N-Me-Tyr

1326 Journal of Natural Products, 2005, Vol. 68, No. 9
von Elert et al.
Research) was grown in 10 L glass bottles in mineral medium25
with aeration (filtered, 0.3 L min-1, without additional CO2).
Cultures were illuminated continuously with fluorescent tubes
at an intensity of 60 µmol m-2‚s-1 at 20 °C for 20-30 days.
Then cells were harvested by centrifugation (4.000g), lyophi-
lized, and kept in a freezer at -20 °C until extraction.
Extraction and Isolation. Portions of the lyophilized
cyanobacterial biomass (1 g) were extracted with MeOH (100
mL) for 12 h in the dark, and the extract was separated by
centrifugation from the residue. The supernatant was diluted
with ultrapure water to a 10% methanol solution and was
allowed to pass under vacuum through an equilibrated reversed-
phase ODS cartridge (10 g sorbent; Varian, Darmstadt,
Germany). Material retained on the cartridge was eluted in
steps with 50 mL each of 20, 40, 60, and 80% methanol in
water and finally with 100% methanol. The fraction eluting
with 60% methanol from the cartridge was evaporated in a
vacuum rotary evaporator at 40 °C. The residue was dissolved
in 50% methanol (1 mL) and fractionated by reversed-phase
HPLC (Nucleosil 150-5 C18, 250 × 4 mm, Macherey-Nagel,
Du¨ren, Germany; DAD at 220 nm, 1 mL min-1 flow rate with
33% solvent B; solvent A: H2O and 0.05% TFA; solvent B: 90:
10 acetonitrile/water and 0.05% TFA). The peaks eluting at
8.8 and 12.7 min were collected in about 50 HPLC runs.
Subsequently the collected volume was diluted with the same
volume of H2O and applied to an ODS cartridge (Varian,
equilibrated with 20% MeOH). The cartridge was washed with
5% MeOH to neutral pH; the inhibitors were eluted with 50
mL of MeOH, evaporated to dryness, and dissolved in MeOH
(1 mL) to obtain 1 in 0.32% and 2 in 0.16% yield based on the
dry weight of the cyanobacterium.
Analysis of the Hydrolysate. The amino acids of the
peptides were determined with GC-MS after hydrolysis of 100
µg of each highly purified cyanopeptolin in 6 M HCl at 110 °C
for 48 h. The derivatization of the dried hydrolysate and of
equivalent amounts of amino acid standards was performed
with MTBSTFA in tetrahydrofurane and TFA (20:25:0.05)
according to Blom et al.26 Analyses were conducted on a
capillary column (30 m DB-1301, 0.32 mm i.d., 0.25 µm film
thickness under the following separation conditions: 1 min
at 120 °C, 120 to 250 °C at a rate of 10 °C min-1. The retention
times (min) of amino acid standards were as follows: Val
(9.84), Leu (10.35), Glu (16.22), Thr (13.58), Phe (14.42),
N-MeTyr (17.12), 3′-chloro-N-MeTyr (19.55).
(7) (Table 1). In Ploutno and Carmeli1 the amide bond
between Phe (6) and N-Me-Tyr (7) is not defined in the text;
however, the structure shows the trans-conformer. Our
results suggest that the peptide isolated from Microcystis
is a different conformer of nostopeptin BN920 and hence
is named nostopeptin BN920 according to Ploutno and
Carmeli.1 In this study all other amide bonds in nostopep-
tin BN920 were determined to be trans. The stereochem-
istry of the amino acids and of Ahp was found to be
identical to those in 1. The stereochemistry of N-Me-l-Tyr
was determined by HPLC analysis with Marfey’s reagent.19
Similar to 1 the lactone ring closure between Thr-3 and
Val-8 was verified by HMBC correlation, and the structure
was determined as 2.
