Cytotoxic veraguamides, alkynyl bromide-containing cyclic depsipeptides from the marine cyanobacterium cf. Oscillatoria margaritifera

Article abstract of DOI:10.1021/np200077f

A family of cancer cell cytotoxic cyclodepsipeptides, veraguamides A-C (1-3) and H-L (4-8), were isolated from a collection of cf. Oscillatoria margaritifera obtained from the Coiba National Park, Panama, as part of the Panama International Cooperative Biodiversity Group program. The planar structure of veraguamide A (1) was deduced by 2D NMR spectroscopy and mass spectrometry, whereas the structures of 2-8 were mainly determined by a combination of ¹H NMR and MS²/MS³ techniques. These new compounds are analogous to the mollusk-derived kulomo'opunalide natural products, with two of the veraguamides (C and H) containing the same terminal alkyne moiety. However, four veraguamides, A, B, K, and L, also feature an alkynyl bromide, a functionality that has been previously observed in only one other marine natural product, jamaicamide A. Veraguamide A showed potent cytotoxicity to the H-460 human lung cancer cell line (LD₅₀ = 141 nM). (Chemical Equation Presented).

Full text of DOI:10.1021/np200077f

ARTICLE
pubs.acs.org/jnp
Cytotoxic Veraguamides, Alkynyl Bromide-Containing Cyclic
Depsipeptides from the Marine Cyanobacterium cf. Oscillatoria
margaritifera
,‡
†
‡
†
§
†
§
Emily Mevers, Wei-Ting Liu, Niclas Engene, Hosein Mohimani, Tara Byrum, Pavel A. Pevzner,
‡
,^
||
,†,^
Pieter C. Dorrestein, Carmenza Spadafora, and William H. Gerwick*
†
Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California San Diego,
La Jolla, California 92093, United States
‡
Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, California 92093, United States
§
Department of Computer Science and Engineering, University of California San Diego, La Jolla, California 92093, United States
^
Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, California 92093, United States
Instituto de Investigaciones Científicas y Servicios de Alta Tecnología, Center of Cellular and Molecular Biology of Diseases, Clayton,
Building 219 P.O. Box 7250, Panam ꢀa 5, Republic of Panama
S Supporting Information
b
ABSTRACT: A family of cancer cell cytotoxic cyclodepsipeptides,
veraguamides AꢀC (1ꢀ3) and HꢀL (4ꢀ8), were isolated from a
collection of cf. Oscillatoria margaritifera obtained from the Coiba
National Park, Panama, as part of the Panama International Co-
operative Biodiversity Group program. The planar structure of
veraguamide A (1) was deduced by 2D NMR spectroscopy and
mass spectrometry, whereas the structures of 2ꢀ8 were mainly
1
2
3
determined by a combination of H NMR and MS /MS techniques.
These new compounds are analogous to the mollusk-derived kulo-
mo’opunalide natural products, with two of the veraguamides (C and
H) containing the same terminal alkyne moiety. However, four veraguamides, A, B, K, and L, also feature an alkynyl bromide, a
functionality that has been previously observed in only one other marine natural product, jamaicamide A. Veraguamide A showed
potent cytotoxicity to the H-460 human lung cancer cell line (LD50 = 141 nM).
arine cyanobacteria are exceptionally prolific producers of
Center of the National Institutes of Health, has enabled unique
opportunities to conduct integrated natural products investiga-
tions, biodiversity inventories and conservation, infrastructure
M
structurally diverse secondary metabolites, of which many
1
have intriguing biological properties. An emerging biosynthetic
theme in cyanobacterial natural products is the frequent combi-
nation of polyketide synthase (PKS) and nonribosomal peptide
synthetase (NRPS) derived portions, and this results in a highly
diverse suite of nitrogen-rich structural frameworks, most of
15
development, and educational training. Moreover, the country
of Panama permits the study of marine cyanobacteria from two
very different tropical environments, the Caribbean Sea in the
Western Atlantic and the Eastern Pacific. Some of these sites are
quite pristine and of exceptional biodiversity, such as Coiba
National Park (CNP), some 15 km off the Pacific coast of
Panama. CNP was formed in 2003 as a result of a developing
recognition of its high number of indigenous and endemic plant,
animal, and microbial species, and in 2005 it was named a World
2
which are lipid soluble. A number of these cyanobacterial
metabolites possess terminal alkyne functionalites in the PKS-
3
4
derived sections, including carmabin A, georgamide, pitipep-
5
6
7
8
tolide A, yanucamides, antanapeptin, trungapeptin A, hantu-
9
10
11
12
peptin, wewakpeptins, dragonamide, and viridamide A.
16
Heritage Site by UNESCO.
Similar metabolites have also been obtained from several species
Several filamentous tuft-forming species of marine cyanobac-
teria were collected from CNP in 2010, and their extracts
evaluated in a number of biological assays. Two reduced com-
plexity fractions from one extract, subsequently tentatively
identified as Oscillatoria margaritifera, were found to be highly
of mollusks, namely, Onchidium sp. and Dolabella auricularia,
13a
13b
14
yielding onchidins A and B and a family of kulolides,
respectively. Due to the strong and distinctive similarity between
these secondary metabolites isolated from mollusks and those of
cyanobacterial origin, it is highly likely that the mollusks obtain
these compounds from their diet of cyanobacteria.
Since 1998, the International Cooperative Biodiversity Group
ICBG) in Panama, a program of the Fogarty International
Received: January 25, 2011
Published: April 13, 2011
(
Copyright r 2011 American Chemical Society and
American Society of Pharmacognosy
928
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Journal of Natural Products
ARTICLE
þ
cytotoxic to H-460 human lung cancer cells in vitro (2% survival
at 3 μg/mL), and these were chosen for further investigation. As
a result of a bioassay-guided fractionation process, one major and
several minor new cytotoxic lipopeptides were isolated and
structurally defined. The major compound, named veraguamide
HRESIMS of 1 gave a [M þ H] at m/z 767.3594 as well as
þ þ
peaks for the [M þ Na] and [M þ K] adducts at m/z
789.3405 and 805.3148, respectively, indicating a molecular
formula of C H N O Br and requiring 10 degrees of unsatura-
37 59 4 8
tion. IR spectroscopy suggested a peptide with a strong absorp-
17
ꢀ1
A (1) (CNP lies within the Panamanian state of Veraguas), was
highly cytotoxic to H-460 cells (LD50 = 141 nM); the minor
compounds were all of lesser potency to this cancer cell line. As
described below, the structure of 1 was fully characterized,
including the absolute configuration at all chiral centers, whereas
the planar structures of the minor compounds were largely
tion band at 1763 cm , and this was supported by observation of
13
six ester or amide type carbonyls by C NMR analysis (δ 173.5,
C
1
172.2, 170.9, 170.7, 169.7, and 166.0). The H NMR spectrum
also suggested a peptide with one amide (NH) proton resonating
at δ 6.28 and two N-methyl groups at δ 3.01 and 2.95. The C
NMR spectrum also revealed the presence of an unusually
1
3
H
H
1
2
3
determined by integrated H NMR and MS /MS analysis.
