Balgacyclamides, antiplasmodial heterocyclic peptides from Microcystis aeruguinosa EAWAG 251

Article abstract of DOI:10.1021/np400814w

The isolation and structural characterization of three new heterocyclic and macrocyclic peptides, balgacyclamides A-C, from Microcystis aeruginosa EAWAG 251 are reported. The constitutions were determined by 2D-NMR methods and mass spectrometry, and the configurations were assigned after ozonolysis and hydrolysis by HPLC-MS methods using Marfey's method as well as GC-MS using authentic standards. Balgacyclamides A and B were active against Plasmodium falciparum K1 in the low micromolar range, while displaying low toxicity to rat myoblasts.

Full text of DOI:10.1021/np400814w

Article
pubs.acs.org/jnp
Balgacyclamides, Antiplasmodial Heterocyclic Peptides from
Microcystis aeruguinosa EAWAG 251
†
‡
‡
§
†
†
Cyril Portmann, Simon Sieber, Silvan Wirthensohn, Judith F. Blom, Laeticia Da Silva, Emilie Baudat,
⊥
⊥
,†,‡
Marcel Kaiser, Reto Brun, and Karl Gademann*
†
Chemical Synthesis Laboratory, SB-ISIC-LSYNC, Swiss Federal Institute of Technology (EPFL), CH-1015 Lausanne, Switzerland
Department of Chemistry, University of Basel, St. Johanns-Ring 19, CH-4056 Basel, Switzerland
‡
§
Limnological Station, Institute of Plant Biology, University of Zu
̈
rich, CH-8802 Kilchberg, Switzerland
Parasite Chemotherapy, Swiss Tropical and Public Health Institute (Swiss TPH), CH-4002 Basel, Switzerland
S Supporting Information
⊥
*
ABSTRACT: The isolation and structural characterization of
three new heterocyclic and macrocyclic peptides, balgacyclamides
A−C, from Microcystis aeruginosa EAWAG 251 are reported. The
constitutions were determined by 2D-NMR methods and mass
spectrometry, and the configurations were assigned after
ozonolysis and hydrolysis by HPLC-MS methods using Marfey’s
method as well as GC-MS using authentic standards. Balgacycla-
mides A and B were active against Plasmodium falciparum K1 in the
low micromolar range, while displaying low toxicity to rat
myoblasts.
yclic peptides and derived macrocycles have recently
series, aerucyclamide B, displayed an antimalarial IC50 value of
0.7 μM against the Plasmodium falciparum K1 strain with a high
selectivity over the L6 rat myoblast cell line. Aerucyclamide B
was taken up as a lead structure for a medicinal chemistry
follow-up program by Serra and co-workers that built on the
first successful total synthesis of this compound. Whereas
these authors were able to briefly delineate the first structure−
activity relationships, the role of the different units in activity
remains unclear. The current main hypothesis suggests that the
C
received significant interest in a variety of fields, ranging
1
from biosynthesis studies to drug discovery and development.
The potentially large, but structurally often well-defined
conformational space sampled, combined with the variety of
amino acids, renders peptides ideally suited to interact with
many receptors or to interfere with protein/protein inter-
1
2
1
actions. Cyanobactins constitute a particular subset of cyclic
peptides produced by cyanobacteria and can be defined as
2
cyclic, ribosomally produced peptides (RiPP) containing
degree of unsaturation of the heterocyclic rings is correlated
3
10−12
heterocyclic modifications. They have been found not only
with biological activitiy,
with higher unsaturation leading
in the producing organism but also in other animals, where they
to more potent compounds. While this hypothesis is supported
4
10−12
have been suggested to be of symbiotic or dietary origin. A
by several examples
(e.g., oxidation of an thiazoline to a
particular subset, the cyclamides, are structurally well-defined
macrocyclic hexapeptides, where often hydrophobic residues
flanked by heterocyclic rings such as oxazol(ine) or thiazol(ine)
thiazole in aerucyclamides A and B led to 10 times increased
11b
activity), more compounds are needed to corroborate these
observations. In this study, we report on the isolation of new
cyclamides from cyanobacteria and evaluate their antimalarial
properties in the P. falciparum K1 assay.