Cyanopeptolin 954 (1) and nostopeptin BN920 (2) inhib-
ited bovine chymotrypsin with IC50’s of 4.1 and 3.0 ng mL-1
(44.5 and 31.2 nM), respectively. Both compounds did not
inhibit bovine trypsin at 15.9 and 10.6 µg mL-1 (17.3 and
11.4 µM), respectively. Thus cyanopeptolin 954 and
nostopeptin BN920 are among the strongest Ahp-contain-
ing chymotrypsin inhibitors. The strongest chymotrypsin
inhibitor so far described is micropeptin T-20 with an IC50
of 2.5 nM,4 while the majority of cyanopeptolins exhibit IC50
values of 2-4 µM. Among the heptadepsipeptides of
Microcystis inhibition of trypsin or chymotrypsin is mutu-
ally exclusive. Compounds that display inhibitory activity
against trypsin but not against chymotrypsin (cyanopep-
tolin A,20 micropeptins A and B,9 micropeptin SF995,21
micropeptins SD944 and SD999,5 micropeptins EI964 and
EI99222) are characterized by a basic amino acid in position
4, while heptadepsipeptides that inhibit chymotrypsin but
not trypsin (micropeptin 88A,23 micropeptins SD979 and
SD1002,5 cyanopeptolin 963A,11 cyanopeptolins 954 and
920, this study) are characterized by a neutral (Leu) or
aromatic (Phe, Tyr) amino acid in position 4. This corre-
sponds to the known specificity of chymotrypsin (preferred
cleavage of peptide bonds at Phe and Tyr) and trypsin
(cleavage at Lys or Arg). Trypsins and chymotrypsins have
recently been shown to be the major digestive proteases
in the most important natural grazer of Microcystis,24 and
it remains to be tested if the natural grazing pressure
drives the evolution of new variants of depsipeptides in
natural ecosystems.
To determine the configuration of the amino acids, the
hydrolysate was dried and subsequently acylated with tri-
fluoroacetic acid anhydride (Fluka, Buchs, Switzerland) at 80
°C for 1 h. Analyses were conducted on a Chirasil-Val column
(Permabond-L-Chirasil-Val; 25 m × 0.25 mm; Macherey-Nagel,
Du¨ren, Germany) under the following separation conditions:
2 min at 80 °C, 80 to 180 °C at the rate of 8 °C min-1, 10 min
at 180 °C. The retention times (min) of enantiomeric amino
acids on the Chirasil Val column were as follows: D-Val (6.54),
L-Val (6.64), D-Thr (6.86), l-Thr (6.98), D-Leu (8.71), L-Leu
(8.92), D-Glu (13.64), L-Glu (13.72), D-Phe (14.81), L-Phe (14.87).
The determination of the absolute configuration of each amino
acid was confirmed by spiking the derivatized hydrolysates
with the derivatized authentic amino acids.
Experimental Section
General Experimental Procedures. Optical rotations
were determined on a Perkin-Elmer 241 polarimeter using a
quartz cell of 10 cm length and 1 mL volume. UV spectra were
recorded on a Cary 50 photometer (Varian). NMR spectra were
recorded on a Bruker DMX500 (Bruker-Daltonics, Fa¨llanden,
Switzerland) spectrometer using a triple inverse probe for the
1D-1H and 2D spectra. The sample concentration was 0.5-1
1
mM in DMSO-d6. H and 13C shifts are referred to DMSO-d6
Oxidation of Cyanopeptolin 954 and Nostopeptin
BN920 by CrO3. CrO3 oxidation was performed according to
Itou et al.17 Compound 1 or 2 (30 µg) was dissolved in 500 µL
of a solution of CrO3 in ethanol (1 mg mL-1) and stirred at
room temperature for 2 h. Then 500 µL of ultrapure water was
added and the sample stored at 4 °C for 12 h. Subsequently
the sample was filled up to 10 mL with 90/10 water/ethanol
and subjected to an ODS cartridge (Varian, equilibrated with
10% MeOH; v/v). The cartridge was flushed with 10% MeOH
and the sample eluted with 100% MeOH. The eluate was
evaporated to dryness (40 °C) and hydrolyzed as described
above. Glu that was obtained after hydrolysis allowed the
determination of the configuration of the R-position of Ahp.
Synthesis of N-Me-L-Tyr. N-methyl-L-tyrosine was pre-
pared according to a slightly modified protocol.27 N-Benzyl-L-
tyrosine: To a stirred solution of 9.06 g (50 mmol) of L-tyrosine
in 30 mL of a 4 M aqueous sodium hydroxide solution was
) 2.49 and 39.9 ppm, respectively The following NMR experi-
ments were carried out: 1D-1H, 1H-1H-COSY, 1H-1H-ROESY,
1H-1H-TOCSY, 1H-13C-COSY (HSQC). A Bruker DRX500
with TXI-CryoProbe was used for the 1H-13C-long-range COSY
(HMBC) and a DRX400 with 13C CryoProbe for the 13C spectra.