Additionally, a new iteration of a recently developed computer
polarized alkyne functionality (δ 79.4 and 38.4), accounting
C
for a further 2 degrees of unsaturation. Thus, 8 of the 10 degrees
of unsaturation were explained and indicated that veraguamide A
must possess two rings.
2
3
algorithm was applied to the MS /MS data and allowed deduc-
tion of the structures of the minor metabolites.
18
During the final stages of this project, a parallel effort in the
Luesch and Paul laboratories in Florida found several of the same
compounds (veraguamides A, B, and C) (1ꢀ3) as well as several
new derivatives from an Atlantic collection, and these form the
substance of a parallel report. It is interesting and potentially
insightful to the origin and evolution of the genetic pathways
responsible for veraguamide biosynthesis that these same dis-
tinctive metabolites have been isolated from cyanobacteria
collected from these two well-separated oceans.
Analysis of 1D and 2D NMR spectra (COSY, TOCSY,
ROESY, HSQC, and HMBC) led to the identifications of four
amino acids [one valine (Val), two N-methylvalines (N-MeVal),
and one proline (Pro)], one hydroxy acid [2-hydroxy-3-methyl-
pentanoic acid (Hmpa)], and one extended chain polyketide.
The proton chemical shifts of the Hmpa residue were very similar
to those reported for isoleucine; however, the carbon chemical
19
shift for the R-carbon was significantly downfield (δ 76.1),
C
consistent with a hydroxy acid. The identity of the extended
polyketide residue was deduced from a combination of COSY
and HMBC correlations. A CHꢀCH constellation formed one
3
spin system, and a deshielded methine adjacent to three sequen-
tial methylene residues formed a second spin system. By HMBC,
the two methine centers were found to be adjacent, and thus a
nearly 90ꢀ angle must exist between their proton substituents.
HMBC between the H-30 methine, as well as its attached
secondary methyl group (H -37), and an amide-type carbonyl
3
at δ 170.9 completed one terminus of this residue. At the other
end, HMBC cross-peaks were observed between the methylene
protons H-34a/H-34b and both carbons C-35 and C-36, whereas
methylene protons H-33a and H-33b showed correlations only
with C-36. The chemical shift of the distal carbon of the alkyne
was quite unusual (δ 38.4), but matched quite well with that
C
reported for the alkynyl bromide present in jamaicamide A, the
20
only other marine natural product reported with this functionality.
Thus, this last residue in veraguamide A (1) was identified as
a derivative of 8-bromo-3-hydroxy-2-methyloct-7-ynoic acid
(
Br-Hmoya).
As the proline residue accounted for one additional degree of
unsaturation, the tenth and final degree of unsaturation must
arise from veraguamide A (1) having an overall cyclic constitu-
tion; this was apparent from the residue connectivities observed
by HMBC and ROESY (Table 1). HMBC correlations from the
two N-Me groups and the NH to their respective adjacent
carbonyls and R-carbons were used to connect three of the
residues in veraguamide A. A correlation from the R-hydroxy
proton of the Br-Hmoya residue (H-31) to the carbonyl of
N-MeVal-1 (C-1) served to connect these two residues. Similarly,
the Hmpa and N-MeVal-2 residues were connected by a HMBC
cross-peak from the R-hydroxy proton of the Hmpa residue
(H-13) to the C-18 carbonyl of the N-MeVal-2 residue. Finally,
a ROESY correlation was used to make the concluding connection
between the Pro and Hmpa residues. Thus, veraguamide A was
deduced to have a cyclo-[N-MeValꢀProꢀHmpaꢀN-MeValꢀ
ValꢀBr-Hmoya] structure.
’
RESULTS AND DISCUSSION
The tentatively identified cyanobacterium O. margaritifera was
collected by hand from shallow waters (1ꢀ5 m deep) in CNP,
Panama, in February 2010. The ethanol-preserved collection was
repetitively extracted (CH Cl /MeOH, 2:1) and fractionated
2
2
using normal-phase vacuum liquid chromatography (VLC). Two
fractions that eluted with 100% EtOAc and 75% EtOAc/MeOH
were cytotoxic to H-460 human lung cancer cells (both exhibit-
ing 2% survival at 3 μg/mL). Further fractionation with reversed-
phase solid-phase extraction (SPE) yielded 2.3 mg of veragua-
mide A (1), a pure amorphous solid, and 0.1 to 0.5 mg of several
analogues, veraguamides B, C, and HꢀL (2ꢀ8).
The absolute configurations of the four R-amino acids in
veraguamide A (1) were determined by LC-MS analysis of the
9
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Journal of Natural Products
ARTICLE
1
13
Table 1. H and C NMR Assignments for Veraguamide A (1) in CDCl3
b
C
a
a
a
residue
position
δ
δ
H
(J in Hz)
HMBC
ROESY
N-MeVal-1
1
2
3
4
5
6
7
8
170.7
65.0
28.3
19.6
19.3
29.5
172.2
57.3
28.7
3.94, d (10.7)
2.28, m
1, 3, 4, 7
8, 9b, 4, 5
4, 6
4
0.98, d (6.8)
0.92, d (6.6)
3.01, s
2, 3, 5
2, 4
2, 7
3
2, 3, 6
2, 3, 5
Pro
4.95, dd (8.5, 6.3)
2.28, m
9, 10, 11
8, 10, 11
7, 8, 10, 11
8, 9, 11
8, 9, 11
8, 9, 10
9, 10
2, 9a, 9b, 10a, 10b
6, 8, 9b
9
9
1
1
1
1
a
b
1.79, m
8, 9a
0a
0b
1a
1b
25.0
47.3
2.03, m
8, 11b, 31
11b
1.99, m
3.85, dt (9.3, 7.1)
3.61, dt (9.3, 7.1)
10a, 11b, 13
10b, 11a, 13
Hmpa
12
166.0
76.1
35.7
24.9
1
1
1
1
1
1
3
4.90, d (9.3)
1.98, m
12, 14, 15, 18
11a, 11b, 14, 16, 31
4
17
13
5a
5b
6
1.54, m
14
15b, 16
15a
1.13, m
14
20.3
10.6
169.7
66.1
28.5
20.4
20.2
30.1
173.5
52.8
32.1
20.2
17.5
1.00, d (6.8)
0.87, t (7.3)
14, 15
13, 14, 15
14
7
15a, 16
N-MeVal-2
18
1
2
2
2
2
9
0
1
2
3
4.15, d (9.8)
2.25, m
18, 20, 22, 23, 24
19
20, 21, 22, 25
19, 23
1.11, d (6.3)
0.99, d (6.8)
2.95, s
19, 20, 22
19, 20, 21
19, 24
19
19, 23
20, 22, 28
Val
24
2
2
2
2
5
6
7
8
4.71, dt (6.3, 8.5)
1.90, m
24, 26, 27, 28, 29
25, 27
19, 26, 27, NH-1
25
0.94, d (6.8)
0.88, d (6.8)
6.28, d (8.5)
25, 26, 28
25, 26, 27
29
25
23
NH-1
25, 32, 37
Br-HMOYA
29
170.9
42.4
76.4
29.7
24.8
3
3
3
3
3
3
3
3
3
3
0
3.12, m
29, 31, 37
1
31, 32
1
4.85, d (10.5)