5
−8
residues are observed.
been investigated,
While cyclamide biosynthesis has
the bioactivity of these compounds both
2
−4,5j
in their ecological niche and in a therapeutic setting has
received much less attention. Early assumptions on the
The balgacyclamides were isolated from aqueous methanolic
extracts of Microcystis aeruguinosa EAWAG 251 (identical to
PCC 7820). These compounds were purified by a combination
of C -SPE, semipreparative, and analytical reversed-phase
7
bioactivity suggested a role as allelochemicals, which has
7
,8
been supported by algicidal and phytotoxic effects. Another
seemingly unrelated activity is related to multidrug-resistance-
reversing activity, which has been reported for several members
18
HPLC methods. The structure determination proved to be
challenging due to the low isolation yield of 0.55, 0.15, and 0.80
9
of this family. Most interestingly, Gerwick and co-workers
10
reported significant antimalarial activity for the venturamides.
Independently, we have determined antimalarial activity for the
Special Issue: Special Issue in Honor of Otto Sticher
Received: October 1, 2013
1
1
aerucyclamides that displayed improved activity when
10
compared to an earlier study. The best compound in this
©
XXXX American Chemical Society and
American Society of Pharmacognosy
A
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Journal of Natural Products
Article
mg per 15 L of culture, for the balgacyclamides A (1), B (2),
and C (3), respectively.
Table 1. NMR Spectroscopic Data (500 MHz, DMSO-d ) for
Balgacyclamide A (1)
6
a
C/N no.
δC, type
δH (J in Hz)
HMBC
1
2
3
4
5
6
7
8
168.9, C
72.4, CH
79.3, CH
20.4, CH3
166.0, C
50.6, CH
30.8, CH
18.1, CH3
15.1, CH3
4.41, dd (6.8, 2.0)
4.95, dq (6.5, 6.4)
1.37, d (6.4)
1, 4, 5
1, 5
2, 3
4.48, ddd (9.0, 2.5, 2.0)
1.98, m
5, 7, 8′
0.62, d (6.9)
0.35, d (6.9)
7.35, d (9.0)
6, 7, 8′
6, 7, 8
5, 9
8′
NH (2)
9
169.7, C
72.7, CH
81.1, CH
21.0, CH3
169.7, C
42.6, CH
19.3, CH3
1
1
1
1
1
1
0
1
2
3
4
5
4.33, d (6.7)
9 or 13, 11, 12
9 or 13
4.79, dq (6.7, 6.3)
1.43, d (6.3)
9 or 13, 10, 11
4.73, dq (7.7, 6.9)
1.47, d (6.9)
13, 15, 16
14
NH (4)
8.30, d (7.7)
1
1
1
1
2
2
2
2
2
2
6
159.1, C
148.0, C
124.6, CH
169.7, C
54.0, CH
40.4, CH
14.2, CH3
25.1, CH2
7
8
8.30, s
17, 19
9
0
5.23, dd (8.4, 5.7)
1.92, m
19, 21, 22
20, 21, 23
1
Balgacyclamide A (1) displayed an exact mass of m/z
2
0.93, d (6.8)
1.44, m
5
33.2552, which supports the molecular formula C H N O S
25 37 6 5
3a
3b
4
+
1
for the [M + H] pseudomolecular ion. The H spectrum
DMSO-d , 500 MHz) displayed the typical pattern of a
1.04, m
21
(
6
11.1, CH3
0.88, dd (7.4, 7.4)
8.21, d (8.4)
21, 23
1
peptide (Table 1). Three NH resonances were present as
doublets. On the basis of characteristic signals and COSY
correlations, the fragments NH-Ala, NH-Val, and NH-Ile were
assigned. HSQC and HMBC experiments were used to assign
NH (6)
a
HMBC correlations are given from proton(s) stated to the indicated
carbon atom.
