The hoiamides, structurally intriguing neurotoxic Lipopeptides from Papua New Guinea marine cyanobacteria

Article abstract of DOI:10.1021/np100468n

Two related peptide metabolites, one a cyclic depsipeptide, hoiamide B (2), and the other a linear lipopeptide, hoiamide C (3), were isolated from two different collections of marine cyanobacteria obtained in Papua New Guinea. Their structures were elucidated by combining various techniques in spectroscopy, chromatography, and synthetic chemistry. Both metabolites belong to the unique hoiamide structural class, characterized by possessing an acetate extended and S-adenosyl methionine modified isoleucine unit, a central triheterocyclic system comprised of two α-methylated thiazolines and one thiazole, and a highly oxygenated and methylated C-15 polyketide unit. In neocortical neurons, the cyclic depsipeptide 2 stimulated sodium influx and suppressed spontaneous Ca²⁺ oscillations with EC₅₀ values of 3.9 μM and 79.8 nM, respectively, while 3 had no significant effects in these assays.

Full text of DOI:10.1021/np100468n

J. Nat. Prod. 2010, 73, 1411–1421
1411
The Hoiamides, Structurally Intriguing Neurotoxic Lipopeptides from Papua New Guinea
Marine Cyanobacteria
Hyukjae Choi,† Alban R. Pereira,† Zhengyu Cao,‡ Cynthia F. Shuman,† Niclas Engene,† Tara Byrum,† Teatulohi Matainaho,§
Thomas F. Murray,‡ Alfonso Mangoni,*,⊥ 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, Discipline of Pharmacology, School of Medicine and Health
Sciences, UniVersity of Papua New Guinea, National Capital District, Papua New Guinea, Department of Pharmacology, School of Medicine,
Creighton UniVersity, Omaha, Nebraska 68178, and Dipartimento di Chimica delle Sostanze Naturali, UniVersita` di Napoli “Federico II”,
Via D. Montesano 49, 80131 Napoli, Italy
ReceiVed July 8, 2010
Two related peptide metabolites, one a cyclic depsipeptide, hoiamide B (2), and the other a linear lipopeptide, hoiamide
C (3), were isolated from two different collections of marine cyanobacteria obtained in Papua New Guinea. Their
structures were elucidated by combining various techniques in spectroscopy, chromatography, and synthetic chemistry.
Both metabolites belong to the unique hoiamide structural class, characterized by possessing an acetate extended and
S-adenosyl methionine modified isoleucine unit, a central triheterocyclic system comprised of two R-methylated thiazolines
and one thiazole, and a highly oxygenated and methylated C-15 polyketide unit. In neocortical neurons, the cyclic
depsipeptide 2 stimulated sodium influx and suppressed spontaneous Ca2+ oscillations with EC50 values of 3.9 µM and
79.8 nM, respectively, while 3 had no significant effects in these assays.
Cyanobacteria are well recognized to be rich producers of
structurally intriguing and biologically active secondary metabolites,
many of which have toxic properties.1 Indeed, freshwater cyano-
bacteria have been studied since the 1930s because their toxins
have impacted both human populations and domestic animals.2 On
the other hand, marine cyanobacteria have been highlighted in the
natural products chemistry field because their metabolites have
interesting structures and pharmacology and are thus of high
potential pharmaceutical utility. Recognition of this fact began more
than 30 years ago with the discovery of majusculamides A and B
by R. E. Moore in 1977.3 To date, more than 700 secondary
metabolites have been reported with various biological properties
including inhibition of microtubules (curacin A),4a,b inhibi-
tion of angiogenesis and promotion of actin polymerization
(hectochlorin),4c sodium channel blocking (kalkitoxin)4d and acti-
vating activities (antillatoxin),4e and G1 cell cycle arrest and
induction of apoptosis (apratoxin A).4f,g Biosynthetically, marine
cyanobacteria produce secondary metabolites of a variety of
structure classes, including peptides, polyketides, terpenoids, and
alkaloids. However, the most predominant structure class are
lipopeptides, which are formed by the integration of polyketide
synthases (PKS) and nonribosomal peptide synthetases (NRPS).5
More than 36% of the known cyanobacterial secondary metabolites
are formed from polyketides transitioning into nonribosomal
peptides, an orientation termed “ketopeptides” (251 compounds),
and 9% are peptides transitioning into polyketides, known as
“peptoketides” (63 compounds).1 Slightly more than 30% of the
ketopeptides and peptoketides are a complex mixture of these
nonribosomal peptide and polyketide components. In addition,
cyanobacterial lipopeptides are highly modified by various biosyn-
thetic enzymes in their complicated biosynthetic pathways, including
by halogenations, unusual oxidations, and a variety of C-, O-, and
N-methylations. In some cases, the complex biosynthetic origin and
extensive secondary modification of cyanobacterial lipopeptides
make their structural elucidation challenging.
