Molluscicidal metabolites from an assemblage of Palmyra Atoll cyanobacteria

Article abstract of DOI:10.1021/np200106b

Molluscicides can play an important role in the control of schistosomiasis because snails of the genus Biomphalaria act as intermediate hosts for the parasite. Schistosomiasis is one of 13 neglected tropical diseases with high morbidity and mortality that collectively affect one billion of the world's poorest population, mainly in developing countries. Thiopalmyrone (1) and palmyrrolinone (2), metabolites isolated from extracts of a Palmyra Atoll environmental assemblage of two cyanobacteria, cf. Oscillatoria and Hormoscilla spp., represent new and potent molluscicidal chemotypes against Biomphalaria glabrata (LC₅₀ = 8.3 and 6.0 μM, respectively). A slight enhancement in molluscicidal effect (LC₅₀ = 5.0 μM) was observed when these two natural products were utilized as an equimolar binary mixture. (Chemical Equation Presented).

Full text of DOI:10.1021/np200106b

ARTICLE
pubs.acs.org/jnp
Molluscicidal Metabolites from an Assemblage of Palmyra Atoll
Cyanobacteria
Alban R. Pereira,†,‡ Lena Etzbach,†,‡,§ Niclas Engene,† Rolf M€uller,§ and William H. Gerwick*,†
†Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, and Skaggs School of Pharmacy and
Pharmaceutical Sciences, University of California, San Diego, La Jolla, California 92093, United States
§Helmholtz-Institute for Pharmaceutical Research Saarland, Helmholtz Center for Infectious Research, Department of Microbial
Natural Products, Saarland University, Campus C2 3, 66123 Saarbr€ucken, Germany
S Supporting Information
b
ABSTRACT: Molluscicides can play an important role in the
control of schistosomiasis because snails of the genus Biompha-
laria act as intermediate hosts for the parasite. Schistosomiasis is
one of 13 neglected tropical diseases with high morbidity and
mortality that collectively affect one billion of the world’s
poorest population, mainly in developing countries. Thiopal-
myrone (1) and palmyrrolinone (2), metabolites isolated from
extracts of a Palmyra Atoll environmental assemblage of two
cyanobacteria, cf. Oscillatoria and Hormoscilla spp., represent
new and potent molluscicidal chemotypes against Biomphalaria
glabrata (LC50 = 8.3 and 6.0 μM, respectively). A slight
enhancement in molluscicidal effect (LC50 = 5.0 μM) was
observed when these two natural products were utilized as an equimolar binary mixture.
eglected tropical diseases (NTDs) are infectious diseases
that collectively affect one billion of the world’s poorest
new water-soluble, inexpensive, and toxicity-specific molluscici-
dal agents that can help reduce the incidence of schistosomiasis
infection and reinfection.4a,7 Using a simple assay8 to screen
marine cyanobacterial extracts for their capacity to kill B. glabrata,
our research group has previously discovered several molluscicidal
agents, including barbamide (LC100 = 21.6 μM),9 tanikolide
(LC50 = 31.6 μM),10 and cyanolide A (LC50 = 1.2 μM).11 Here,
we describe the isolation and structure elucidation of two new
molluscicides, thiopalmyrone (1, LC50 = 8.3 μM) and palmyrro-
linone(2, LC50 = 6.0 μM), fromanassemblageof twoundescribed
species of cyanobacteria collected from Palmyra Atoll.
N
population in developing countries near the equator1 and con-
tribute to approximately 500 000 deaths per year.2 Commonly
associated with long-term illness, impaired childhood develop-
ment, disfigurement, decreased productive capabilities, and
social stigma, NTDs are perpetuated by diverse factors including
geographical isolation, lack of education, poor sanitation, and
substandard housing conditions.1,3 Additionally, since NTDs
generally do not represent a threat to the developed world, there
is little financial incentive for the pharmaceutical industry to
engage in research toward developing new treatments.4
Although there is some variation in what constitutes a NTD, the
most comprehensive account encompasses 37 conditions, with
13 core diseases causing the most significant morbidity and
mortality.1a,b,3 Three of the 13 core NTDs are protozoal
(African sleeping sickness, Chagas disease, and leishmaniasis)
and three are bacterial (Buruli ulcer, leprosy, and trachoma). The
remaining seven diseases are caused by worms (elephantiasis,
guinea worm, hookworm, river blindness, roundworm, whip-
worm, and schistosomiasis).3,4b As part as our International
Cooperative Biodiversity Group (ICBG) program in Panama,5
we have screened natural product materials for activities against
malaria, leishmaniasis, Chagas disease, and schistosomiasis. In
the case of schistosomiasis, molluscicides constitute an important
method for the control of the disease because snails of the genus
Biomphalaria (e.g., B. glabrata and B. alexandrina) are inter-
mediate hosts for the parasite.6 Thus, there is an urgent need for
’ RESULTS AND DISCUSSION
A dark brown mat of cyanobacteria was collected by hand from
Palmyra Atoll in the Central Pacific Ocean. The cyanobacterial
collection was repeatedly extracted with CH2Cl2/MeOH (2:1),
and a portion (4.0 g) was fractionated by silica gel vacuum liquid
chromatography (VLC) to produce nine subfractions (AꢀI).