1.26, m
10a, 30, 32, 33a, 33b, 34a, 34b, 37
2
30, 31
NH-1
31
3a
3b
4a
4b
5
1.59, m
32, 34, 35
34, 35
1.42, m
31
19.5
2.20, m
33, 35, 36
33, 35, 36
31
1.97, m
31
79.4
38.4
6
7
13.9
1.25, m
13
29, 30, 31
31, NH-1
a
1
b
500 MHz for H NMR, HMBC, and ROESY. 125 MHz for C NMR.
acid hydrolysate appropriately derivatized with Marfey’s reagent
D-FDAA). The six standards, L-Pro, D-Pro, L-Val, D-Val, L-N-
The absolute configuration of the Hmpa residue was deter-
mined by comparing the GC-MS retention time of the methy-
lated residue liberated by acid hydrolysis with authentic
standards. The four standards, L-allo-Hmpa, L-Hmpa, D-allo-Hmpa,
and D-Hmpa, were synthesized from L-allo-Ile, L-Ile, D-allo-Ile,
and D-Ile, respectively, following literature procedures. The four
standards each possessed distinctly different retention times by GC-
MS [44.86 (L-allo-Hmpa), 45.06 (D-allo-Hmpa), 45.26 (D-Hmpa),
(
MeVal, and D,L-N-MeVal, were also reacted with D-FDAA and
compared to the derivatized hydrolysate by LC-MS. From the
retention times and co-injections it was clear that all four of the
amino acids, Pro, Val, and two N-MeVal residues, were of the
L configuration (see Experimental Section and Supporting
Information).
2
1
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Journal of Natural Products
ARTICLE
and 45.63 min (L-Hmpa)]. The methylated residue from the acid
hydrolysate gave a single peak at 45.63 min, thus indicating its
configuration as L-Hmpa.
To determine the absolute configuration of the Br-Hmoya
residue, compound 1 was hydrogenated with 10% Pd/C to
remove simultaneously the bromine atom and fully reduce the
terminal alkyne functionality. This hydrogenation product was
then hydrolyzed with 6 N HCl in a microwave reactor to yield the
free residues. An aliquot of the methylated hydrolysate was
treated with the S-Mosher acid chloride [S-(þ)-R-methoxy-
R-(trifluoromethyl)phenylacetyl chloride, S-(þ)-MTPA-Cl]
and compared to four synthetic standards, as described below.
Two core standards, 2S,3S-Hmoaa and 2S,3R-Hmoaa, were
synthesized using a published procedure. To create the four
chromatographic standards, 2S,3S-Hmoaa and 2S,3R-Hmoaa
were each separately treated with S-MTPA-Cl and R-MTPA-
Cl, yielding four diastereomeric compounds. These four stan-
dards were then compared to the S-MTPA-Cl-derivatized hydro-
lysate of veraguamide A (1). Two of the standards (2S,3S-Hmoaa
and 2S,3R-Hmoaa reacted with S-MTPA-Cl) are each identical
to a possible configuration of the natural residue, whereas the
other two standards (2S,3S-Hmoaa and 2S,3R-Hmoaa reacted
with R-MTPA-Cl) are enantiomeric to the other two possible
configurations of the natural residue (2R,3R-Hmoaa and 2R,3S-
Hmoaa, respectively). A GC-MS instrument equipped with a
DB5-MS column was then used to compare the retention times
of the four diastereomeric standards with the derivatized hydro-
lysate. The retention time of the hydrolysate product (47.13 min)
matched 2S,3R-Hmoaa that was reacted with S-MTPA-Cl,
identifying that the absolute configuration of the Hmoya residue
in 1 is 30S,31R. In summary, the above experiments established
that veraguamide A (1) has a 2S, 8S, 13S, 14S, 19S, 25S, 30S, and
Figure 1. Selected 2D NMR data for veraguamide A (1).
2
3
for 2, containing both MS and a series of MS spectra, was
13b
18
subjected to the comparative dereplication algorithm. This
algorithm compares the MS data set to the Norine database plus
any user inputted sequences (such as veraguamide A); as expec-
ted, 1 was the top hit, with the 14 Da difference located in the
22
2
Hmpa residue. To verify this assignment, the MS spectra for
compounds 1 and 2 were compared manually, and this also
indicated that the 14 Da structural difference was present in the
Hmpa residue (Figure 2). Thus, the Hmpa residue in veragua-
mide A (1) was replaced by a 2-hydroxy-3-methylbutanoic acid
(Hmba) residue in veraguamide B (2). Due to the small amount
of compound obtained and the desire to explore the biological
properties of these veraguamide A analogues (discussed below),
the absolute configuration was not established for compound 2, but
we speculate that it is likely identical to that of veraguamide A (1).
In a similar fashion, the structures for compounds 3, 4, 5, and 6
were also determined, with each possessing only a single
modified residue in comparison with either veraguamide A (1)
or veraguamide B (2). Veraguamides C (3) and H (4) were
found to be analogues of compounds 1 and 2, respectively;
however, they lacked the alkynyl bromine atom but retained the
alkyne functionality. Veraguamides I (5) and J (6) also proved to
be analogues of compounds 1 and 2, respectively; in this case
they lack both the bromine atom and the alkynyl functionality in
the polyketide section of the molecule. Again, due to the low
yields of compounds 3ꢀ6, their absolute configurations were not
determined experimentally; it may be that they are the same as
veraguamide A (1).
3
1R absolute configuration.
Several analogues of compound 1 were isolated from the more
polar chromatographic fraction (eluted with 75% EtOAc/MeOH)
of the crude extract. Because these analogues were obtained in
quite small yield (0.1ꢀ0.5 mg), we were motivated to examine
their structures using a newly reported computer analysis of
2
3
18
MS /MS data obtained for cyclic peptides. Additionally,
1
because H NMR analysis of several of these analogues showed
them to be similar in overall structure to veraguamide A (1), the
position of structural modifications could be determined on the
basis of mass shifts in characteristic fragments. With the structure
of 1 rigorously determined by a full spectrum of spectroscopic
and chemical techniques, it was possible to use this parent
structure to determine the characteristic fragmentation pattern
for this family of metabolites. Thus, by both a manual comparison
Two additional veraguamides, K (7) and L (8), were isolated
from the more polar and biologically active VLC fraction;
however, their structures could not be determined by the
2
3
MS /MS method because the algorithm is currently designed
specifically for the analysis of cyclic peptides. Additional devel-
opment of the algorithm is underway to expand its ability to
distinguish between linear and cyclic peptides using mass spec-
trometry data, as this is a long-standing problem in the proteo-
mics and peptidomics fields. Nevertheless, using 600 MHz
cryoprobe NMR it was possible to obtain a nearly complete
2D NMR data set for 8 (HSQC, HMBC, and TOCSY).