The voltage-gated sodium channel (VGSC) is an important drug
target and is the site of action for several classes of pharmaceutical
agents, including local anesthetics (bupivacaine, cocaine, and
lidocaine), antiarrhythmics (flecainide and propafenone), anticon-
vulsants (carbamazepine, phenytoin, and valproic acid), and
analgesics (ziconotide); new treatments for neurodegenerative
disorders are also being developed for this target.6 The VGSC
allows passage of Na+ across membranes and thus plays an
important role in the generation of action potentials in excitable
cells such as neurons.7a In the open state, the pore of the VGSC is
open and allows Na+ influx into the cytoplasm of the cell, which
then produces a regenerative membrane depolarization. The VGSC
complex consists of a large R-subunit, critical for channel pore
formationandvoltagesensing,andoneortwoauxiliaryR-subunits.7a-c
VGSCs represent the molecular targets for toxins that act at six or
more distinct neurotoxin sites on the channel protein.7d Tetrodot-
oxin, saxitoxin, and δ-conotoxins GIIIA and GIIIB, binding to
neurotoxin site 1, block the channel pore and inhibit sodium flux.
Batrachotoxin and veratridine, acting on neurotoxin site 2, inhibit
VGSC inactivation. R-Scorpion, δ-conotoxin, and the sea anemone
toxins, targeting neurotoxin site 3, also retard inactivation of the
VGSC, whereas ꢀ-scorpion toxins, interacting with neurotoxin site
4, shift the voltage dependence of activation to more negative
membrane potentials without an effect on channel inactivation.
Brevetoxins and ciguatoxins link to neurotoxin site 5 and stimulate
channel activity by shifting the activation potential to more negative
values and blocking channel inactivation. δ-Conotoxin interacting
with neurotoxin site 6 delays channel inactivation. Pyrethroids and
DDT are coupled to neurotoxin site 7 and produce effects similar
to site 5 toxins.
Recently, we reported a cyclic depsipeptide named hoiamide A
(1), which illustrates a new chemotype within the natural products
of cyanobacteria and possesses potent neuropharmacological prop-
erties.8 It was found that compound 1 inhibits the binding of
[3H]batrachotoxin to site 2 of the mammalian VGSC and stimulates
sodium influx in neocortical neurons as a partial agonist. Structur-
ally, it possesses a stereochemically complex structure (15 chiral
centers) with highly modified peptide and polyketide units as well
as an unusual triheterocyclic section.
* To whom correspondence should be addressed. Tel: (858) 534-0578.
Fax: (858) 534-0529. E-mail: wgerwick@ucsd.edu; alfonso.mangoni@
unina.it.
† University of California, San Diego.
‡ Creighton University.
§ University of Papua New Guinea.
⊥ Universita` di Napoli “Federico II”.
10.1021/np100468n  2010 American Chemical Society and American Society of Pharmacognosy
Published on Web 08/05/2010

1412 Journal of Natural Products, 2010, Vol. 73, No. 8
Choi et al.