Received: February 2, 2011
Published: April 07, 2011
r
Copyright
2011 American Chemical Society and
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Table 1. NMR Spectroscopic Data (600 MHz, CDCl3) for Thiopalmyrone (1)
carbon
δC, mult.a
δH, mult. (J in Hz)b
HMBCc
COSY
NOESY
7
1
189.0, C
2
101.1, CH
174.2, C
5.46, s
3, 4a, 4b, 5
4a, 4b
3
4a
4b
5
31.3, CH2
2.86, dd (17.1, 4.8)
2.76, dd (17.1, 7.5)
3.59, dddd (7.5, 6.6, 6.6, 4.8)
3.81, dd (11.4, 6.6)
3.77, dd (11.4, 6.6)
3.72, s
2, 3, 5, 6a, 6b
2, 3, 5, 6a, 6b
1, 3, 4a, 4b, 6a, 6b
4a, 4b, 5
2, 4b, 5
2, 4a, 5
4a, 4b, 6a, 6b
5, 6b
6a, 6b
6a, 6b
42.8, CH
6a
6b
7
64.1, CH2
4a, 4b
4a, 4b
2
4a, 4b, 5
5, 6a
55.8, CH3
2, 3, 4a, 4b
a Recorded at 125 MHz. b Recorded at 600 MHz. c From proton to the indicated carbon.
1
Fraction F (0.170 g) displayed intriguing H NMR resonances
and was thus subjected to 1H NMR-guided fractionation invol-
ving gradient silica gel column chromatography, preparative
TLC, and normal-phase HPLC. Two new compounds were
isolated from this process, thiopalmyrone (1, 2.6 mg, 0.06%) and
palmyrrolinone (2, 5.9 mg, 0.15%). In addition to these two new
cyanobacterial natural products, fraction F was also found to
contain the known metabolites lyngbic acid and the carotenoid
zeaxanthin.12,13
HRESIMS of 1 yielded an [M þ H]þ peak at m/z 175.0421,
with a complex isotopic pattern composed of m/z 175/176/177
(100:7:5 ratio), suggesting the presence of one sulfur atom.
Combined with NMR data (Table 1), it was possible to derive a
molecular formula of C7H10O3S (three degrees of unsaturation).
Intense IR absorptions at 3420 (broad), 1732, and 1609 cmꢀ1
were consistent with a hydroxy group and an R,β-unsaturated
carbonyl functionality in 1; the latter moiety was corroborated by
UV absorptions at 237 and 265 nm. The 1H NMR spectrum of 1
(Table 1) displayed resonances assignable to one vinyl singlet,
one methoxy singlet, two diastereotopic methylene multiplets,
and one methine multiplet. In agreement with these assignments,
the 13C NMR spectrum of 1 showed one sp2 methine carbon and
one methoxy resonance, as well as two methylene and one
methine sp3 carbons. This spectrum was completed by two
deshielded quaternary carbonyl-type resonances. Extensive anal-
ysis by 2D NMR, including HSQC, HMBC, COSY, and NOESY,
revealed the planar structure of thiopalmyrone (1) as
described below.
COSY and coupling constant data revealed that the methine
proton at δ 3.59 (H-5) was flanked by two diastereotopic
methylene pairs, giving rise to the sole spin system present in
1. One of these pairs was comprised of protons at δ 2.86 (H-4a)
and 2.76 (H-4b), which showed an additional long-range COSY
correlation with the broadened singlet proton at δ 5.46 (H-2).
This vinylic methine was a component of a trisubstituted double
bond with an oxygenated quaternary carbon at δ 174.2 (C-3), as
revealed by HMBC correlations between the methoxy singlet at δ
3.72 (H-7) and carbon resonances at δ 101.1 (C-2), 174.2 (C-3),
and 31.3 (C-4). Furthermore, an intense NOE correlation
between H-2 and H-7 allowed assignment of this double bond
as E. The second diastereotopic methylene was comprised of
deshielded protons with almost isochronous chemical shift
values at δ 3.81 (H-6a) and 3.77 (H-6b, δC 64.1), suggesting
the presence of an attached oxygen atom. In agreement with the
COSY-derived spin system above, HMBC correlations sup-
ported the carbon sequence C2dC3(OC7)ꢀC4ꢀC5ꢀC6ꢀO
(I in Figure 1). An informative HMBC correlation between
Figure 1. Partial structures for thiopalmyrone (1) and palmyrrolinone
(2) derived from NMR data and their assembly by HMBC correlations.
methine H-5 (δ 3.59) and the remaining carbonyl resonance at δ
189.0 (C-1) led us to postulate a thioester linkage on the basis of
the relatively shielded 13C chemical shift for C-5 (δ 42.8). With
all atoms in the molecular formula assigned and one unsaturation
degree unaccounted for, we assembled an R,β-unsaturated
thioester moiety that formed a six-membered ring, thus complet-
ing the planar structure of thiopalmyrone (1). This unique
functional group was in agreement with the intense IR absorp-
tions at 1732 and 1609 cmꢀ1, and the 1H and 13C chemical shifts
were consistent with those of structurally similar synthetic model
compounds.14
The preferred conformation between the diastereomeric
methylene pair H-4a (δ 2.86)/H-4b (δ 2.76) and the adjacent
methine H-5 (δ 3.59) was determined via J-based configuration
analysis (Figure 2).15 Homonuclear coupling constants were
1
obtained from the H NMR spectrum, whereas heteronuclear
coupling values were obtained from HETLOC16 and
HSQMBC17 experiments. Interestingly, intermediate values for
3
the homonuclear JH-5,H-4b (7.5 Hz) and three out of four
heteronuclear coupling constants led us to consider two major
envelope-like conformers about the C-4ꢀC-5 carbon bond
where the planes of the C-1dO and C-2dC-3 double bonds
are coplanar and H5/H4b exhibit both anti and gauche orienta-
tions (Figure 2A). The strong NOE correlation observed be-
tween H-2 and H-7 suggested that the methoxy group at C-7 is
coplanar with the double bonds. Fortunately, the six possible
rotamer pairs with the different prochiral assignments for the
methylene protons were unequivocally distinguished using these
various J values.15 The only combination in agreement with our
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Table 2. NMR Spectroscopic Data (600 MHz, CDCl3) for
Palmyrrolinone (2)
δH, mult.