2
of MS fragmentation pattern for each of the analogues to that of
1
and application of this newly developed computer algorithm
for cyclic peptides, the location and nature of the structural
modifications to the veraguamide A (1) parent structure were
1
þ
determined readily. In most cases, confirmatory H NMR data
Additionally, HRESIMS of 8 gave a [M þ Na] peak at m/z
were also obtained.
821.3673, indicating a molecular formula of C H N O Br
38 63 4 9
Compound 2 was isolated as a slightly more polar secondary
metabolite in approximately 0.3 mg yield and by HRESIMS
indicated a molecular formula of C H N O Br. This mass is 14
(9 degrees of unsaturation), differing from veraguamide B (2)
13
by C H O and one less degree of unsaturation. C NMR shifts
2
6
were deduced by a combination of HMBC and HSQC data and
revealed the presence of six ester- or amide-type carbonyls (δC
176.0, 172.5, 172.5, 171.0, 170.0, and 166.7) and an alkynyl
bromide (δC 79.7 and 38.0), accounting for 8 degrees of
unsaturation. As detailed below, a proline in 8 accounted for
the ninth and final degree of unsaturation in veraguamide L,
signifying that 8 is a linear depsipeptide.
36 57 4 8
Da less than that of veraguamide A (1), and thus, veraguamide B
2) possesses one fewer fully saturated carbon atom. Consistent
(
1
with this observation, H NMR analysis showed a spectrum
nearly identical with that obtained for veraguamide A, with only
small differences observed in the high-field methyl and methy-
lene regions. To localize this mass offset, the MS ion data set tree
9
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Journal of Natural Products
ARTICLE
Figure 2. Sequencing by ESIMS/MS fragmentations. To localize the residue reduced by 14 Da relative to veraguamide A (1), fragments that bear the 14
Da shift are labeled in red, with the nonshifting fragments labeled in blue. By comparing the shifted and nonshifted ions, the offset mass was located on
residue “d”. All of the resulting fragments agreed with this new mass annotation.
1
The NMR spectra of veraguamide L (8) possessed similar H
Taxonomy of the Veraguamide-Producing Strain. A taxo-
nomic investigation of the veraguamide-producing cyanobacter-
ium (PAC-17-FEB-10-2) showed that the morphology agreed
relatively well with the current definition of Oscillatoria margar-
itifera (for morphological description, see Supporting Informa-
1
3
and C NMR shifts to most of the resonances present for
veraguamide A (1). Analysis of the 1D and 2D NMR spectra led to
the assignments of four amino acids [valine (Val), two N-methyl-
valines (N-MeVal), and proline (Pro)] as well as one hydroxy acid
23
[
2
2-hydroxy-3-methylbutyric acid (Hmba)] and 8-bromo-3-hydroxy-
-methyloct-7-ynoic acid (Br-Hmoya). In addition, HMBC correla-
tion). O. margaritifera was described initially from brackish and
23
marine environments of northern Europe, which makes it
geographically and environmentally unlikely that tropical marine
tions were observed from a deshielded methylene (δ 4.15) to
H
24
both a methyl carbon (δ 14.3) and a carbonyl (δ 171.0), features
PAC-17-FEB-10-2 would belong to the same taxon. Moreover,
C
C
not observed for compound 1. By TOCSY, this same deshielded
methylene was directly adjacent to the new methyl group, thus
defining an ethyl ester at the carboxylic acid terminus of veraguamide
specimens of Oscillatoria have overlapping morphological char-
acters with the genus Lyngbya, and phylogenetic analysis is
25
therefore essential to delineate these morphologically similar but
2
26
L (8). Subsequently, comparison of the MS data for compounds 7
evolutionarily unrelated genera. Phylogenetic inferences of the
and 8 revealed that the only difference between these two com-
pounds is in the hydroxy acid residue. In 7, this residue is Hmpa
SSU (16S) rRNA gene of PAC-17-FEB-10-2 revealed that this
strain nested within the Oscillatoria lineage with O. sancta PCC
24
(comparable to 1), while in 8 it is Hmba (comparable to 2). At this
7515 as the closest related reference strain. However, the
Oscillatoria lineage forms two distinct sister clades, one tempe-
rate sensu stricto (including PCC 7515) and one tropical marine
(including PAC-17-FEB-10-2). The DNA bar-coding gap be-
tween the two clades was 4.2 (mean p-distance: interclade =
2.3%; intraclade = 0.6%), which may support the separation of
temperate and tropical marine Oscillatoria into two distinct
genera. However, because such a revision in the taxonomy of
tropical marine Oscillatoria has not yet occurred, at the present
time the best taxonomic definition of the veraguamide-producing
strain PAC-17-FEB-10-2 is cf. Oscillatoria margaritifera.
point, we are uncertain if veraguamides K (7) and L (8) are artifacts
of the preservation of the original sample in ethanol or if they
represent true natural products of the cyanobacterium.
Only compounds 1, 2, 3, 7, and 8 were available in sufficient
quantity for evaluation in the H-460 cytotoxicity assay. Com-
pound 1 showed potent activity (LD50 = 141 nM), while
compounds 2, 3, 7, and 8 all exhibited activity in the low
micromolar range, but due to insufficient quantities, no further
evaluation of theseanalogues waspossible. However, twostructural
analogues of veraguamide A, kulomo’opunalide-1 and -2, have
similar or identical NRPS portions of the molecule but lack the
alkynyl bromide in the PKS portion. These two compounds were
previously tested against P388 cells, but were reported to exhibit
’
EXPERIMENTAL SECTION
14
only moderate cytotoxicity, suggesting that the alkynyl bromide
may be an essential structural feature for the potent cytotoxic
activity observed for veraguamide A (1).
General Experimental Procedures. Optical rotations were
measured on a JASCO P-2000 polarimeter, UV spectra on a Beckman
Coulter DU-800 spectrophotometer, and IR spectra using a Nicolet
9
32
dx.doi.org/10.1021/np200077f |J. Nat. Prod. 2011, 74, 928–936

Journal of Natural Products
ARTICLE
Figure 3. Evolutionary tree of the veraguamide-producing strain PAC-17-FEB-10-2 (highlighted with a red arrow). Note that PAC-17-FEB-10-2 nests
within the tropical marine Oscillatoria clade (blue box) and that the evolutionarily distance from the Oscillatoria sensu stricto (red box) suggests that
tropical marine Oscillatoria should be revised as a new taxa. Tropical marine Lyngbya, which are often confused with the genus Oscillatoria, are highlighted
with a green box. The cladogram is based on SSU (16S) rRNA gene sequences using the maximum-likelihood (GARLI) method, and the support values
T
are indicated as bootstrap at the nodes. The specimens are indicated as species, strain, and access number in brackets. Specimens designated with ( )
represent type-strains obtained from Bergey’s Manual. The scale bar is indicated at 0.04 expected nucleotide substitutions per site.