structures A-F, and extensive HMBC analysis provided connec-
tions between these six partial structures to afford the planar
structure of hoiamide B (2) (Figure 2). The first two peptidic
fragments, A and B, were found to be threonine (Thr) and
2-hydroxy-3-methylpentanoic acid (Hmpa) by COSY and HMBC
spectra analysis. Similarly, two additional spin systems could be
constructed, one composed of a methyl doublet (H-19, δH 0.84),
two methines (H-12, δH 2.32; H-13, δH 3.77), and a hydroxy proton
(OH-13, δH 2.32), and the other composed of a methyl doublet
(H-18, δH 0.92), a methyl triplet (H-17, δH 0.83), two methine
protons (H-14, δH 3.52; H-15, δH 1.56), one pair of methylene
protons (H-16a, δH 1.42; H-16b, δH 1.05), and one exchangeable
proton from an amide (14-NH, δH 6.83). The HMBC correlations
from H-13 to C-14 and from NH-14 to C-13 allowed the
combination of fragments C and D into a new partial structure
comprising 4-amino-3-hydroxy-2,5-dimethylheptanoic acid (Ah-
dhe). A fifth fragment (E) was revealed as possessing three
consecutive heterocyclic rings. The 1H and 13C NMR chemical shifts
of C-22 (δH 3.82/3.15; δC 41.1) and C-26 (δH 3.52/3.43; δC 42.8)
were at shifts consistent with placement of heteroatoms at these
two positions. HMBC correlations from H-22a/H-22b/H-23 to C-20/
C-21, from H-26a/H-26b/H-27 to C-24/C-25, and from H-30 to
C-28/C-29/C-31 allowed assignments of two R-methylated thiazo-
lines and one thiazole. The additional HMBC correlations from
H-22a/H-22b to C-24 and from H26a/H26b to C-28 identified that
the three heterocyclic rings were successively connected to one
another. The final partial structure of compound 2 was elucidated
as a 5,7-dihydroxy-3-methoxy-4,6,8-trimethylundecanoyl-derived
residue (Dmetua). The methoxy group was placed at C-33 (δC 79.3)
by an HMBC correlation from H-45 (δH 3.24) to C-33. COSY
correlations helped to assign the terminal chain of four carbons
(C-32/C-33/C-34/C-35) with a methyl branch (C-44) at C-34. A
second section of partial structure F, assigned by the COSY and
HMBC correlations, was composed of a seven-carbon chain (C-
43/C-36/C-37/C-38/C-39/C-40/C-41) with hydroxy (C-37) and
methyl branches (C-38). These two sections were linked into partial
structure F on the basis of HMBC correlations from H-35 to C-36/
C-37 and from H-43 to C-35. Finally, the HMBC correlations from
NH-2 to C-5, from H-6 to C-11, from NH-14 to C-20, from H-30
to C-31, from H-32/H-33 to C31, and from H-35 to C-1 allowed
connection of these six partial structures, thereby forming the planar
structure of hoiamide B (2) (Figure 2).
The absolute configuration of the 16 chiral centers in hoiamide
B (2) was assigned by various means, including degradation
reactions to yield chiral fragments followed by chromatographic
analysis and additional NMR spectroscopic analyses. The absolute
configuration of the Thr residue was assigned as L by acid
hydrolysis, derivatization with L-FDLA, and LC-ESIMS analysis.
The absolute configuration of the Hmpa residue was determined
as 2S, 3S by comparing the retention time of Hmpa released by
acid hydrolysis of 2 with the four synthetic stereoisomers of Hmpa
by chiral HPLC.9 In order to assign the absolute configuration of
the modified cysteic acids, 2 was subjected to ozonolysis, oxidative
workup, and acid hydrolysis to yield 2-methylcysteic acid (MeCysA).
The reaction products were then analyzed by chiral HPLC and
compared with the retention times of synthetic 2S-MeCysA and
2R-MeCysA standards; only 2S-MeCysA was detected by this
analysis. Therefore, the absolute configuration of C-21 and C-25
was assigned as S and R, respectively.
Figure 1. Structures of natural hoiamides A-C (1-3) and hoiamide
A analogues 48 and 5.8
As part of an ongoing neuropharmacological screening program
aimed at discovering additional neurotoxins produced by marine
organisms, we investigated the extracts of two independent col-
lections of Papua New Guinea marine cyanobacteria. The first
extract, prepared from an assemblage of Symploca sp. and Oscil-
latoria cf. sp., exhibited potent inhibition of calcium oscillation
1
and activation of sodium influx in mouse neocortical neurons. H
NMR-guided fractionation of this material afforded the new cyclic
depsipeptide hoiamide B (2), along with the known metabolite
hoiamide A (1). The second extract, derived from a Symploca sp.
specimen, showed mild brine shrimp toxicity, and its fractionation
led to the discovery of the linear hoiamide C (3). Herein, we report
the isolation, structure elucidation, and biological activity of
hoiamides B (2) and C (3) as the newest members of this unique
structural class, the hoiamides.