carbon δC, mult.a
(J in Hz)b
HMBCc
2, 3, 4, 5
COSY NOESY
1
63.1, CH2 4.35, dd (4.9, 1.5)
137.5, CH 6.67, dt (15.8, 4.9)
123.2, CH 7.53, dt (15.8, 1.5)
167.8, C
2, 3
1, 3
1, 2
2
1, 3, 4, 5
1, 2, 4
3
4
5
94.2, CH 6.81, s
1, 2, 3, 4, 6
12
6
164.9, C
7
170.1, C
8
125.8, CH 6.07, dd (6.0, 1.6)
153.1, CH 7.21, dd (6.0, 1.9)
6, 7, 9, 10, 11 9, 10
7, 8, 10, 11 8, 10
9
11
10
11
12
58.2, CH 4.88, qdd (6.6, 1.9, 1.6) 6, 7, 8, 9, 11 8, 9, 11
17.9, CH3 1.46, d (6.6)
8, 9, 10
10
9
5
55.8, CH3 3.82, s
3, 4, 5
a Recorded at 125 MHz. b Recorded at 600 MHz. c From proton to the
indicated carbon.
with the presence of a hydroxy group and an R,β-unsaturated
carbonyl moiety. The latter, however, differed from that of 1 as
shown by a UV absorption at 293 nm, which suggested additional
1
conjugation. The H NMR spectrum of 2 (Table 2) showed
signals for five vinylic protons, deshielded multiplets for one sp3
methine and one methylene, a methoxy singlet, and one final
methyl doublet. Consistent with these assignments, the 13C
NMR spectrum of 2 possessed three oxygenated quaternary
sp2 carbons, five vinylic methine carbons, one sp3 methine and
one oxymethylene, a methoxy carbon, and a final high-field
methyl carbon.
Two partial structures were assembled for 2 using the full set
of 2D NMR data (II and III in Figure 1). A spin system
comprising part of substructure II possessed a deshielded doub-
let methyl group (δ 1.46, H-11, d, J = 6.6 Hz) adjacent to three
contiguous methine protons. The first of these possessed a
chemical shift of δ 4.88 (H-10, qdd, J = 6.6, 1.6, 1.9 Hz),
consistent with its attachment to a carbon bearing a heteroatom
(C-10, δ 58.2). This proton was coupled vicinally and allylically
to both protons of an adjacent polarized Z double bond
constituted by protons at δ 7.21 (H-9, dd, J = 1.9, 6.0 Hz) and
6.07 (H-8, dd, J = 1.6, 6.0 Hz), thus suggesting an R,β-
unsaturated amide functionality. This moiety was confirmed by
HMBC correlations from protons H-8, H-9, and H-10 to a
carbonyl carbon at δ 170.1, a chemical shift consistent with the
amide of a pyrrolinone ring (II).20
Partial structure III possessed a spin system that included an
oxymethylene at δ 4.35 (H-1, dd, J = 1.5, 4.9 Hz, δC 63.1) that was
placed adjacent to a second polarized double bond (δ6.67, H-2, dd,
J = 4.9, 15.8 Hz and δ 7.53, H-3, dd, J = 1.5, 15.8 Hz) by COSY.
These protons in turn displayed HMBC correlations with an
oxygenated sp2 quaternary carbon at δ 167.8 (C-4) and a vinylic
methine carbon at δ 94.2 (C-5), thus defining a conjugated
trisubstituted double bond. This second double bond was sup-
ported by HMBC correlations from the O-methyl singlet (H-12) as
well as the vinyl proton singlet (H-5) to carbon resonances at δ
123.2 (C-3) and 167.8 (C-4), among others. The latter H-5 proton
possessed an additional HMBC correlation with an amide-type
carbonyl at δ 164.9 (C-6), giving rise to an R,β,γ,δ-unsaturated
amide, thus completing partial structure III. The geometries of
both double bonds were determined to be E on the basis of a 3JH2,
Figure 2. (A) J-based configuration analysis for thiopalmyrone (1) [S,
small; M, medium; L, large; H4a is δ 2.86 (low field) and H4b is δ 2.76
(high field)]. aRotamer designation according to Murata and co-workers.15
(B) Application of Snatzke’s enone sector rules for prediction of the sign of
the nfπ* transition (320ꢀ350 nm) in the CD spectrum of 1.18
coupling constant and NOE data corresponded to the rotamer
pair D-1/D-2. Because D-2 is thermodynamically disfavored due
to two gauche interactions (C-6/C-3 and C-3/S), as well as
repulsive electronic 1,3-diaxial interactions between the axial
C-6ꢀOH and the π bonds at C-1 and C-3 (giving rise to the
carbonꢀoxygen and carbonꢀcarbon double bonds, re-
spectively), the equilibrium appears to be displaced toward the
half-chair D-1 conformer. This conclusion was substantiated by
medium large 3JH-5,H-4b and medium small 3JH-5,H-4a values of 7.5
and 4.8 Hz, respectively. This favored half-chair conformer with
methine H-5 and methylene H-6 out of the plane was subjected
to Snatzke’s sector rules for planar enones.18 This analysis
predicts a positive Cotton effect for 1 exhibiting an R configura-
tion at C-5 (Figure 2B), whereas the opposite could be expected
for the S enantiomer. The CD curve for 1 showed a positive
Cotton effect for the nfπ* transition (320ꢀ350 nm), indicating
the R configuration at C-519 and thus completing the structure of
thiopalmyrone (1).