ESIMS spectra were recorded in positive and negative ion
mode, using a 9.4 T Apex-III FT-MS (Bruker Daltonics,
Fa¨llanden, Switzerland). HPLC-ESI-MS-derived mass spectra
were obtained on a LC-MS (LCQ Duo mass spectrometer,
Finnigan Thermoquest) equipped with an electrospray source
(ESI). The composition of the derivatized hydrolysate was
determined using GC-MS (Finnigan GCQ). HPLC separations
were performed on a Shimadzu 10AVP system equipped with
a diode array detector and an autosampler.
Culture of Microcystis. M. aeruginosa NIVA Cya 43
(Culture Collection of Algae, Norwegian Institute for Water

Cl-Containing Chymotrypsin Inhibitor of M. aeruginosa
Journal of Natural Products, 2005, Vol. 68, No. 9 1327
added 5 mL (50 mmol) of freshly distilled benzaldehyde as a
single portion. After 30 min of stirring, the mixture became
homogeneous and 570 mg (150 mmol) of sodium borohydride
was added carefully in small portions, keeping the temperature
below 15 °C. After stirring for another 30 min the same
procedure was repeated with the above given amounts of
benzaldehyde and sodium borohydride. After stirring for an
additional 2 h, the reaction mixture was extracted two times
with 30 mL of diethyl ether each and neutralized with 1 M
aqueous hydrochloric acid to give a precipitate. The crude
N-benzyl-L-tyrosine was filtered off, washed with water, dried
under vacuum (yield 88%), and used for the methylation
without further purification. N-Benzyl-N-methyl-L-tyrosine:
5.15 g (20 mmol) of finely powdered N-benzyl-L-tyrosine was
treated with a mixture of 2.3 mL (60 mmol) of formic acid and
2 mL (24 mmol) of aqueous formaldehyde solution (38-40%)
at 100 °C. After 3 h the reaction was finished as monitored by
TLC. Evaporation to dryness and recrystallization of the
residue from water/ethanol gave the desired compound as a
colorless solid (yield: 78%). N-Methyl-L-tyrosine: A solution
of 4 g (14.7 mmol) of N-benzyl-N-methyl-L-tyrosine was dis-
solved in 60 mL of a mixture consisting of 58 mL of glacial
acetic acid and 2 mL of concentrated hydrochloric acid. The
mixture was hydrogenated with 200 mg of palladium on
charcoal (5 mol % Pd). After hydrogenation the catalyst was
filtered off and the solution was evaporated to dryness.
Recrystallization from water/ethanol gave the desired com-
measured for 5 min at 20 °C at 390 nm in 1 mL with a Cary
50 photometer.
Cyanopeptolin 954 (1): amorphous powder; UV (MeOH)
1
λmax 282 (ꢀ 2000) nm; H and 13C NMR, see Table 1; ESIMS
(positive mode) m/z 976.9 (100%), 497.2 (80%), 936.9 (20-25%),
940.8 (20-25%), 993.9, 489.3; ESIMS (negative mode) m/z
250.1 (100%), 953.0 (20%), 988.9 (15%); HRESIMS (positive)
m/z 977.4141 (C46H63N8O12ClNa, relative mass error ∆m ) 0.5
ppm).
Nostopeptin BN920 (2): amorphous powder; UV (MeOH)
λmax 279 (ꢀ 1600) nm; ESIMS (positive mode) m/z 942.9 (100%),
480.4 (50%), 903.0 (10-15%), 940.8, 958.9, 472.2, ESIMS
(negative mode) m/z 250.1 (100%), 919.1 (20%), 955.0 (15%);
HRESIMS (positive) m/z 943.4537 (C46H64N8O12Na, relative
mass error ∆m ) 0.5 ppm).
Acknowledgment. Technical and scientific advice during
the chemical analysis and helpful comments on an earlier
version of this paper by F. Ju¨ttner are deeply acknowledged.