Results and Discussion
Isolation and Structure Elucidation of Hoiamide B (2). A
collection of tuft-forming marine cyanobacteria was obtained via
scuba at Gallows Reef, Papua New Guinea, in 2006 (PNG-4-28-
06-1). The collection was extracted repeatedly with CH2Cl2-MeOH
(2:1) and further fractionated by silica gel vacuum column
chromatography (VLC) to produce nine fractions (A-I). The F
fraction was found to possess potent sodium channel activating
activity in neuro-2a cells and inhibited calcium oscillations in mouse
cerebrocortical neurons. This fraction was thus subjected to RP
HPLC to afford hoiamide B (2, 8.9 mg, 1.2%) as well as the
previously reported hoiamide A (1, 25 mg, 3.4%) (Figure 1).8
Hoiamide B (2) was obtained as a pale yellow oil, and its
LRESIMS showed a molecular ion cluster at m/z 962.6/963.6/964.5/
965.5/966.4 in a ratio of 100:50:29:10:4, indicating the likely
presence of three sulfur atoms in the molecule, as found for
hoiamide A (1). The molecular formula of 2 was determined as
C45H73N5O10S3 by interpretation of HRESITOFMS data ([M + H]+
m/z 940.4584). The IR spectrum of 2 displayed absorption bands
at 3375, 1738, and 1604 cm-1, indicating the presence of hydroxy,
ester, and amide functionalities, respectively. The 1H and 13C NMR
spectrum of 2 in DMSO-d6 showed peptide and oxygenated
polyketide features and included seven downfield-shifted carbons
without attached protons (δC 176.2, 174.4, 173.3, 170.3, 169.8,
165.8, and 161.7), five exchangeable protons (δH 7.84, 6.83, 5.15,
4.92, and 3.97), eight N- or O-substituted methines (δH 5.18, 4.94,
4.51, 4.24, 3.79, 3.77, 3.52, and 3.18), three methyl triplets (δH
0.835, 0.831, and 0.70), seven methyl doublets (δH 1.15, 0.92, 0.87,
0.86, 0.84, 0.82, and 0.73), and three methyl singlets including one
methoxy group (δH 3.22, 1.61, and 1.55), as shown in Table 1.
The analysis of 1D and 2D NMR data, including COSY,
TOCSY, HSQC, and HMBC, allowed the construction of partial
The relative configuration of the Ahdhe (C-11-C-20) and
Dmetua (C-31-C-45) units was revealed by J-based configurational
analysis (Figure 3). Homonuclear coupling constants were measured
from 1H NMR and 1D-TOCSY spectra, whereas heteronuclear
coupling constants were measured using a combination of the
HETLOC and HSQMBC experiments. The large coupling constant
between H-12 and H-13 (3JH-12,
relationship between these protons, and the ROESY correlations
) 7.9 Hz) indicated an anti
H-13

Neurotoxic Lipopeptides from Marine Cyanobacteria
Journal of Natural Products, 2010, Vol. 73, No. 8 1413
Table 1. NMR Spectroscopic Data for Hoiamide B (2) in DMSO-d6 at 600 MHz (1H) and 150 MHz (13C)
residue
Thr
position
δC
δH mult (J in Hz)
COSY
NH, 3
HMBCa
ROESY
1
2
170.3
58.5
4.51 dd (7.7, 2.8)
NH, 7.84 d (7.7)
4.24 m
1, 3
3, 5
4, 8
4, 6, 7, 10
2
3
66.3
2, 4, OH
1, 2, 4
8, 37, 43, 3-OH
OH, 4.92 d (5.6)
1.15 d (6.3)
3
3
4
5
6
7
8a
8b
9
10
11
12
13
20.2
169.8
76.3
36.4
22.5
2, 3
2, 37
Hmpa
Ahdhe
4.94 d (3.5)
1.91 m
1.28 m
1.09 m
0.70 t (7.4)
0.87 d (7.6)
7
5, 11
5
8a, 8b, 9, 10, 2-NH
9
6
2, 3
7
6, 8, 10
7, 8b
7, 8a
11.5
14.9
174.4
44.4
7, 8
6
7
2.32 m
3.77 m
OH, 5.15 d (4.6)
3.52 dd (10.2, 6.9)
NH, 6.83 d (9.6)
1.56 m
1.42 m
1.05 m
0.834 t (7.0)
0.92 d (6.9)
0.84 d (7.2)
13, 19
12, OH
13
NH, 15
11, 13, 14
11, 12, 14
12, 13, 14
20
13, 14, 20
14,
14, 13-OH, 14-NH
15, 18, 19
12, 13, 14, 15, 16, 17, 14-NH
12, 18, 19
12, 14, 15, 23, 27, 13-OH
13, 13-OH, 14-NH
71.1
14
52.7
14
15
35.7
25.2
14, 16a, 16b, 19
15, 16b, 17
16a, 17
16
15
16a
16b
17
18
19
20
21
22a
22b
23
17
14, 15, 17, 18
10.6
13.7
15.6
173.3
84.7
41.1
14, 15
11, 12, 13
13, 14
12, 13, 16
12
MoCys1
MoCys2
3.82 m
3.15 d (11.3)