13
the C chemical shifts. Furthermore, two Thr residues missing
the NH resonance were assigned as methyloxazoline (MeOzn)
moieties. The last fragment was assigned as a thiazole (Tzl)
+
for the [M + H] pseudomolecular ion. Its molecular formula
based on its characteristic signals (H-18 Tzl, singlet, δ 8.30
suggests that 2 is an H O adduct of balgacyclamide A (1). The
H
2
13
ppm, C-18 Tzl δ 126.4 ppm). The sequence of the amino
constitution of 2 was obtained utilizing the same methods as for
balgacyclamide A (1). As for 1, NH-Ala, NH-Val, and NH-Ile
were identified, but instead of two MeOzn in 1, one MeOzn
and one NH-Thr were present, accounting for the difference in
the molecular formula. The sequence was established by
HMBC long-range correlations (Figure 1). Correlations were
observed from H-2 of MeOzn to C-5 of Val, from H-6 of Val to
C-9 of Thr, from H-10 of Thr to C-13 of Ala, from H-14 of Ala
to C-16 of Tzl, from H-18 of Tzl to C-19 of Ile, and from H-20
of Ile to C-1 of MeOzn. As the 13C chemical shifts of the two
carbonyls and of the thioimidate were close to each other (C-1
MeOzn δ 169.7, C-9 Thr δ 169.4, and C-19 Ile δ 169.6), the
C
acids was challenging to determine because of the presence of
1
13
resonance overlap in the H and C spectra. HMBC long-
range correlations were measured in two different solvents,
DMSO-d and CDCl , to establish the sequence (Figure 1;
6
3
additional NMR data in CDCl are available in the Supporting
3
Information). From the HMBC spectrum in DMSO-d6,
correlations from H-2 of MeOzn1 to C-5 of Val and from
NH-6 of Ile to C-1 of MeOzn1 were unambiguous.
Additionally, a long-range J coupling of 2.0 Hz was observed
between the C(α)-H of the MeOzn1 and the C(α)-H of the
Val residue. These long-range J couplings are frequently found
C
C
C
1
1,14
in cyclamides with oxazoline ring systems.
From the
sequence was confirmed by ROESY correlations. Correlations
were observed between H-4 of MeOzn and H-6 of Val,
between NH-2 of Val and H-10 and H-11 of Thr, between NH-
3 of Thr and H-14 and H-15 of Ala, and between NH-6 of Ile
and H-2 of MeOzn. As for cyclamide 1, a long-range J coupling
of 1.6 Hz was observed between the C(α)-H of MeOzn and the
C(α)-H of the Val residue. Only the link between C-16 and C-
17 of Tzl could not be established by the different NMR
experiments. Again, according to the molecular formula,
balgacyclamide B (2) was required to be tricyclic, and therefore
C-16 and C-17 needed to be connected, thus closing the
macrocyclic ring of cyclo-(-MeOzn-Val-Thr-Ala-Tzl-Ile-)-.
HMBC spectrum recorded in CDCl , correlations from NH-2
3
of Val to C-9 of MeOzn2 and from H-18 of Tzl to C-19 of Ile
established the partial sequence -Tzl-Ile-MeOzn1-Val-MeOzn2-
(Figure 1). The position of Ala was not directly established by
the NMR data, but based on the molecular formula,
balgacyclamide A (1) is required to be tetracyclic; therefore
the peptide had to be macrocyclic. As a result, the sequence of
the peptide was assigned as cyclo-(-MeOzn1-Val-MeOzn2-Ala-
Tzl-Ile-)-.
Balgacyclamide B (2) displayed an exact mass of m/z
5
51.2657, which supports the molecular formula C H N O S
25 39 6 6
B
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Journal of Natural Products
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Figure 1. Key COSY, ROESY, and HMBC correlations in balgacyclamides A (1), B (2), and C (3). Note the observed long-range J coupling
between H-2 and H-6 in both balgacyclamides A (1) and B (2).