IR-100 FT-IR spectrophotometer using KBr plates. NMR spectra were
recorded with chloroform as internal standard (δ 77.0, δ 7.26) on a
Varian Unity 500 MHz spectrometer (500 and 125 MHz for H and
(fraction H) were separated further using RP SPE [500 mg SPE,
stepwise gradient solvent system of decreasing polarity starting with
C
H
1
13
C
20% CH
3
CN in H
2
O to 100% CH
2
Cl
2
, to produce four fractions (1ꢀ4)
NMR, respectively) and on a Varian VNMRS (Varian NMR System) 500
each] to yield pure veraguamide A (1). Further fractionation by RP
HPLC using a Phenomenex 4 μm Synergi Fusion analytical column,
1
MHz spectrometer equipped with a cold probe (500 and 125 MHz for H
13
and C NMR, respectively). Also used were a Bruker 600 MHz spectro-
3 2 3
with a gradient from 50% CH CN/H O to 100% CH CN over 30 min,
1
meter equippedwith a 1.7 mm MicroCryoProbe(600 and 150 MHzfor H
yielded pure veraguamides B, C, K, and L (2ꢀ8).
13
22
D
and C NMR, respectively) and a JEOL 500 MHz spectrometer (500 and
Veraguamide A (1): amorphous solid; [R]
ꢀ14.7 (c 0.33, CH2
1
13
1
25 MHz for H and C NMR, respectively). LR- and HRESIMS were
Cl
2
); UV (MeCN) λmax (log ε) 204 (4.00), 266 (2.83) nm; IR (neat)
ꢀ
1 1
obtained on a ThermoFinnigan LCQ Advantage Max mass detector and a
Thermo Scientific LTQ Orbitrap-XL mass spectrometer, respectively.
νmax 3327, 2964, 2930, 1734, 1700, 1456, 1272, 1194, 1128 cm ; H
1
3
NMR (500 MHz, CDCl ) and C NMR (500 MHz, CDCl ), see
3
3
2
3
80
36 61 4 7 31 50
MS /MS spectra were obtained on a Biversa Nanomate with nanoelec-
trospray ionization on a ThermoFinnigan LTQ-MS, which utilized Tune
Plus software version 1.0. HPLC was carried out using a Waters 515 pump
systemwithaWaters 996 PDAdetector. Allsolventswere either distilledor
of HPLC quality. Acid hydrolysis was performed using a Biotage (Initiator)
microwave reactor equipped with high-pressure vessels.
Table 1; ESIMS/MS m/z 741.26 (C H N O Br), 654.13 (C H -
7
8
80
80
5
N
(C20
3
O
7
Br), 574.13 (C26
H
44
N
3
O
H
6
Br), 542.20 (C25
), 438.24 (C23
(C H N O ), 325.12 (C H N O ), 297.19 (C H N O ), 228.12
H
40 3
N O
Br), 463.20
80
H
35
N
2
O
5
Br), 456.26 (C23
42
N
3
O
6
40 3 5
H N O ), 343.17
17
31
2
5
17 29
2
4
16 29 2 3
þ
(C12
O
8
H
22NO
3
); HRESIMS [M þ H] m/z 767.3594 (calcd for C37
Br 767.3594).
Veraguamide B (2): amorphous solid; [R]
61 4
H N
7
8
2
3
Cyanobacterial Collections and Morphological Identifica-
tion. The veraguamide-producing cyanobacterium PAC-17-FEB-10-2
was collected by hand using snorkel gear in shallow water off Isla Canales
D
ꢀ13.1 (c 0.25,
1
CH Cl ); H NMR (600 MHz, CDCl ) δ 0.90 (3 H, d, J = 6.7 Hz),
2
2
3
0.94 (3H, d, J = 7.3 Hz), 0.95 (3H, d, J = 6.7 Hz), 0.96 (3H, d, J = 5.8 Hz),
1.00 (3H, d, J = 7.6 Hz), 1.01 (3H, d, J = 7.3 Hz), 1.04 (3H, d, J = 6.7 Hz),
1.12 (3H, d, J = 6.7 Hz), 1.27 (3H, d, J = 4.7 Hz), 1.27 (1H, m), 1.45
(1H, m), 1.81 (2H, m), 1.97ꢀ2.12 (4H, m), 2.14ꢀ2.39 (5H, m), 2.96
(3H, s), 3.02 (3H, s), 3.14 (1H, m), 3.62 (1H, q, J = 7.4 Hz), 3.81 (1H, q,
J = 7.4 Hz), 3.95 (1H, d, J = 10.3 Hz), 4.16 (1H, d, J = 9.3 Hz), 4.73 (1H,
t, J = 6.2 Hz), 4.86 (2H, d, J = 8.1 Hz), 4.96 (1H, t, J = 6.1 Hz), 6.27 (1H,
0
0
de Afuera on the Pacific coast of Panama (7ꢀ41.617 N, 81ꢀ38.379 E).
Morphological characterization was performed using an Olympus IX51
epifluorescent microscope (1000ꢁ) equipped with an Olympus
U-CMAD3 camera. Morphological comparison and putative taxonomic
identification of the cyanobacterial specimen was performed in accor-
25,27
dance with modern classification systems.
80
Extraction and Isolation. The cyanobacterial biomass (9.75 g, dry
59 4 7
d, J = 8.2 Hz); ESIMS/MS m/z 727.21 (C35H N O Br), 642.12
8
0
80
80
wt) was extracted with 2:1 CH Cl /CH OH to afford 1.8 g of dried
(C30
463.20 (C20
329.13 (C16
214.11 (C11
H
48
N
3
O
H
7
Br), 574.13 (C26
H
44
N
3
O
6
Br), 542.20 (C25
), 424.19 (C22
), 283.15 (C15
H
40
N
H
H
3
O
5
Br),
),
),
); HRESIMS [M þ Na] m/z 775.3257 (calcd for
BrNa 775.3252).
2
2
3
80
extract. A portion of the extract was fractionated by silica gel VLC using a
stepwise gradient solvent system of increasing polarity starting from
35
N
2
O
5
Br), 442.21 (C22
H
40
N
O
2
3
O
6
38
N O
3
5
H
29
N O ), 311.06 (C16
2 5
H
N
27
N O
2
4
þ
27
3
1
00% hexanes to 100% MeOH (nine fractions, AꢀI). The two fractions
H
20NO
3
7
8
eluting with 100% EtOAc (fraction G) and 75% EtOAc in MeOH
C
36
H
57
N
4
O
8
9
33
dx.doi.org/10.1021/np200077f |J. Nat. Prod. 2011, 74, 928–936

Journal of Natural Products
ARTICLE
2
3
Veraguamide C (3): amorphous solid; [R]
D
ꢀ13.0 (c 0.17,
Hydrogenation, Acid Hydrolysis, and Marfey’s Analysis.