1.55 s
22b
22a
20, 21, 24
20, 21, 24
20, 21, 22
23
25.6
176.2
83.4
13, 14, 13-OH, 14-NH, 22a, 22b
24
25
26a
26b
27
28
29
30
31
32
33
34
35
36
37
42.8
3.52 d (11.2)
3.43 d (11.2)
1.61 s
26b
26a
24, 25, 28
24, 25, 28
24, 25, 26
27
27
26a, 26b
24.1
161.7
147.7
122.8
165.8
33.2
79.3
35.8
75.2
38.2
MoCys3
Dmetua
7.94 s
28, 29, 31
2.98 d (7.8)
3.79 m
2.35 m
5.18 d (9.7)
1.72 m
3.18 d (8.4)
OH, 3.97 d (6.0)
1.49 m
1.25 m
1.15 d (6.3)
1.24 m
0.831 t (6.9)
0.73 d (6.7)
0.82 d (6.8)
0.86 d (7.2)
3.24 s
33
31, 33
31, 35, 45
33, 35
1, 33, 34, 36, 37, 43
37
35
37
40
37
37
35, 44
35, 44, 45
36, 43, 45
32, 33, 37, 38, 44, 37-OH
34, 38, 42, 37-OH
3, 35, 39a, 39b, 43
35, 36, 39a, 39b, 42
36, 43
32, 34
33, 35, 44
34
37, 43
36, 38
72.2
38
33.7
36.6
37, 39a, 42
38, 39b
38
39a
39b
40
41
42
43
44
45
37, 37-OH
37
20.0
14.4
12.0
9.9
10.5
56.8
39, 40
37, 39
35, 36, 37
33, 35
33
38
36
34
36, 39a, 37-OH
3, 34, 37. 38
33, 35
33, 34
a
1
From H to the indicated 13C.
3
between H-19 and H-13/H-14 led to the assignment of the relative
configuration of C-12-C-13 as an erythro rotamer B-3. The small
homonuclear and heteronuclear coupling constants between H-13/
H-14, H-13/C-15, and H-12/C-14 were indicative of the relative
configuration between C-13-C-14 being the threo rotamer A-1,
and this was consistent with a series of ROESY correlations (H-
12/H-14, H-14/H-13, H-13/H-15, H-15/OH-13, and OH-13/NH-
14). The relative configuration of C-14/C-15 was assigned as an
erythro rotamer B-3 by the large J value between H-14 and H-15
centers, namely, C-35/C-36 and C-37/C-38, possessed small JHH
and large 3JHC couplings (3JH-35C-43 ) 8.7 Hz; 3JH-37C-42 ) 8.8 Hz),
and thus their configurations were both assigned as threo rotamers
A-1.
Observation of ROESY correlations between protons of the
Ahdhe unit and MoCys1, the latter of which was now of assigned
absolute configuration, allowed identification of the four stereo-
centers present in the Ahdhe unit. Specifically, the exchangeable
proton NH-14 correlated with two methine protons of the Ahdhe
unit (H-12 and H-14) as well as the methyl singlet (H-23) of
MoCys1. Thus, the absolute configuration of the Ahdhe unit was
deduced as 12R, 13S, 14S, and 15S.8
The absolute configuration of the Dmetua portion was revealed
by analysis of the Mosher esters produced by esterification of the
C-37 hydroxy group of hoiamide B (2). However, the Mosher
esterification reactions that yielded the 37-(S/R)-MTPA esters were
(3JH-14,
) 9.7 Hz) and another series of ROESY correlations
H-15
(H-14/H-18, H-18/H-13, H-13/H-15, and H-15/14-NH).
On the basis of the large coupling constants between H-33/H-
34, H-34/H-35, and H-36/H-37, in combination with ROESY
correlations between the protons associated with these adjacent
chiral centers, C-33/C-34, C-34/C-35, and C-36/C-37 were assigned
as erythro rotamers B-3. The remaining two pairs of methine

1414 Journal of Natural Products, 2010, Vol. 73, No. 8
Choi et al.
were made by COSY, such as between C-3 and C-4, as revealed
by correlations between H-3 (δH 4.43) and H-4 (δH 4.13). An
HMBC correlation between H-36 (δH 4.17/4.11) and C-1 (δC 176.3),
along with the chemical shift of C-36 (δC 60.5), indicated the
presence of an ester linkage between C-1 and C-36. The chemical
shifts of C-3 (δC 71.7), C-23 (δC 81.8), C-25 (δC 71.6), and C-27
(δC 76.4) suggested directly linked oxygen atoms to each of these
methines. In contrast, the chemical shift of C-4 (δC 54.1) was more
appropriate for a nitrogen-linked carbon, and this was confirmed
by observation of vicinal coupling between the NH doublet at δH
7.57 (NH at C-4) and the methine proton at δH 4.13 (H-4) by COSY.