Balgacyclamides A (1) and B (2) differ in only one structural
variation, i.e., in the opening of the MeOzn residue to Thr. We
think that this is not an artifact of the isolation procedure, as
acidic conditions were avoided throughout the workup, and the
presence of balgacyclamide B (2) was detected in the original
extract.
experiments therefore established the structures of balgacycla-
mides A−C (1−3).
Balgacyclamides A (1) and B (2) were evaluated for their
antiparasite activity. Compounds 1 and 2 displayed micromolar
IC50 activity (9.0 and 8.2 μM, respectively) against the
chloroquine-resistant strain K1 of P. falciparum, whereas 1
and 2 showed only moderate activity against other parasites,
IC50 = 59 and 51 μM, respectively, against Trypanosoma brucei
rhodesiense STIB 900 and IC50 = 28 μM for 2 against
Leishmania donovani MHOM-ET-67/L82. Balgacyclamides A
and B also displayed selectivity with respect to their cytotoxicity
to the L6 rat myoblast cell line, for which no activity was
detected up to 150 μM. These results are in agreement with
previous antiparasite evaluations of cyclamide compounds.
Venturamides A and B displayed IC values of 8.2 and 5.2 μM
Balgacyclamide C (3) displayed an exact mass of m/z
5
85.2512, which supports the molecular formula C H N O S
28 37 6 6
+
for the [M + H] pseudomolecular ion. The six fragments
MeOzn, NH-Phe, NH-Thr, NH-Gly, Tzl, and NH-Ile were
identified as described above. HMBC spectroscopy allowed the
establishment of the sequence through a series of long-range
correlations: from H-2 of MeOzn to C-5 of Phe, from H-6 of
Phe to C-12 of Thr, from NH-3 of Thr to C-16 of Gly, from H-
17 of Gly to C-18 of Tzl, from H-20 of Tzl to C-21 of Ile, and
5
0
10
from NH-6 of Ile to C-1 of MeOzn, which established the
sequence as cyclo-(-MeOzn-Phe-Thr-Gly-Tzl-Ile-)-.
When comparing balgacyclamide C (3) to its congeners, the
replacement of a Val by a Phe residue and of the Ala by a Gly
residue becomes apparent. Again, the Thr residue is present,
which we attribute to the original metabolite (corroborated by
its presence in the extract).
against P. falciparum, while aerucyclamides A, B, C, and D
displayed IC values of 5.0, 0.7, 2.3, and 6.3 μM, respectively.
11
50
It is interesting to note that opening of the oxazoline ring in 1
did not result in significantly lower bioactivity.
In conclusion, we have reported the isolation and structure
determination of balgacyclamides A (1), B (2), and C (3) from
Microcystis aeruguinosa EAWAG 251. Balgacyclamides A (1)
and B (2) were evaluated for their antiparasite activity and
displayed micromolar IC50 activity against P. falciparum with
good selectivity compared to their cytotoxicity.
The configurations of balgacyclamides A−C (1−3) were
11
determined by the same strategy as for the aerucyclamides.
First, the compounds were ozonized to avoid the epimerization
of the Ile residues that are next to the thiazole residues, as it is
known that amino acids next to thiazole or oxazole residues are
EXPERIMENTAL SECTION
15
■
prone to epimerization during hydrolysis. The compounds
were then hydrolyzed, and half the amount of the hydrolyzed
General Experimental Procedures. UV experiments were
obtained with a Shimadzu UV-1650PC double-beam spectropho-
tometer using a 1 cm path length cell. ECD spectra were measured in
MeOH with a Chirascan CD spectrometer in a 0.1 cm path length cell.
NMR spectra were acquired on Bruker Avance 500, Bruker Avance III
TCI 1.7 mm MicroCryoProbe, Bruker DRX-600, and Bruker Avance II
800 spectrometers, all equipped with cryoprobes. The spectra were
16
compounds was derivatized with Marfey’s reagent while the
other half was derivatized with trifluoroacetic acid anhydride.