Veraguamide A (1, 1 mg) was dissolved in 1 mL of EtOH and treated
with a small amount of 10% Pd/C and then placed under an atmosphere
1
CH Cl ); H NMR (600 MHz, CDCl ) δ 0.86ꢀ0.89 (6H, m), 0.94
2
2
3
(
(
(
(
(
3H, d, J = 6.4 Hz), 0.96 (3H, d, J = 6.6 Hz), 1.00 (3H, d, J = 6.4 Hz), 1.01
3H, d, J = 7.1 Hz), 1.03 (3H, d, J = 7.0 Hz), 1.12 (3H, d, J = 6.6 Hz), 1.26
3H, s), 1.81 (1H, m), 1.93ꢀ2.12 (7H, m), 2.15ꢀ2.40 (4H, m), 2.95
3H, s), 3.02 (3H, s), 3.12 (1H, m), 3.63 (1H, m), 3.86 (1H, m), 3.95
1H, d, J = 10.7 Hz), 4.15 (1H, d, J = 10.8 Hz), 4.71 (1H, m), 4.88 (1H,
of H (g) for 5 h. The reaction product was treated with 1.5 mL of 6 N
2
HCl in a microwave reactor at 160 ꢀC for 5 min. An aliquot (∼300 μg) of
the hydrolysate was dissolved in 300 μL of 1 M sodium bicarbonate, and
then 16 μL of 1% D-FDAA (1-fluoro-2,4-dinitrophenyl-5-D-alanine
amide) was added in acetone. The solution was maintained at 40 ꢀC
for 90 min, at which time the reaction was quenched by the addition of
50 μL of 6 N HCl. The reaction mixture was diluted with 200 μL of
m), 4.90 (1H, d, J = 9.2 Hz), 4.96 (1H, m), 6.27 (1H, m); ESIMS/MS
m/z 661.35 (C36
H
61
N
4
O
7
), 576.23 (C31 ), 496.25 (C26
H
50
N
3
O
7
H
46
-
-
N
3
O
6
), 462.30 (C25
H
40
N
3
O
5
), 456.25 (C23H N O ), 438.22 (C23
42 3 6
H N O ), 383.26 (C H N O ) 343.17 (C H N O ), 325.12
3
CH CN, and 10 μL of the solution was analyzed by LC-ESIMS.
The Marfey’s derivatives of the hydrolysate and standards were
analyzed by RP HPLC using a Phenomenex Luna 5 μm C18 column
4
0
3
5
20 35
2
4
17 31 2 5
þ
(
7
C
17
H
29
N
2
O
4
), 297.19 (C16
H
29
N
2
O
3
); HRESIMS [M þ Na] m/z
Na 711.4303).
Veraguamide H (4): amorphous solid; ESIMS/MS m/z 647.33
60 4 8
11.4302 (calcd for C37H N O
(4.6 ꢁ 250 mm). The HPLC conditions began with 10% CH
O acidified with 0.1% formic acid (FA) followed by a gradient profile
to 50% CH CN/50% H O acidified with 0.1% FA over 85 min at a flow
3
CN/90%
(
C H N O ), 562.21 (C H N O ), 496.25 (C H N O ),
H
2
3
5
59
4
7
30 48
3
7
26 46 3 6
4
3
2
C
62.30 (C25
65.24 (C19
H
H
40
N
3
O
5
), 442.23 (C22
H
40
N
3
O
O
5
6
), 424.20 (C22
H
H
38
N
3
O
5
),
),
3
2
33
N
2
O
4
) 329.14 (C16
H
29
N
2
), 311.07 (C16
27
N
2
O
4
of 0.4 mL/min, monitoring from 200 to 600 nm. The retention times of
authentic acid D-FDAA derivatives were D-Pro (66.49), L-Pro (69.30),
D-Val (78.45), D-N-Me-Val (86.61), L-Val (88.00), and L-N-Me-Val
(91.66); the hydrolysate product gave peaks with retention times of
69.49, 88.07, and 91.74 min, according to L-Pro, L-Val, and L-N-Me-Val,
respectively.
Preparation and GC-MS Analysis of 2-Hydroxy-3-methyl-
pentanoic Acid (Hmpa). Veraguamide A (1, 1 mg) was dissolved in
1 mL of ethanol and treated with a small amount of 10% Pd/C and
þ
83.18 (C H N O ); HRESIMS [M þ Na] m/z 697.4141 (calcd for
15 27 2 3
H N O
36 58 4 8
Na 697.4147).
Veraguamide I (5): amorphous solid; ESIMS/MS m/z 665.37
(
(
(
(
C H N O ), 580.28 (C H N O ), 500.28 (C H N O ), 466.34
36
65
44
35
4
3
2
7
5
4
31 54
42
31
3
7
26 50
40
29
3
6
C
25
H
H
N
N
O
O
), 456.25 (C23
H
N
3
O
6
), 438.22 (C23
H
N
3
O
O
4
5
), 383.26
), 297.19
C
20
) 343.17 (C17
H
N
2
O
5
), 325.12 (C17
H
N
2
þ
C H N O ); HRESIMS [M þ Na] m/z 715.4619 (calcd for
1
6
29
2
3
C
37 64 4 8
H N O Na 715.4616).
Veraguamide J (6): amorphous solid; ESIMS/MS m/z 651.35
), 566.20 (C30 ), 500.25 (C26 ), 467.05
H
2
(g). The reaction product was then treated with 1.5 mL of 6 N HCl at
110 ꢀC for 16 h. The reaction product was dried under N (g), then
dissolved in 0.5 mL of MeOH and Et O and treated with diazomethane.
L-Ile (20 mg) was dissolved in 5 mL of cold (0 ꢀC) 0.2 N HClO , and
then 2 mL of NaNO (aq) was added with rapid stirring. The reaction
(
(
(
(
C H N O
35 63 4 7
H N O
52 3 7
H N O
50 3 6
2
C H N O ), 442.23 (C H N O ), 424.20 (C H N O ), 365.24
2
2
5
45
33
27
3
2
2
5
4
3
22 40
3
6
22 38
3 5
C
C
19
H
H
N
N
O
O
29
) 329.14 (C16H N
2
O
5
), 311.07 (C16
27
H N
2
O
4
), 283.18
4
þ
15
); HRESIMS [M þ Na] m/z 699.4298 (calcd for
2
C H N O Na 699.4303).
mixture was stored at room temperature for 1 h. The solution was boiled
for 3 min, cooled to room temperature, and then saturated with NaCl.
3
6 62 4 8
2
3
Veraguamide K (7): amorphous solid; [R]
CH
ꢀ21.4 (c 0.33,
D
1
Cl
2
); H NMR (600 MHz, CDCl
3
) δ 0.85 (3H, d, J = 6.9 Hz),
The mixture was extracted three times with Et
then dried under N (g) to yield the oily 2S,3S-Hmpa. An aliquot was
dissolved in 1.5 mL of MeOH and Et O and treated with diazomethane.