In addition, HMBC correlations involving both the NH proton (δH
7.57) and H-4 (δH 4.13) with the carbonyl carbon atom C-10 (δC
174.7) suggested an adjacent amide functional group.
Structure elucidation of the central triheterocyclic partial structure
of compound 3 was greatly facilitated by a combination of HMBC
and 15N HMBC experiments. The H-13 methyl protons, resonating
as a singlet at δH 1.75, exhibited HMBC correlations with the amide
carbon atom C-10 (δC 174.7), a quaternary carbon atom at δC 85.7
(C-11), and a methylene carbon atom at δC 41.9 (C-12). In addition,
an 15N HMBC experiment displayed a correlation between H3-13
and a nitrogen atom resonating at δN -76.1 (see Supporting
Information). The chemical shift of protons at C-12 (δH 4.18/3.31)
suggested an adjacent heteroatom, which, on the basis of the
chemical shift of C-12 (δC 41.9), was deduced to be a sulfur atom.
Both protons at C-12 showed reciprocal HMBC correlations with
C-13 and C-11 and also with a quaternary sp2 carbon atom at δC
178.9 (C-14). The deshielded chemical shift of the latter carbon
atom suggested a link with the δN -76.1 nitrogen atom and thus
described a methylthiazolene ring. Likewise, HMBC correlations
of H3-17 (δH 2.03) with C-14 (δC 178.9), C-15 (δC 85.0), C-16 (δC
43.0), and a second nitrogen at δN -70.7, as well as those of H2-
16 (δH 4.13/3.52) with C-17 (δC 27.0), C-14 (δC 178.9), C-15 (δC
85.0), and C-18 (δC 163.5), were indicative of a second methylthi-
azolene ring. The last heterocycle in the system, a thiazole ring,
was indicated by HMBC correlations of the aromatic proton H-20
(δH 8.27) with C-18 (δC 163.5), C19 (δC 148.3), and C-21 (δC
170.4). With this latter thiazole ring described, all of the required
degrees of unsaturation were satisfied. COSY provided evidence
of vicinal coupling between H-23 (δH 4.41) and both diastereotopic
protons at H2-22 (δH 3.49/3.13); in turn, these methylene protons
also displayed HMBC correlations with the sp2 carbon atom at δC
170.4 (C-21) of the adjacent thiazole ring. These latter connections
linked the triheterocyclic ring portion with the polyketide section
and thus completed the planar structure of hoiamide C (3) (Figure
5).
Owing to the limited quantity of hoiamide C (3) at this point in
the structure elucidation, we prioritized evaluation of its biological
properties over chemical degradation studies. However, we envi-
sioned a possible semisynthetic strategy to produce 3 from hoiamide
A (1) via regioselective hydrolysis of both ester bonds using LiOH,
followed by esterification of the resulting free carboxylic acid with
ethanol in the presence of catalytic HCl (Figure 6a). This sequence
of reactions was performed with hoiamide A (1), and the resulting
semisynthetic compound proved to be identical by 1H and 13C NMR
(Figure 6b), IR, UV, MS, and HPLC comparison with the material
extracted from Symploca sp., confirming the structure proposed for
the natural product 3. Furthermore, positive specific rotation values
and comparable circular dichroism curves (Supporting Information)
for both semisynthetic and natural hoiamide C (3) confirmed that
both hoiamides A and C possess the same configuration at their
comparable stereocenters. Thus, hoiamide C (3) was shown to
possess 2R, 3S, 4S, 5S, 11S, 15R, 23S, 24R, 25R, 26S, 27S, and
28R absolute configuration.
Figure 2. Partial structures of hoiamide B (2) derived from 2D
NMR data and their assembly by key HMBC correlations.
accompanied by dehydration of the threonine residue, producing
the 2,3-dehydrohoiamide B derivatives 6 and 7. Calculation of ∆S-R
values around C-37 allowed assignment of its absolute configuration
as S, and thus, the absolute configuration of the Dmetua residue
was determined as 33S, 34S, 35R, 36S, 37S, and 38R (Figure 4).