The second metabolite isolated, palmyrrolinone (2), analyzed
for C12H15NO4 (six degrees of unsaturation) by HRESIMS of an
[M þ Na]þ peak at m/z 260.0895. As with 1, intense IR bands at
3423 (broad), 3105, 1715, 1663, and 1570 cmꢀ1 were consistent
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H3 value of 15.8 Hz anda strongNOEcorrelation between H-5 and
H-12. At this point, the planar structure of 2 could be assembled by
connecting partial structures II and III via HMBC correlations
from H-8 and H-10 to C-6.
To determine the absolute configuration at C-10 in the
pyrrolinone ring, a sample of 2 was treated with ozone followed
by oxidative workup and acid hydrolysis. Chiral-phase HPLC
analysis of this product revealed the presence of L-(S)-Ala in
comparison with commercial standards, thus completing the
structure of palmyrrolinone (2).
PAL 8/1/09-2.1 was morphologically in agreement with the
current definition of the genus Oscillatoria Vaucher ex Gomont,
1892 (for morphological description see Supporting
Information).23 However, PAL 8/1/09-2.1 (GenBank accession
number JF262061) was phylogenetically relatively distant (p-
distance = 3.9%) to the genus reference strain O. sancta PCC
7515.24 Instead, PAL 8/1/09-2.1 was closely related to the
ecologically distinct genus Trichodesmium (reference strain =
IMS 101), and this paraphyly of the genus Oscillatoria highlights
the need to taxonomically revise PAL 8/1/09-2.1 and related
specimens as a new genus. Thus, in advance of such a taxonomic
revision, PAL 8/1/09-2.1 is identified as cf. Oscillatoria sp.
The associated finely filamentous cyanobacterium PAL 8/1/
09-2.2 (GenBank acc. no. JF262062) was evolutionarily most
closely related to the obligate sponge-symbiont Hormoscilla
(formerly Oscillatoria) spongeliae Anagnostidis et Komꢀarek,
1988,25 although no reference strain has been defined for this
genus.24 The phylogenetic similarity (p-distance = 2.8%) to H.
spongeliae suggests that PAL 8/1/09-2.2 should also belong to
the genus Hormoscilla. However, due to the lack of Hormoscilla
species corresponding to PAL 8/1/09-2.2, this cyanobacterium
likely represents a new species, hence, the nomenclature Hor-
moscilla sp. nov (for a morphological description see Supporting
Information). Morphologically and phylogenetically similar cya-
nobacterial assemblages have been observed in geographically
disperse tropical marine habitats, such as Curac-ao, Netherlands
Antilles (unpublished data). Thus, these two types of cyanobac-
teria appear to commonly form assemblages with one another in
Nature. The fact that the strain PAL 8/1/09-2.2 represents the
closest related free-living basal clade to the obligate sponge-
symbiont H. spongeliae suggests a possible evolutionary adapta-
tion toward symbiosis in this group of cyanobacteria.
Pure thiopalmyrone (1) and palmyrrolinone (2) exhibited
toxicity against B. glabrata with LC50 values of 8.3 and 6.0 μM,
respectively. Interestingly, the molluscicidal effect was slightly
enhanced [(LC50 = 5.0 μM) = 2.5 μM of 1 þ 2.5 μM of 2] by
exposing the snails to an equimolar mixture of 1 and 2. Thus, it is
possible that thiopalmyrone (1) and palmyrrolinone (2) act
synergistically by interacting with more than one cellular target.
Neither metabolite was active in antifungal (Candida albicans) or
antibacterial (MRSA) assays and were both noncytotoxic to
H-460 human lung adenocarcinoma and neuro-2a murine neu-
roblastoma cell lines (up to a maximum test concentration of 100
μM). Thus, thiopalmyrone (1) and palmyrrolinone (2) repre-
sent two new chemotypes of relatively selective molluscicides for
potential use in treating waterways infested with schistosomiasis-
carrying snails of the genus Biomphalaria.
Biosynthetic dissection of the chemical structure of 1 suggests
that it derives from a PKS pathway, whereas 2 appears to be
produced by a combination of PKS and NRPS biosynthetic routes.
Interestingly, thiopalmyrone (1) and the C1ꢀC6 appendage of 2
share the same oxidation pattern and a similar R,β-unsaturated
carbonyl moiety with E double-bond geometry, suggesting that
they may derive from the same nascent triketide with similar
elongation, reduction, and dehydration processing. The origin of
the sulfur atom in 1 is uncertain, as it is difficult to envision a direct
incorporation of cysteine or methionine, the usual mechanism of
sulfur incorporation into cyanobacterial secondary metabolites.21
Interestingly, thiopalmyrone (1) represents only the second thioe-
ster reported from a marine cyanobacterium, the other being the
potent antiproliferative depsipeptide largazole.22 Both 1 and 2
possess methoxy groups that likely derive from S-adenosyl methio-
nine. However, there is a possibility that these are isolation artifacts,
as one might envision them arising from the addition of MeOH to
R,β-unsaturated ester or amide precursors in a Michael fashion,
followed by H2O elimination.14b Nevertheless, we believe these are
natural features of 1 and 2, as similar β-methoxy esters/amides are
quite common structural features in other cyanobacterial metabo-
lites (e.g., malyngamide A, barbamide, jamaicamide A).21
The relative composition of the cf. Oscillatoria sp. and Hormos-
cilla sp. nov cyanobacteria in the consortium was approximately
equivalent. Attempts toconclusivelyidentifythe actual producer of
1 and 2 in the assemblage (e.g., via MALDI imaging techniques26)
were not successful, due in part to a lack of live cultures of either
the conglomerate or the two cyanobacteria separately.
’ EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were
measured on a JASCO P-2000 polarimeter, whereas UV and circular
dichroism data were obtained using a Beckman DU800 spectrophot-
ometer and a JASCO J-815 CD spectrometer, respectively. IR spectra
were recorded on a Nicolet 100 FT-IR spectrometer. 1H, 13C, and 2D
NMR spectra were collected at a 1H resonance frequency of either 600
MHz (Bruker Avance III DRX600 equipped with a 1.7 mm TCI
cryoprobe) or 500 MHz (JEOL ECA500 and Varian VX500 equipped
with a 3 mm Varian XSens cold probe). Chemical shifts were calibrated
internally to the residual signal of the solvent in which the sample was
dissolved (CDCl3, δH 7.26, δC 77.0; CD2Cl2, δH 5.30, δC 53.52). High-
Taxonomy of the Cyanobacterial Strains. Microscopic char-
acterization of the thiopalmyrone (1)- and palmyrrolinone (2)-
producing specimen PAL 8/1/09-2 revealed that it was composed
of an assemblage of two different types of filamentous cyanobacter-
ia (Figure S17). The cyanobacterial assemblage was carefully
dissected into single filaments of each type. Genomic DNA from
each single filament was amplified by multiple displacement
amplification (MDA) and gene sequences PCR-amplified directly
from the different MDA-DNA products. Phylogenetic inference of
the SSU (16S) rRNA genes of the two cyanobacteria supported
their morphological differentiation and revealed that they were
evolutionarily unrelated to one another (p-distance = 9.9% 16S
rRNA gene sequence divergence, Figure 3).
resolution mass spectra were obtained on
a ThermoFinnigan
MAT900XL mass spectrometer. HPLC was carried out using a dual
Waters 515 pump system equipped with a Waters 996 photodiode array
detector. Vacuum and flash chromatographic separations were per-
formed using type H (10ꢀ40 μm, Aldrich) silica and silica gel 60
(40ꢀ63 μm, EMD), respectively. Merck TLC sheets (silica gel 60 F254)
were used for analytical TLC (aluminum-supported, layer thickness 200
μm) and preparative TLC (glass-supported, layer thickness 250 μm). L-
and D-Alanine were acquired from Fluka and Sigma-Aldrich, respec-
tively. All solvents were purchased as HPLC grade.
Taxonomically, the two cyanobacteria both represent novel
and undescribed taxa. The larger filamentous cyanobacterium
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Figure 3. Evolutionary tree for the thiopalmyrone (1) and palmyrrolinone (2) producers PAL 8/1/09-2.1 and PAL 8/1/09-2.2 (highlighted with
arrows). The cladogram is based on SSU (16S) rRNA gene sequences using Bayesian inference (MrBayes), and the support values are indicated as
posterior probability at the nodes. The specimens are indicated as species, strain, and access number in brackets. The scale bar is indicated at 0.04
expected nucleotide substitutions per site. In the cladogram, the evolutionarily distance between the tropical marine Oscillatoria (green box) and the
Oscillatoria sensu stricto (gray box), as well as the paraphyly of the genus Oscillatoria, indicates a need to taxonomically revise PAL 8/1/09-2.1 and related
specimens as a new group. PAL 8/1/09-2.2 can be noted as a basal clade to the genus Hormoscilla and likely belongs to this group as a new species.
Cyanobacterial Collection and Taxonomic Identification.
Samples of the cyanobacterial assemblage (voucher specimen available
from W.H.G. as collection number PAL 8/1/09-2) were collected by
hand at a depth of 0.5ꢀ1.5 m on flat rocks at the end of a runway at
North Beach, Palmyra Atoll, in August 2009 (05°530.831 N, 162°040.272
W). Samples were stored in 70% EtOH at ꢀ20 °C prior to extraction.
Morphological characterization was performed using an Olympus IX51
epifluorescent microscope (1000ꢁ) equipped with an Olympus
U-CMAD3 camera. Morphological comparisons and putative taxo-
nomic identification of the cyanobacterial specimen were performed
in accordance with modern classification systems.24,25
Extraction and Isolation. Approximately 1.96 kg (dry wt) of
cyanobacterial mat was extracted repeatedly with CH2Cl2/MeOH (2:1)
to afford 4.43 g of extract. A portion of this material (4.0 g) was
fractionated by silica gel vacuum liquid chromatography using a stepwise
gradient of increasing polarity, starting with 10% EtOAc in hexanes and
finishing with 100% MeOH, to produce nine fractions (BꢀI). Fraction F
(eluted with 80% EtOAc in hexanes, 0.170 g) was subjected to 1H NMR-
guided fractionation using gradient silica gel column chromatography
(20% to 60% EtOAc in hexanes), followed by preparative TLC (55%
EtOAc in hexanes) and normal-phase HPLC (Phenomenex Luna 5 μm
silica 100 Å, 250 ꢁ 10 mm, 50% EtOAc in hexanes at 3 mL/min,
detection at 250 and 290 nm) to yield 2.6 mg of compound 1 and 5.9 mg
of compound 2. The preparative TLC step above additionally provided
19.5 mg of lyngbic acid and 3.0 mg of zeaxanthin.
Thiopalmyrone (1): colorless oil; [R]23D þ7.6 (c 1.1, CH3CN); UV
(MeCN) λmax 237 nm (log ε 4.06), 265 nm (log ε 3.83); CD (c 0.06,
CH3CN) λmax (Δε) 450 (0.03), 410 (0.04), 290 (0.04), 250 (0.06), 210
(0.22); IR (neat) 3420, 2934, 1732, 1609, 1383, 1167, 1030, 818 cmꢀ1
;
1H, 13C, and 2D NMR data, see Table 1; HRESIMS m/z [M þ H]þ
175.0421 (calcd for C7H11O3S, 175.0423).