The product was then dried under N (g). Correspondingly, 2R,3R-
2 2
O, and the Et O layer was
2
0.88 (3H, d, J = 7.7 Hz), 0.90 (3H, t, J = 6.5 Hz), 0.91 (3H, d, J = 6.5 Hz),
0.99 (3H, d, J = 6.5 Hz), 1.00 (6H, d, J = 6.9 Hz), 1.04 (3H, d, J = 6.5 Hz),
1.16 (1H, m), 1.20 (3H, d, J = 6.9 Hz), 1.25 (3H, t, J = 7.0 Hz), 1.47 (1H,
2
2
2
m), 1.47 (2H, m), 1.74 (1H, m), 1.89 (1H, m), 2.13ꢀ2.30 (7H, m), 2.41
1H, m), 2.93 (1H, d, J = 5.5 Hz), 3.10 (3H, s), 3.13 (3H, s), 3.68 (1H,
m), 3.79 (1H, t, J = 6.5 Hz), 3.89 (1H, m), 4.15 (1H, m), 4.17 (1H, m)
.81 (1H, m), 4.86 (1H, t, J = 7.0 Hz), 4.88 (1H, d, J = 10.8 Hz), 4.90
1H, m), 6.36 (1H, d, J = 8.6 Hz); ESIMS/MS m/z 769.24
Hmpa, 2S,3R-Hmpa, and 2R,3S-Hmpa were synthesized with the same
procedure from D-Ile, L-allo-Ile, and D-allo-Ile, respectively.
(
Each authentic stereoisomer of Hmpa was dissolved in CH Cl with
2 2
4
(
(
retention times measured by GC using a Cyclosil B column (Agilent
Technologies J&W Scientific, 30 m ꢁ 0.25 mm) under the following
conditions: the initial oven temperature was 35 ꢀC, held for 15 min,
followed by a ramp from 35 to 60 ꢀC at a rate of 1 ꢀC/min and another
80
80
C H N O Br), 656.21 (C H N O Br), 559.14 (C H
3
7
60
4
8
31 49
3
7
26 42-
8
0
78
4
N O Br), 484.22 (C H N O ), 443.10 (C H N O Br), 438.22
2
6
25 46
3
6
20 33
2
o
(
(
C
C
23
H
H
40
N
N
3
O
O
5
), 371.06 (C19
H
35
N
2
O
5
), 325.11 (C17
H
29
N
2
O
4
), 297.20
ramp to 170 C at a rate of 10 ꢀC/min, and held at 170 ꢀC for 5 min. The
þ
); HRESIMS [M þ Na] m/z 835.3831 (calcd for
retention time of the Hmpa residue in acid hydrolysate of 1 matched
with 2S,3S-Hmpa (45.63 min; 2S,3R-Hmpa, 44.86 min; 2R,3S-Hmpa,
45.06; 2R,3R-Hmpa, 45.26).
16
29
2
3
7
8
C H N O BrNa 835.3827).
3
8 65 4 9
22
D
Veraguamide L (8): amorphous solid; [R]
ꢀ27.9 (c 0.50,
1
CH
2
Cl
2
); H NMR (600 MHz, CDCl
3
) δ 0.85 (3H, d, J = 6.7 Hz),
Preparation and GC-MS Analysis of Methyl 3-Hydroxy-2-
Methyloctanoate (Hmoaa). 2S,3S-Hmoaa and 2S,3R-Hmoaa were
0
0
.88 (3H, d, J = 6.7 Hz), 0.89 (3H, d, J = 6.6 Hz), 0.95 (3H, d, J = 6.6 Hz),
.97 (3H, d, J = 6.5 Hz), 0.98 (3H, d, J = 6.2 Hz), 1.00 (3H, d, J = 6.9
1
3b
synthesized following literature conditions. A sample of 5 mg of each
product was dissolved in 2 mL of dry CH Cl and treated with 0.122 mmol
Hz), 1.04 (3H, d, J = 1.04 Hz), 1.17 (3H, d, J = 7.1 Hz), 1.23 (3H, t, J =
2
2
7
1
.l Hz), 1.46 (2H, dt, J = 7.1, 6.9 Hz), 1.51 (1H, m), 1.72 (1H, m),
.87 (1H, m), 2.00 (1H, m), 2.06 (1H, m), 2.15 (1H, m), 2.19 (1H,
of triethylamine and 16.4 mmol of DMAP, and each was separately
treated with 0.126 mmol of both R-MTPA-Cl and S-MTPA-Cl for 17 h
at room temperature. Each reaction was quenched with 2.5 mL of 1 N
m), 2.20 (1H, m), 2.22 (1H, m), 2.24 (1H, m), 2.26 (1H, m), 2.39
(
(
7
4
1H, m), 2.93 (1H, d, J = 3.81 Hz), 3.09 (3H, s), 3.13 (3H, s), 3.67
1H, dt, J = 7.7, 7.5 Hz), 3.78 (1H, t, J = 6.6 Hz), 3.85 (1H, dt, J = 7.7,
.5 Hz), 4.14 (1H, m), 4.17 (1H, m), 4.81 (1H, dt, J = 6.2, 7.8 Hz),
.82 (1H, d, J = 8.6 Hz), 4.85 (1H, d, J = 10.5 Hz), 4.87 (1H, d, J = 10.3
2
HCl and extracted with Et O to produce the four diastereomeric
standards. An aliquot of the hydrolysate of veraguamide A (1, 0.3 mg)
was dissolved in 1 mL of CH Cl and treated with 7.32 μmol of
2
2
triethylamine, 0.964 mol of DMAP, and 7.56 μmol of S-MTPA for 18 h
at room temperature.
Hz), 4.90 (1H, dd, J = 8.5, 6.3 Hz), 6.37 (1H, d, J = 8.8 Hz); ESIMS/
MS m/z 755.24 (C36
5
N
(
8
8
0
80
H
58
N
4
O
8
Br), 642.21 (C30
H
47
N
3
O
7
Br),
The four stereoisomeric standards of Hmoaa as well as the derivatized
hydrolysate product of compound 1 were dissolved in CH Cl and
80
45.14 (C H N O Br), 470.21 (C H N O ), 445.11 (C H -
25 40 2 6 24 44 3 6 20 33
2
2
8
0
2
O
4
Br), 424.21 (C22
), 283.20 (C15
21.3673 (calcd for C H N O BrNa 821.3671).