Isolation, Structure Elucidation, and Semisynthesis of
Hoiamide C (3). A second mixed collection of marine cyanobac-
teria, obtained by scuba on a reef wall near Pigeon Island, Papua
New Guinea, was extracted repeatedly with CH2Cl2-MeOH (2:1)
and then fractionated by silica gel vacuum-column chromatography
(VLC) to produce nine fractions (A-I). The F fraction, eluting with
80% EtOAc-hexanes, was found to possess potent brine shrimp
toxicity (LC50, ca. 5 µg/mL). This material was thus subjected to
RP HPLC and yielded a small quantity of hoiamide C (3, 2.9 mg,
0.02%). Pure hoiamide C (3) exhibited an LC50 of 1.3 µM in the
brine shrimp toxicity assay.
LRESIMS of hoiamide C (3) yielded an [M + H]+ peak at m/z
771.22 as part of a complex isotopic pattern [m/z 771/772/773/
774/775 (100:52:26:11:5)] and thus suggested once again the
presence of three sulfur atoms as in hoiamide A (1). This
interpretation was confirmed by HREIMS of 3, which gave a
molecular ion peak at m/z 770.3743 for a molecular formula of
C37H62N4O7S3 (calcd 770.3775) with nine degrees of unsaturation.
IR absorptions at 3389 (broad), 1731, and 1656 (broad) cm-1 were
consistent with the presence of hydroxy, ester, and amide func-
tionalities, respectively. Furthermore, a UV absorption maximum
at 250 nm was essentially identical to that measured for hoiamides
A (1) and B (2). On combining this information with a complete
NMR data set for 2 (Table 2), it was clear that compound 3 was
related to hoiamides A (1) and B (2), but of an overall truncated
size.
The 1H NMR spectrum of 3 (pyridine-d5) possessed resonances
for a methoxy singlet at δH 3.32, three methyl triplets at δH 1.11,
0.91, and 0.87, five methyl doublets between δH 1.32 and 0.93,
and multiple oxymethine resonances between δH 4.50 and 2.50,
suggesting polyketide-derived substructures closely related to Ahdhe
and Dmetua. Additionally, two methyl singlets at δH 2.03 and 1.75,
as well as an aromatic proton singlet at δH 8.27, confirmed the
presence of a cysteine-based triheterocyclic ring system as found
in hoiamides A (1) and B (2). Extensive analysis by HSQC, HMBC,
COSY, and z-TOCSY experiments revealed the planar structure
of a new linear hoiamide analogue, named hoiamide C (3).
A first inspection of the HMBC data involved analysis of long-
range correlations to the various methyl group protons, each of
which showed a full complement of two- and three-bond correla-
tions with their neighboring carbon atoms, and led to the identifica-
tion of partial structures A-E (Figure 5). Additional connections
Biosynthetic Prediction of Hoiamides B (2) and C (3).
Hoiamides A-C (1-3) are interesting representatives of a new
natural product class deriving from integration of PKS and NRPS

Neurotoxic Lipopeptides from Marine Cyanobacteria
Journal of Natural Products, 2010, Vol. 73, No. 8 1415
Figure 3. Depiction of homonuclear and heteronuclear coupling constants and ROE correlations used to assign the relative stereochemistry
of the Ahdhe (C-11-C-19) and Dmetua (C-31-C-45) residues in hoiamide B (2).
most interesting in this section of the molecule is the observation
that the Dmetua fragment consists of an 11-carbon unbranched
chain. While conceivably deriving from a propionate starter unit
followed by four acetate extensions (from malonyl CoA), its
origination from SAM methylation of a acetate starter unit is favored
because (a) utilization of propionate is essentially unknown in
cyanobacterial polyketides10 and (b) there is precedence for this
type of transformation from biosynthetic labeling experiments with
homoanatoxin A11 as well as unpublished work on apratoxin A
biosynthesis in our laboratory. The three consecutive heterocyclic
rings are likely created from three cysteine residues by heterocy-
clization followed by either dehydration to form the thiazole or
stereoselective methylation of the R-carbon to produce the two
R-methyl thiazolines. Following this section is the Ahdhe fragment,
which is likely created from an isoleucine residue extended by an
acetate unit. In this case, it is predicted that the C-2 position of the
acetate unit is methylated by SAM followed by reduction of the
carbonyl group to a secondary alcohol.5 While the Ahdhe residue
of hoiamide C (3) is capped as the ethyl ester (possibly an artifact
from EtOH extraction), in hoiamides A (1) and B (2) this group is
extended by connection to the hydroxy acid hydroxyisovaleric acid
(Hiva) or hydroxymethylpentanoic acid (Hmpa), respectively. From
Figure 4. ∆δS-R values around C-37 of the Mosher esters of
hoiamide B (6 and 7).
biosynthetic pathways. The Dmetua residue is a highly oxidized
and branched polyketide chain and is predicted to be the initial
biosynthetic unit formed in the hoiamides. The remnant oxidations
on the chain are likely reflections of C-1 positions in the acetate
subunits. Conversely, the methyl groups all occur at predicted C-2
positions of the acetate subunits and thus are likely incorporated
from the methyl group of S-adenosyl methionine (SAM). Perhaps

1416 Journal of Natural Products, 2010, Vol. 73, No. 8
Choi et al.