The Marfey’s derivatives were analyzed by HPLC-MS to
establish the configurations of the Ala, Thr, Val, and Phe amino
acids by comparing the retention times with authentic
derivatized amino acids as standards. Co-injection of authentic
and natural samples was performed when necessary. The TFA
derivatives were analyzed by GC-MS to establish the
configuration of the Ile amino acids. It was necessary to
perform a co-injection analysis with Ile standards to differ-
entiate between the D-Ile and D-allo-Ile residues. This
methodology established the presence of D-Ala, L-Thr, L-Val,
and D-allo-Ile for balgacyclamides A and B (1 and 2) and L-Thr,
L-Phe, and D-allo-Ile for balgacyclamide C (3). These
referred to residual solvent proton and carbon signals (δ
2.50, δ 39.5
H
C
for DMSO-d and δH 7.26, δC 77.2 for CDCl ). Accurate mass ESI
6
3
spectra were recorded on a Micromass (ESI) Q-TOF Ultima API
instrument. HPLC purification and analyses were performed on a
Dionex HPLC system equipped with a P680 pump, an ASI-100
automated sample injector, a TCC-100 thermostated column
compartment, a PDA-100 photodiode array detector, a Foxy Jr.
fraction collector, and a MSQ-ESI mass spectrometric detector. The
determination of the Ile enantiomers was carried out on a GC-FID
instrument (Thermo Focus with a Thermo AS 3000 autosampler).
C
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Table 2. NMR Spectroscopic Data (500 MHz, DMSO-d ) for
Balgacyclamide B (2)
Table 3. NMR Spectroscopic Data (500 MHz, DMSO-d ) for
Balgacyclamide C (3)
6
6
a
a
C/H no.
δC, type
δH (J in Hz)
HMBC
ROESY
C/H no.
δC, type
δH (J in Hz) HMBC
ROESY
1
2
169.7, C
73.4, CH
1
2
169.0, C
73.8, CH
4.34, dd (6.2, 1, 4, 5
.6)
4
4.28, dd
1, 3, 4, 5
1, 2, 5
4
1
(7.6, 1.7)
3
78.0, CH
4.84, dq (6.2, 1, 2, 5
.2)
3
78.3, CH
4.88, dq
(7.6, 6.4)
6
4
5
6
20.0, CH3 1.29, d (6.2)
165.7, C
2, 3
4
5
6
20.3, CH3 1.36, d (6.4) 2, 3
165.0, C
51.1, CH
4.43, ddd
8.6, 3.7,
.6)
5, 7, 8, 8′, 9 4, 8, 8′
48.0, CH
4.76, m
5, 7, 8, 12 7a
(
7
7
8
a
36.7, CH2 2.98, dd
5, 6, 8,
9/9′
1
(
13.6, 5.2)
7
8
30.3, CH
1.95, m
5, 6, 8, 8′
6, 7, 8′
6, 7, 8
8, 8′
b
2.86, dd
(13.6, 6.0)
5, 6, 8,
9/9′
17.8, CH3 0.67, d (6.9)
15.8, CH3 0.53, d (6.9)
6.95, d (8.6)
8′
135.4, C
NH (2)
5, 6, 9
6, 7, 8, 8′, 10, 11
9/9′
10/10′
129.0, CH
127.4, CH
7.01, d (7.4) 7, 9/9′, 11 3, 6, 7b, OH
9
169.4, C
6.94, dd
8, 9/9′,
3, 6, 7b, OH
(
7.4, 7.4)
10/10′
1
0
58.5, CH
4.23, dd (8.7, 9, 11, 13
.5)
12
12
3
11
126.0, CH
6.82, t (7.4) 9/9′
1
1
1
2
65.0, CH