The technical assistance of C. Gebauer is gratefully acknowl-
edged. The authors are indebted to C. Guenat for providing
the HR-MS data. We thank Y. Shimizu and an anonymous
reviewer for helpful comments on an earlier version of the
paper.
Supporting Information Available: NMR data of 3-amino-6-
hydroxy-2-piperidone (Ahp) in previously published depsipeptides. This
material is available free of charge via the Internet at http://pubs.ac-
s.org.
pound in 71% yield, [R]20 +30.4 [c 1, 1:1 6 M HCl/HOAc (v:
D
v)].
Synthesis of 3′-Chloro-N-Me-Tyr. N-Me-D-Tyr was ob-
tained from Bachem (Bubendorf, Switzerland). A mixture of
60 mg of N-Me-D-Tyr or N-Me-L-Tyr and 1 mL of freshly
distilled sulfuryl chloride was warmed at 80 °C (3 min)
according to Ishida et al.13 until gas no longer evolved. Then
2 mL of sulfuryl chloride was added and warmed at 80 °C (1
h). Thereafter, excess sulfuryl chloride was removed by
evaporation and freeze-drying.
HPLC Analysis of N-Me-Tyr and chloro-N-Me-Tyr. The
enantiomers of N-Me-Tyr and chloro-N-Me-Tyr were deter-
mined by HPLC using Marfey’s method.19 To the acid hydroly-
sate (6 M HCl at 110 °C for 24 h) of 100 µg of 1 or 2 was added
50 µL of 1-fluoro-2,4-dinitrophenyl-5-L-alanineamide (L-FD-
AANaHCO3, Novabiochem, La¨ufelfingen, Switzerland) in ac-
etone (10 mg mL-1). The mixtures were heated at 80 °C for 3
min. After cooling, 120 µL of 1 M HCl and 300 µL of 50%
acetonitrile were added. The derivatives were analyzed on a
C-18 Supelco-Sil ODS reversed-phase column (4.6 × 250 mm;
Supelco; Bellefonte, PA); the mobile phase was acetonitrile/
H2O/trifluoroacetic acid (23/77/0.05; v/v/v). The retention times
of standards (min) were as follows: N-Me D-Tyr L-FDAA (29.7
min), N-Me L-Tyr L-FDAA (30.2 min), chloro-N-Me D-Tyr
L-FDAA (49.8 min), and chloro-N-Me L-Tyr L-FDAA (51.9 min).
The retention times of N-Me Tyr L-FDAA in the acid hydroly-
sate of nostopeptin BN920 was 30.3 min, and that of chloro-
N-Me Tyr L-FDAA in the acid hydrolysate of cyanopeptolin
954 was 51.8 min.
References and Notes
(1) Ploutno, A.; Carmeli, S. Tetrahedron 2002, 58, 9949-9957.
(2) Carmichael, W. W. J. Appl. Bacteriol. 1992, 72, 445-459.
(3) Agrawal, M. K.; Bagchi, D.; Bagchi, S. N. Hydrobiologia 2001, 464,
37-44.
(4) Okano, T.; Sano, T.; Kaya, K. Tetrahedron Lett. 1999, 40, 2379-2382.
(5) Reshef, V.; Carmeli, S. Tetrahedron 2001, 57, 2885-2894.
(6) Harada, K. I.; Mayumi, T.; Shimada, T.; Suzuki, M.; Kondo, F.;
Watanabe, M. F. Tetrahedron Lett. 1993, 34, 6091-6094.
(7) Murakami, M.; Kodani, S.; Ishida, K.; Matsuda, H.; Yamaguchi, K.
Tetrahedron Lett. 1997, 38, 3035-3038.
(8) Martin, C.; Oberer, L.; Ino, T.; Koenig, W. A.; Busch, M.; Weckesser,
J. J. Antibiot. 1993, 46, 1550-1556.
(9) Okino, T.; Murakami, M.; Haraguchi, R.; Munekata, H.; Matsuda,
H.; Yamaguchi, K. Tetrahedron Lett. 1993, 34, 8131-8134.
(10) Tsukamoto, S.; Painuly, P.; Young, K. A.; Yang, X.; Shimizu, Y. J.
Am. Chem. Soc. 1993, 115, 11046-11047.