Palmyrrolinone (2): colorless oil; [R]23D þ36 (c 0.18, CH3CN); UV
(MeCN) λmax 293 nm (log ε 4.13), 212 nm (log ε 3.97); IR (neat) 3423,
1
3105, 2927, 2852, 1715, 1663, 1570, 1335, 1201, 822, 756 cmꢀ1; H,
13C, and 2D NMR data, see Table 2; HRESIMS m/z [M þ Na]þ
260.0895 (calcd for C12H15NO4Na, 260.0893).
Lyngbic acid: pale yellow oil; [R]23D ꢀ8.7 (c 0.04, CHCl3); UV, IR,
1H and 13C NMR spectroscopic data were in accordance with those
previously reported for the natural product;12 HRESIMS m/z [M þ
Na]þ 279.1934 (calcd for C15H28O3Na, 279.1931).
Zeaxanthin: orange solid; [R]23 ꢀ33 (c 0.1, CHCl3); UV, IR, 1H
D
and 13C NMR spectroscopic data were in accordance with those
previously reported for the natural product;13 HREIMS m/z [M]þ
568.4268 (calcd for C40H56O2, 568.4275).
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Journal of Natural Products
ARTICLE
Ozonolysis, Acid Hydrolysis, and Chiral-Phase HPLC Anal-
ysis of 2. Ozone was bubbled through a sample of 2 (0.3 mg) dissolved
in CH2Cl2 (3 mL) at 25 °C for 30 min. The solvent was evaporated, and
the residue treated with 1 mL of H2O2/HCOOH (1:2) at 70 °C for 20
min. After removal of solvent, the ozone-treated and oxidized sample
of 2 was hydrolyzed in degassed 6 M HCl (1 mL) at 110 °C for 18 h.
The hydrolysate was concentrated to dryness and then dissolved in
an aqueous solution of 2 mM CuSO4. An aliquot of this solution
was analyzed by chiral-phase HPLC [Phenomenex Chirex 3126 (D),
4.6 ꢁ 250 mm; 2 mM CuSO4 in H2O at 0.7 mL/min; detection at
254 nm] to give L-alanine (tR 13.2 min). The retention times of authentic
L-Ala and D-Ala were 12.9 and 20.6 min, respectively.
under a dissecting microscope (20ꢁ magnification), the snails were
assessed as dead.
’ ASSOCIATED CONTENT
Supporting Information. 1H NMR, 13C NMR, and 2D
S
b
NMR spectra in CDCl3 or CD2Cl2 for 1 and 2, concentra-
tionꢀresponse toxicity profiles for B. glabrata, morphological
description of cyanobacterial specimen PAL 8/1/09-2. This
material is available free of charge via the Internet at http://
pubs.acs.org.
Multiple Displacement Amplification and PCR Amplifica-
tion from Single Filaments. The cyanobacterial assemblage was
dissected, and single filaments of the cyanobacterial components were
isolated under an Olympus VMZ dissecting microscope. The single
filaments were washed twice in 0.5 μL of sterile SWBG-11 medium and
twice in 0.5 μL of D2O before transfer into 0.2 mL PCR tubes. DNA was
amplified from the single filaments by MDA using the REPLI-g mini kit
(Qiagen), following the manufacturer’s specifications. All MDA reac-
tions were performed in 50 μL reaction volume for 16 h at 30 °C. The
16S rRNA genes were PCR-amplified from isolated DNA using the
modified lineage-specific primers OT106F 50-GGACGGGTGAG-
TAACGCGTGA-30 and OT1445R 50-AGTAATGACTTCGGGCG-
TG-30. 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, and 20.5 μL of D2O. The PCR
reactions were performed in an Eppendorf Mastercycler gradient as
follows: initial denaturation for 2 min at 95 °C, 25 cycles of amplification,
followed by 20 s at 95 °C, 20 s at 55 °C, and 1.5 min at 72 °C, and final
elongation for 3 min at 72 °C. PCR products were purified using a
MinElute PCR purification kit (Qiagen) before subcloning using the
Zero Blunt TOPO PCR cloning kit (Invitrogen) following the manu-
facturer’s specifications. Plasmid DNA was isolated using the QIAprep
Spin miniprep kit (Qiagen) and sequenced with M13 primers. The 16S
rRNA gene sequences are available in the DDBJ/EMBL/GenBank
databases under acc. nos. JF262061 (PAL 8/1/09-2.1) and JF262062
(PAL 8/1/09-2.2).
’ AUTHOR INFORMATION
Corresponding Author
*Tel: (858)-534-0578. Fax: (858)-534-0529. E-mail: wgerwick@
ucsd.edu.
Author Contributions
‡A.R.P. and L.E. contributed equally to this work.
’ ACKNOWLEDGMENT
We thank A. Jones for his help in the collection of the
cyanobacterial assemblage as well as The Nature Conservancy
staff at Palmyra Atoll. We thank Y. Su (UCSD Mass Spectro-
metry) for HRMS data acquisition, T. Byrum for running
mammalian cell line cytotoxicity measurements, and Prof. C.
Bayne (Oregon State University) for originally supplying B.
glabrata snails. We acknowledge the San Diego Supercomputer
Center for statistical analyses using the CIPRES cluster. Support
was partially provided by Fogarty International Center’s Inter-
national Cooperative Biodiversity Group program (NIH
TW006634) and NIH CA100851. We further acknowledge the
NSF CHE-0741968 for support of the JEOL and 13C-sensitive
Varian NMR spectrometers in the Department of Chemistry and
Biochemistry, UCSD.