4.19, m
12
NH (2)
12
7.45, d (7.5) 12
9/9′, 6, 13, 14
20.0, CH3 1.07, d (6.3)
4.76, d (5.6)
10, 11
169.2, C
58.8, CH
OH
10, 11, 12
10, 11, 13
12
13
4.19, dd
12, 14, 15 15
(
9.0, 4.5)
NH (3)
8.47, d (8.7)
10, 11, 12, 14, 15,
OH, NH(2)
14
65.4, CH
4.09, m
12
15
1
1
1
3
4
5
172.4, C
15
19.8, CH3 0.92, d (6.3) 13, 14
47.8, CH
4.80, m
13, 15, 16
13, 14
OH
NH (3)
4.91, d (5.4) 13, 14, 15 14, 15
17.4, CH3 1.30, d (6.5)
8.22, d (7.9)
7.81, d (9.0) 13, 16
9/9′, 13, 14, 15, 17a,
OH, NH(2)
NH (4)
13, 14, 16
14, 15, NH(2)
1
1
6
169.6, C
1
1
1
1
2
6
7
8
9
0
159.6, C
7a
42.9, CH2
4.39, dd
16, 18
16, 18
147.9, C
(
15.3, 7.5)
123.6, CH
169.6, C
8.22, s
17, 19
1
7b
3.63, dd
(
15.3, 3.3)
54.4, CH
5.13, dd (8.5, 1, 19, 21,
.5) 22, 23
22, 23b
NH (4)
8.26, dd
17a, 17b, NH(2),
NH(3), NH(6)
5
(
7.5, 3.3)
2
1
39.5, CH
1.91, m 19, 20, 22, 24
3, 24
1
1
2
2
2
8
9
0
1
2
159.9, C
147.5, C
123.7, CH
168.6, C
54.1, CH
2
2
2
2
14.1, CH3 0.90, d (6.8)
25.2, CH2 1.44, m
20, 21, 23
20, 21, 22,
8.29, s
18, 19, 21
3a
2
4
5.18, dd
21, 23, 24, 23, 26
25
2
2
3b
4
1.11, m
20, 21, 22,
(
8.1, 5.2)
2
4
2
3
39.8, CH
1.92, m
26
10.9, CH3 0.88, dd (7.3, 20, 21, 23
.3)
7
24
13.9, CH3 0.84, d (6.9) 22, 23, 25
NH (6)
8.43, d (8.5)
1, 20
2, 6, 20, 21, 23a,
25a
25.0, CH2 1.45, m
22, 23, 24,
26
2
4
a
2
5b
0.98, m
22, 23, 24
23, 25
HMBC correlations are given from proton(s) stated to the indicated
carbon atom.
26
11.2, CH
3
0.87, dd
7.4, 7.4)
(
NH (6)
7.98, d (8.2) 1, 22
2, 22, 23, 24, 25a
Sources and Cultivation of Cyanobacteria. Microcystis
aeruginosa EAWAG 251 was obtained from the Eawag (Swiss Federal
Institute of Aquatic Science and Technology) Culture Collection of
Cyanobacteria, currently at the University of Basel. This strain is
identical to the M. aeruginosa PCC7820 from the Pasteur Culture
Collection of Cyanobacteria, Paris, France. The strain was grown in a
a
HMBC correlations are given from proton(s) stated to the indicated
carbon atom.
Table 4. Biological Evaluation of Balgacyclamides A (1) and
B (2) against Plasmodium falciparum K1, Trypanosoma brucei
rhodesiense STIB 900, Trypanosoma cruzi Tulahuen C4,
Leishmania donovani MHOM-ET-67/L82, and Rat Myoblast
Cells L6 (IC50 values are reported as μM concentrations)
17
2
0 L batch reactor (Zehnder medium ) with continuous aeration and
a light/dark cycle of 12:12 h. The biomass was isolated by
centrifugation and subsequently kept frozen until extraction.
Extraction and Isolation. The biomass was extracted first with
a
b
c
d
e
P.f.