38 3 5
H N O ), 357.06 (C18
H
33
N
2
O
5
), 311.10
analyzed by GC-MS as described below. A DB-5MS GC column
(Agilent Technologies J&W Scientific, 30 m ꢁ 0.25 mm) was used
with the following conditions: initial oven temperature was 35 ꢀC, held
þ
C
16
H
27
N
O
2 4
H
27
N
2
3
O ); HRESIMS [M þ Na] m/z
7
8
3
8 63 4 9
9
34
dx.doi.org/10.1021/np200077f |J. Nat. Prod. 2011, 74, 928–936

Journal of Natural Products
ARTICLE
2
9
for 2 min, followed by a ramp from 35 to 140 ꢀC at a rate of 25 ꢀC/min,
followed by another ramp to 165 ꢀC at a rate of 1 ꢀC/min, and held for
SSU secondary structures model for Escherichia coli J01695 without
data exclusion. The best-fitting nucleotide substitution model optimized
by maximum likelihood was selected using corrected Akaike/Bayesian
Information Criterion (AIC /BIC) in jModeltest 0.1.1. The evolu-
C
tionary histories of the cyanobacterial genes were inferred using max-
imum likelihood (ML) and Bayesian inference algorithms. The ML
inference was performed using GARLI 1.0 for the GTRþIþG model
assuming a heterogeneous substitution rate and gamma substitution of
variable sites (proportion of invariable sites (pINV) = 0.494, shape
parameter (R) = 0.485, number of rate categories = 4) with 1000
bootstrap replicates. Bayesian inference was conducted using MrBayes
3.1 with four Metropolis-coupled MCMC chains (one cold and three
heated) run for 3 000 000 generations. The first 25% were discarded as
burn-in, and the following data set was sampled with a frequency of every
1
5 min before it was finally ramped up to a temperature of 190 ꢀC at
ꢀC/min. The retention time of the Hmoaa residue from the derivatized
3
0
1
hydrolysate mixture of 1 matched that of 2S,3R-Hmoaa that was reacted
with S-MTPA-Cl (47.13 min; 2S,2S-Hmoaa reacted with S-MTPA-Cl,
3
1
48.17 min; 2S,3R-Hmoaa reacted with R-MTPA-Cl, 48.13 min; 2S,3S-
Hmoaa reacted with R-MTPA-Cl, 47.63 min).
Tandem Mass Spectrometry Data Acquisition and Pre-
processing. For the ion trap data acquisition, each compound was
2
prepared as a 1 μM solution using 50:50 MeOH/H O with 1% AcOH as
3
2
solvent and underwent nanoelectrospray ionization on a Biversa Nano-
mate (pressure 0.3 psi, spray voltage 1.4ꢀ1.8 kV). Ion trap spectra were
acquired on a Finnigan LTQ-MS (Thermo-Electron Corporation)
running Tune Plus software version 1.0. Ion tree data sets were collected
33
100 generations. The MCMC convergence was detected by AWTY.
þ
using automatic mode, in which the [M þ H] of each compound was
n
set as the parent ion. MS data were collected with the following
’
ASSOCIATED CONTENT
n
parameters: maximum breadth, 50; maximum MS depth, 3. At n = 2,
1
13
isolation width, 4; normalized energy, 50. At n = 3, isolation width, 4;
normalized energy, 30. The Thermo-Finnigan files (in RAW format)
were then converted to an mzXML file format using ReAdW (http://
tools.proteomecenter.org/) and subject to analysis using algorithms as
S
Supporting Information. H NMR, C NMR, COSY,
TOCSY, NOESY, HSQC, and HMBC spectra in CDCl for
veraguamide A (1). H NMR spectra in CDCl for veraguamides B
2) and K (3). H NMR, TOCSY, HSQC, and HMBC spectra in
b
3
1
3
1
(
18
well as manual interpretation.
2
CDCl for veraguamide L (8). MS chromatograms for veraguamides
3
Cytotoxicity Assay. H-460 cells were added to 96-well plates at
2
3
AꢀC and HꢀL (1ꢀ8). MS /MS algorithm results for veragua-
mides B, C, H, and I. Biological assay results for veragumide A.
Morphological description of the veraguamide producer. This material
is available free of charge via the Internet at http://pubs.acs.org.
4
3
.33 ꢁ 10 cells/mL of Roswell Park Memorial Institute (RPMI) 1640
medium with fetal bovine serum (FBS) and 1% penicillin/streptomycin.
The cells, in a volume of 180 μL per well, were incubated overnight
2
(37 ꢀC, 5% CO ) to allow recovery before treatment with test
compounds. Compounds were dissolved in DMSO to a stock concen-
tration of 10 mg/mL. Working solutions of the compounds were made
in RPMI 1640 medium without FBS, with a volume of 20 μL added to each
well to give a final compound concentration of either 30 or 3 μg/mL. An
equal volume of RPMI 1640 medium with FBS was added to wells
designated as negative controls for each plate. Plates were incubated for
approximately 48 h before staining with MTT. Using a ThermoElectron
Multiskan Ascent plate reader, plates were read at 570 and 630 nm.
DNA Extraction, Amplification, and Sequencing. Algal
biomass (∼50 mg) was partly cleaned under an Olympus VMZ
dissecting microscope. The biomass was pretreated using TE (10 mM
Tris; 0.1 M EDTA; 0.5% SDS; 20 μg/mL RNase)/lysozyme (1 mg/mL)
at37 ꢀCfor 30minfollowedbyincubation withproteinaseK(0.5mg/mL)
at 50 ꢀC for 1 h. Genomic DNA was extracted using the Wizard
Genomic DNA purification kit (Promega) following the manufacturer’s
specifications. DNA concentration and purity was measured on a DU
’
AUTHOR INFORMATION
Corresponding Author
Tel: (858) 534-0578. Fax: (858) 534-0576. E-mail: wgerwick@
*
ucsd.edu.
’
ACKNOWLEDGMENT
We thank the government of Panama for permission to make
the cyanobacterial collections, K. Tidgewell, H. Choi, P. Boudreau,
M. Balunas, and J. Nunnery for helpful discussions, and the
UCSD mass spectrometry facilities for their analytical services.
13
The 500 MHz NMR C cold probe was supported by NSF
CHE-0741968. We are grateful for data analysis using the NSF-
funded CIPRES cluster at the San Diego Supercomputer Center.
Financial support was provided by the Fogarty International
Center’s International Cooperative Biodiversity Groups (ICBG)
program in Panama (ICBG TW006634). Funding of the Dor-
restein Laboratory was provided by the Beckman Foundation
and NIGMS grant NIHGM 086283 (to P.C.D. and P.A.P.). W-T.
L. was supported, in part, by study abroad grant SAS-98116-
2-US-108 from Taiwan.
800 spectrophotometer (Beckman Coulter). The 16S rRNA genes were
PCR-amplified from isolated DNA using the modified lineage-specific
0
0
primers, OT106F 5 -GGACGGGTGAGTAACGCGTGA-3 and
0
0
OT1445R 5 -AGTAATGACTTCGGGCGTG-3 . The PCR reaction
volumes were 25 μL containing 0.5 μL (∼50 ng) of DNA, 2.5 μL of 10ꢁ
PfuUltra IV reaction buffer, 0.5 μL (25 mM) of dNTP mix, 0.5 μL of
each primer (10 μM), 0.5 μL of PfuUltra IV fusion HS DNA polymerase,
2
and 20.5 μL of dH O. The PCR reactions were performed in an
Eppendorf Mastercycler gradient as follows: initial denaturation for
’
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