Phylogenetic Inference. The 16S rRNA gene sequences of PAL-
8/1/09-2.1 and PAL-8/1/09-2.2 were aligned with evolutionarily in-
formative cyanobacteria using the L-INS-I algorithm in MAFFT 6.71727
and refined using the SSU secondary structures model for Escherichia coli
J0169528 without data exclusion. The best-fitting nucleotide substitution
model optimized by maximum likelihood (ML) was selected using
corrected Akaike/Bayesian Information Criterion (AICC/BIC) in jMo-
deltest 0.1.1.29 The evolutionary histories of the cyanobacterial genes
were inferred using maximum likelihood and Bayesian inference algo-
rithms. The ML inference was performed using GARLI 1.030 for the
GTRþIþG model assuming a heterogeneous substitution rate and
gamma substitution of variable sites (proportion of invariable sites
(pINV) = 0.422, shape parameter (R) = 0.455, number of rate categories
= 4) with 1000 bootstrap replicates. Bayesian inference was conducted
using MrBayes 3.131 with four Metropolis-coupled MCMC chains (one
cold and three heated) ran for 3 000 000 generations. The first 25% were
discarded as burn-in, and the following data set was being sampled with a
frequency of every 100 generations. The MCMC convergence was
detected by AWTY.32
’ REFERENCES
(1) (a) Payne, L.; Fitchett, J. R. Trends Parasitol. 2010, 26, 421–423.
(b) Hotez, P. J.; Fenwick, A.; Savioli, L.; Molyneux, D. H. Lancet 2009,
373, 1570–1575.(c) World Health Organization Control of neglected
tropical diseases (NTD). http://www.who.int/neglected_diseases/en/.
Accessed October 28, 2010.
(2) Molyneux, D. H.; Hotez, P. J.; Fenwick, A. PLoS Med. 2005,
2, 1064–1070.
(3) Reddy, M.; Gill, S. S.; Kalkar, S. R.; Wu, W.; Anderson, P. J.;
Rochon, P. A. JAMA 2007, 298, 1911–1924.
(4) (a) Domling, A.; Khoury, K. ChemMedChem 2010, 5, 1420–
1434. (b) Feasey, N.; Wansbrough-Jones, M.; Mabey, D. C. W.; Solo-
mon, A. W. Brit. Med. Bull. 2010, 93, 179–200. (c) Trouiller, P.; Olliaro,
P.; Torreele, E.; Orbinski, J.; Laing, R.; Ford, N. Lancet 2002,
359, 2188–2194.
(5) Kursar, T. A.; Caballero-George, C. C.; Capson, T. L.; Cubilla-
Rios, L.; Gerwick, W. H.; Gupta, M. P.; Iba~nez, A.; Linington, R. G.;
McPhail, K. L.; Ortega-Barria, E.; Romero, L. I.; Solis, P. N.; Coley, P. D.
BioScience 2006, 56, 1005–1012.
Molluscicidal Assay. Molluscicidal activity was evaluated according
to a previously outlined procedure using the test organism Biomphalaria
glabrata.8 In all assays, a stock solution of 20 mg/mL of pure compound in
EtOH was prepared, of which 35 μL was taken and diluted to 7 mL with
distilled H2O. The snails were placed in wells of varying concentrations and
observed after 24 h. If no heartbeat could be detected upon examination
(6) (a) Perrett, S.; Whitfield, P. J. Parasitol. Today 1996, 12,
156–159. (b) World Health Organization. Report of the Scientific
Working Group Meeting on Schistosomiasis; Geneva, Switzerland,
November 14ꢀ16, 2005.
(7) (a) Bergquist, R. Acta Trop. 2008, 108, 65–68. (b) Ribeiro, K. A.;
de Carvalho, C. M.; Molina, M. T.; Lima, E. P.; Lopez-Montero, E.; Reys,
1180
dx.doi.org/10.1021/np200106b |J. Nat. Prod. 2011, 74, 1175–1181

Journal of Natural Products
ARTICLE
J. R.; de Oliveira, M. B.; Pinto, A. V.; Santana, A. E.; Goulart, M. O. Acta
Trop. 2009, 111, 44–50.
(8) Hostettmann, K.; Kizu, H.; Tomimori, T. Planta Med. 1982,
44, 34–35.
(9) Orjala, J.; Gerwick, W. H. J. Nat. Prod. 1996, 59, 427–430.
(10) Singh, I. P.; Milligan, K. E.; Gerwick, W. H. J. Nat. Prod. 1999,
62, 1333–1335.
(29) Posada, D. Mol. Biol. Evol. 2008, 25, 1253–1256.
(30) Zwickl, D. J. Ph.D. Thesis, University of Texas at Austin, TX,
2006.
(31) Ronquist, F.; Huelsenbeck, J. P. Bioinformatics 2003, 12, 1572–
1574.
(32) Nylander, J. A. A.; Wilgenbusch, J. C.; Warren, D. L.; Swofford,
D. L. Bioinformatics 2008, 15, 581–583.
(11) Pereira, A. R.; McCue, C. F.; Gerwick, W. H. J. Nat. Prod. 2010,
73, 217–220.
(12) (a) Cardellina, J. H.; Dalietos, D.; Marner, F. J.; Mynderse, J. S.;
Moore, R. E. Phytochemistry 1978, 17, 2091–2095. (b) Kwan, J. C.;
Teplitski, M.; Gunasekera, S. P.; Paul, V. J.; Luesch, H. J. Nat. Prod. 2010,
73, 463–466. (c) Chen, J.; Shi, Z. F.; Zhou, L.; Xie, A. L.; Cao, X. P.