T. b. rhod.
T. cruzi
L. donovani
L6
1
00% MeOH and then twice with 60% MeOH. The extract was
separated from the biomass by centrifugation. MeOH was removed
from the combined extracts by evaporation under reduced pressure,
and the mixture was then dried by lyophilization. The resulting powder
was dissolved in 80% MeOH and centrifuged to remove remaining
particles. The solutions were then sequentially loaded to a C18 SPE
column (Supelco Discovery DSC-18, 10 g, conditioned with 10%
aqueous MeOH) and eluted with 30%, 60%, 80%, and 100% aqueous
1
2
9.0
8.2
59
51
>150
>150
>150
28
>150
>150
a
b
Plasmodium falciparum K1. Trypanosoma brucei rhodesiense STIB
00. Trypanosoma cruzi Tulahuen C4. Leishmania donovani MHOM-
ET-67/L82. Rat myoblast L6 cells.
c
d
9
e
D
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Journal of Natural Products
Article
MeOH solutions. MeOH was removed from the combined fractions
by evaporation under reduced pressure, and the mixture was then
dried by lyophilization. The resulting oil from the 80% MeOH fraction
was redissolved in 80% MeOH, and the compounds were isolated
using multiple C18 RP-HPLC runs (Phenomenex Gemini C18 5 μm;
12 h and quenched with 0.03 mL of MeOH. The configuration of
isoleucine was determined after analyses on a chiral-phase GC column
(Permabond-L-Chirasil-Val; 25 m, 0.25 mm, Macherey-Nagel) with
the following conditions: 2 min at 80 °C, 80 to 180 °C at a rate of 8
°C min and 10 min at 180 °C with a split ratio 10:1, at a flow of 1
mL/min and an FID detector at 250 °C.
The four commercially available stereoisomers of isoleucine were
derivatized with the same method as presented above, and the
retention times (min) were D-allo-Ile 6.20, L-allo-Ile 6.35, D-Ile 6.39,
and L-Ile 6.45.
−1
1
3
1
50 × 10 mm). The gradient MeOH/H O at a flow of 5 mL/min was
5% MeOH to 100% over 30 min. The column was then washed for
0 min with 100% MeOH and stabilized for the next cycle over 10 min
2
with 35% MeOH. MeOH was removed under reduced pressure, and
the compounds were then dried with a stream of N . Balgacyclamides
2
A, B, and C eluted at 17.4, 13.2, and 14.3 min, respectively.
Balgacyclamides B and C were further purified by HPLC using
multiple runs on a C18 RP-HPLC analytical column (Phenomenex
Gemini C18 5 μm; 150 × 4.6 mm). The gradient CH CN/H O at a
Configuration of Compounds 1−3. Balgacyclamide A (1): from
ozonolysis−hydrolysis−Marfey D-Ala, L-Thr (confirmed by a co-
injection with L-allo-Thr), L-Val, and D-Ile or D-allo-Ile and from
ozonolysis−hydrolysis−TFA D-allo-Ile (confirmed by a co-injection
with D-Ile).
3
2
flow of 1 mL/min was 30% CH CN to 100% over 60 min. The
3
column was then washed for 10 min with 100% CH CN and stabilized
Balgacyclamide B (2): from ozonolysis−hydrolysis−Marfey D-Ala,
L-Thr, L-Val, and D-Ile or D-allo-Ile and from ozonolysis−hydrolysis−
TFA D-allo-Ile (confirmed by a co-injection with D-Ile).
Balgacyclamide C (3): from ozonolysis−hydrolysis−Marfey L-Thr,
L-Phe, and D-Ile or D-allo-Ile and from ozonolysis−hydrolysis−TFA D-
allo-Ile (confirmed by a co-injection with D-Ile).
3
for the next cycle over 10 min with 30% CH CN. Balgacyclamides B
3
and C eluted at 10.0 and 11.2 min, respectively. The compounds were
dried as described above. The isolation yields of the balgacyclamides A
(1), B (2), and C (3) were 0.55, 0.15, and 0.80 mg per 15 L of culture,
respectively.
Balgacyclamide A (1): colorless, amorphous solid; UV (CH CN)
Biological Assays. Plasmodium falciparum, Trypanosoma b.