(11) Bister, B.; Keller, S.; Baumann, H.; Nicholson, G.; Weist, S.; Jung,
G.; Su¨ssmuth, R. D.; Ju¨ttner, F. J. Nat. Prod. 2004, 67, 1755-1757.
(12) Matern, U.; Oberer, L.; Falchetto, R. A.; Erhard, M.; Koenig, W. A.;
Herdman, M.; Weckesser, J. Phytochemistry 2001, 58, 1087-1095.
(13) Ishida, K.; Matsuda, H.; Murakami, M.; Yamaguchi, K. J. Nat. Prod.
1997, 60, 184-187.
(14) Matern, U.; Oberer, L.; Erhard, M.; Herdman, M.; Weckesser, J.
Phytochemistry 2003, 64, 1061-1067.
(15) Bonjouklian, R.; Smitka, T. A.; Hunt, A. H.; Occolowitz, J. L.; Perun,
T. J.; Doolin, L.; Stevenson, S.; Knauss, L.; Wijayaratne, R.; Szewczyk,
S.; Patterson, G. M. L. Tetrahedron 1996, 52, 395-404.
(16) Jakobi, C.; Oberer, L.; Quiquerez, C.; Koenig, W. A.; Weckesser, J.
FEMS Microbiol. Lett. 1995, 129, 129-133.
(17) Itou, Y.; Ishida, K.; Shin, H. J.; Murakami, M. Tetrahedron 1999,
55, 6871-6882.
Molar Absorption Coefficient. The molar absorption
coefficient was determined by quantitative analysis of L-Leu
after acidic hydrolysis of 1 and 2. L-U-13C6 Leu (Euriso-top,
Saarbru¨cken, Germany) was added as an internal standard
before hydrolysis. After derivatization with MTBSTFA and
GC-EIMS, the integrals of the fragment ions m/z 200, 274, 302,
and 360 were used for quantitative analysis of Leu and m/z
205, 279, 308, and 366 for the 13C-labeled Leu. These values
were correlated to the UV absorption of 1 and 2 measured
before hydrolysis.
(18) Matern, U.; Schleeberger, C.; Jelakovic, S.; Weckesser, J.; Schulz, G.
Chem. Biol. 2003, 10, 997-1001.
(19) Harada, K. I.; Fujii, K.; Mayumi, T.; Hibino, Y.; Suzuki, M. Tetra-
hedron Lett. 1995, 36, 1515-1518.
(20) Weckesser, J.; Martin, C.; Jakobi, C. Syst. Appl. Microbiol. 1996, 19,
133-138.
(21) Banker, R.; Carmeli, S. Tetrahedron 1999, 55, 10835-10844.
(22) Ploutno, A.; Shoshan, M.; Carmeli, S. J. Nat. Prod. 2002, 65, 973-
978.
(23) Ishida, K.; Matsuda, H.; Murakami, M. Tetrahedron 1998, 54, 5545-
5556.
(24) Von Elert, E.; Agrawal, M. K.; Gebauer, C.; Jaensch, H.; Bauer, U.;
Zitt, A. Comp. Biochem. Physiol. B 2004, 137, 287-296.
(25) Ju¨ttner, F.; Leonhardt, J.; Mo¨hren, S. J. Gen. Microbiol. 1983, 129,
407-412.
(26) Blom, J. F.; Bister, B.; Bischoff, D.; Nicholson, G.; Jung, G.; Su¨ssmuth,
R. D.; Ju¨ttner, F. J. Nat. Prod. 2003, 66, 431-434.
(27) Quitt, P.; Hellerbach, J.; Vogler, K. Helv. Chim. Acta 1963, 46,
327-333.
Inhibition of Proteases. The inhibition of trypsin and
chymotrypsin was measured according to Von Elert et al.24
Solutions of N-R-benzoyl-DL-arginine 4-nitroanilide hydrochlo-
ride (Sigma, 187 µM) and N-succinyl-L-alanyl-L-alanyl-L-prolyl-
L-phenylalanine 4-nitroanilide (Sigma, 125 µM) in potassium
phosphate buffer (0.1 M, pH 7.5) were used as substrates for
both trypsin (T-4665, Sigma) and R-chymotrypsin (C-4129,
Sigma) from bovine pancreas. The absorption change was
NP050079R