Tetrahedron 2010, 66, 3499–3507.
(13) (a) Wegerski, C. J.; France, D.; Cornell-Kennon, S.; Crews, P.
Bioorg. Med. Chem. 2004, 12, 5631–5637. (b) Eisenreich, W.; Bacher, A.;
Berry, A.; Bretzel, W.; Humbelin, M.; Lopez-Ulibarri, R.; Mayer, A. F.;
Yeliseev, A. J. Org. Chem. 2002, 67, 871–875. (c) Englert, G.; Noack, K.;
Broger, E. A.; Glinz, E.; Vecchi, M.; Zell, R. Helv. Chim. Acta 1991,
74, 969–982. (d) Moss, G. P. Pure. Appl. Chem. 1976, 47, 97–102. (e)
Maoka, T.; Arai, A.; Shimizu, M.; Matsuno, T. Comp. Biochem. Physiol.
1986, 83B, 121–124. (f) Buchecker, R.; Liaaen-Jensen, S.; Borch, G.;
Siegelman, H. W. Phytochemistry 1976, 15, 1015–1018.
(14) (a) Ward, D. E.; Gai, Y.; Zoghaib, W. M. Can. J. Chem. 1991,
69, 1487–1497. (b) Schweiger, K. Monatsh. Chem. 1982, 113, 1283–1297.
(15) Matsumori, N.; Kaneno, D.; Murata, M.; Nakamura, H.;
Tachibana, K. J. Org. Chem. 1999, 64, 866–876.
(16) Uhrin, D.; Batta, G.; Hruby, V. J.; Barlow, P. N.; Kover, K. E.
J. Magn. Reson. 1998, 130, 155–161.
(17) Williamson, R. T.; Marquez, B. L.; Gerwick, W. H.; Kover, K. E.
Magn. Reson. Chem. 2000, 38, 265–273.
(18) (a) Lightner, D. A.; Gurst, J. E. Organic Conformational Analysis
and Stereochemistry from Circular Dichroism Spectroscopy; Wiley-VCH:
New York, 2000; pp 337ꢀ394. (b) Snatzke, G. Tetrahedron 1965,
21, 439–448. (c) Snatzke, G. Tetrahedron 1965, 21, 413–419.
(d) Snatzke, G. Tetrahedron 1965, 21, 421–438. (e) Suga, T.; Imamura,
K. Bull. Chem. Soc. Jpn. 1972, 45, 2060–2064.
(19) When the sulfur atom in thiopalmyrone (1) is replaced by an
oxygen atom, the specific rotation of the oxygenated analogue
(exhibiting also the R configuration) increases in magnitude but remains
positive {i.e., [R]23D þ187.3 (c 0.95, CHCl3)}. See: Pospisil, J.; Marko,
I. E. Tetrahedron Lett. 2008, 49, 1523–1526.
(20) (a) Meickle, T.; Matthew, S.; Ross, C.; Luesch, H.; Paul, V.
Planta Med. 2009, 75, 1427–1430. (b) Edwards, D. J.; Marquez, B. L.;
Nogle, L. M.; McPhail, K.; Goeger, D. E.; Roberts, M. A.; Gerwick, W. H.
Chem. Biol. 2004, 11, 817–833. (c) Koehn, F. E.; Longley, R. E.; Reed,
J. K. J. Nat. Prod. 1992, 55, 613–619. (d) Moore, R. E.; Entzeroth, M.
Phytochemistry 1988, 27, 3101–3103.
(21) Tidgewell, K., Clark, B. T., Gerwick, W. H. In Comprehensive
Natural Products Chemistry, 2nd ed.; Moore, B.; Crews, P., Eds.;
Pergamon Press: Oxford, 2010; pp 141ꢀ188.
(22) (a) Taori, K.; Paul, V. J.; Luesch, H. J. Am. Chem. Soc. 2008,
130, 1806–1807. (b) Taori, K.; Paul, V. J.; Luesch, H. J. Am. Chem. Soc.
2008, 130, 13506–13506.
(23) Gomont, M. M. Ann. Sci. Nat. Bot. Ser. 1892, 16, 91–264.
(24) Castenholz, R. W.; Rippka, R.; Herdman, M. In Bergey’s Manual
of Systematic Bacteriology; Boone, D. R.; Castenholz, R. W., Eds.;
Springer: New York, 2001; Vol. 1, pp 473ꢀ599.
(25) Komꢀarek, J.; Anagnostidis, K. In S€usswasserflora von Mitteleur-
opa; B€udel, B.; G€artner, G.; Krienitz, L.; Schagerl, M., Eds.; Elsevier:
M€unchen, 2005; Vol 19/2, pp 441ꢀ625.
(26) Taniguchi, M.; Nunnery, J. K.; Engene, N.; Esquenazi, E.;
Byrum, T.; Dorrestein, P. C.; Gerwick, W. H. J. Nat. Prod. 2010,
73, 393–398.
(27) Katoh, K.; Toh, H. Briefings Bioinf. 2008, 9, 286–298.
(28) Cannone, J. J.; Subramanin, S.; Schnare, M. N.; Collett, J. R.;
D’Souza, L. M.; Du, Y.; Feng, B.; Lin, N.; Madabusi, L. V.; Muller, K. M.;
Pnde, N.; Schang, Z.; Yu, N.; Gutell, R. R. BMC Bioinf. 2002, 3, 1471–2105.
1181
dx.doi.org/10.1021/np200106b |J. Nat. Prod. 2011, 74, 1175–1181