3
−
4
λmax (log ε) 224 nm (3.54); ECD (MeOH, c = 3.5 × 10 ) λ (Δε)
44 (1.92), 207 (13.5); H and C NMR data (DMSO-d ), see Table
; H and C NMR data (CDCl ), see Supporting Information;
rhodesiense, and in vitro cytotoxicity assays were performed according
max
1
1b
1
13
2
1
to procedures previously described.
6
1
13
3
+
HRESI-QqTof-MS m/z 533.2552 [M + H] (calcd for C H N O S,
ASSOCIATED CONTENT
Supporting Information
2
5
37
6
5
■
5
33.2546).
Balgacyclamide B (2): colorless, amorphous solid; UV (CH CN)
*
S
3
Copies of NMR spectra and the different HPLC and GC
experiments for the determination of configuration are
provided. This material is available free of charge via the
Internet at http://pubs.acs.org.
−3
λmax (log ε) 237 nm (2.61); ECD (MeOH, c = 5.9 × 10 ) λ (Δε)
2
2
max
1
13
31 (0.33), 209 (0.77); H and C NMR data (DMSO-d ), see Table
6
+
; HRESI-QqTof-MS m/z 551.2657 [M + H] (calcd for
C H N O S, 551.2652).
2
5
39
6
6
Balgacyclamide C (3): colorless, amorphous solid; UV (CH CN)
3
−4
λmax (log ε) 242 nm (2.74); ECD (MeOH, c = 8.4 × 10 ) λ (Δε)
AUTHOR INFORMATION
Corresponding Author
Tel: ++41 61 267 11 44. Fax: ++41 61 267 11 45. E-mail: karl.
gademann@unibas.ch.
max
■
*
1
13
2
48 (0.45); H and C NMR data (DMSO-d ), see Table 3; HRESI-
6
+
QqTof-MS m/z 585.2512 [M + H] (calcd for C H N O S,
28
37
6
6
5
85.2495).
Ozonolysis of Compounds 1−3. Each compound (0.1 mg, 0.2
μmol) was dissolved in 0.3 mL of dry CH Cl and ozonized at room
Notes
2
2
temperature for 5 min, and the solvent was removed under a stream of
The authors declare no competing financial interest.
N .
2
Hydrolysis of Compounds 1−3. Each ozonized compound was
hydrolyzed in a closed microwave tube by adding 0.5 mL of freshly
prepared 6 N HCl at 108 °C for 20 h. The solvent was concentrated
ACKNOWLEDGMENTS
■
Part of this research was supported by the NCCR Chemical
Biology and by the R’Equip grant (206021_150760), funded by
the Swiss National Science Foundation. We thank PD Dr. D.
by a stream of N and removed by lyophilization, and then the residue
2
was dissolved in 0.06 mL of H O.
2
Hau
̈
ssinger for NMR measurement support.
Marfey’s Analysis of Compounds 1−3. Half of the solution of the
hydrolyzed compounds was treated with 0.03 mL of a 1% N-(2,4-
dinitro-5-fluorophenyl)-L-alaninamide (FDAA) solution in acetone
and 0.03 mL of 6% Et N in H O at 50 °C for 1 h in a closed HPLC
DEDICATION
■
3
2
Dedicated to Prof. Dr. Otto Sticher, of ETH-Zurich, Zurich,
Switzerland, for his pioneering work in pharmacognosy and
phytochemistry.
̈
̈
vial. The reaction was quenched with 0.03 mL of 5% acetic acid, and
the solvent was removed by lyophilization. The residue was dissolved
in 0.03 mL of MeOH and analyzed by RP-HPLC using an Agilent
Zorbax SB-C18 column (3.55 μm 150 × 2.1 mm). The mobile phases
were A, 5% acetic acid in H O, and B, CH CN/MeOH (9:1). The
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the column oven at 50 °C.
By comparison of the retention times of the derivatized
commercially available amino acids the configurations can be
determined except for isoleucine. The retention times (min) for the
derivatized standard amino acids were L-Ala 28.25; D-Ala 32.21; L-Val
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