Table 2. NMR Spectroscopic Data for Hoiamide C (3) in Pyridine-d5 at 700 MHz (1H) and 175 MHz (13C)
a
residue
Ahdhe
position
δC
δH mult (J in Hz)
COSY
HMBCb
ROESY
1
2
3
4
176.3
45.3
71.7
54.1
2.90, m
3, 9
2, 4
1, 3
3
4.43, br d (9.7)c
4.13, br t (9.7)c
NH 7.57, d (9.7)
2.00, m
2, 4, 8, 9
3, 8, 9, NH
4
3, 5, NH
4
5, 8, 10
10
5
6a
6b
7
8
36.7
26.4
4, 8
6b, 7
6a, 7
6a, 6b
5
1.76, m
1.37, m
0.91, t (7.5)
0.95, d (6.7)
1.32, d (7.0)
6b
6a, 8
7, 8
5, 6
4, 5, 6
1, 2, 3
11.5
16.0
14.7
174.7
85.7
41.9
3, 4, 6b
3, 4
9
2
MoCys1
MoCys2
10
11
12a
12b
13
14
15
16a
16b
17
18
19
20
21
22a
22b
23
24
25
26
27
28
29a
29b
30
31
32
33
34
35
36a
36b
37
4.18, d (11.5)
3.31, d (11.5)
1.75, s
12b
12a
10, 11, 13, 14
10, 11, 13, 14
10, 11, 12
12b
12a, 13
12b
26.5
178.9
85.0
43.0
4.13, d (11.3)
3.52, d (11.3)
2.03, s
16b
16a
14, 15, 17, 18
14, 15, 17, 18
14, 15, 16
16b
16a, 17
16b
27.0
163.5
148.3
122.1
170.4
34.2
MoCys3
Dmetua
8.27, s
18, 19, 21
3.49, dd (15.4, 2.2)
3.13, dd (15.4, 10.6)
4.41, ddd (10.6, 3.5, 2.2)
2.50, m
4.39, dd (10.3, 1.3)
2.03, m
3.90, dd (7.1, 4.5)
1.86, m
1.57, m
22b, 23
22a, 23
22a, 22b, 24
23, 25, 34
24, 26
21
22b, 23, 25
22a, 34
21, 23
81.8
37.4
71.6
37.9
76.4
35.5
37.3
24, 35
23, 33, 34, 35
22a, 26, 27, 28
25, 27, 32, 34
25, 26, 28, 29a, 29b, 33
25, 27, 33
26, 33, 34
27, 33
25, 26, 29, 32
25, 27, 33
26, 28
24, 29a, 29b, 32
28, 29b, 30
28, 29a
29a, 31
30
29
30
30
29
27, 29b
27, 29a
1.36, m
1.37, m
20.8
14.9
14.2
10.5
10.4
56.8
60.5
0.87, t (7.0)
1.14, d (6.7)
1.19, d (6.9)
0.93, d (7.0)
3.32, s
4.17, m
4.11, m
1.11, t (7.1)
29, 30
27, 28, 29
25, 26, 27
22, 24, 25
23
28
26
24
26
24, 27, 28
22b, 24, 26
23, 24
36b, 37
36a, 37
36a, 36b
1
14.6
36
b
a Derived from HSQC and HMBC data. From H to the indicated 13C. c Multiplicity from the z-TOCSY spectrum.
1
Figure 5. Selected HMBC, 15N HMBC, and COSY correlations involved in building and interconnecting partial structures A-E of hoiamide
C (3).
our genetic work with the hectochlorin biosynthetic pathway, it is
predicted that these two residues are selected initially for incorpora-
tion as the corresponding R-keto acids and then reduced while
tethered to the corresponding peptidyl carrier protein (PCP).12 The

Neurotoxic Lipopeptides from Marine Cyanobacteria
Journal of Natural Products, 2010, Vol. 73, No. 8 1417
Figure 6. (a) Route for the semisynthesis of hoiamide C (3) from hoiamide A (1); (b) ∆δC of natural and semisynthetic hoiamide C (3).
predicted pathways to both hoiamides A (1) and B (2) conclude
with incorporation of the standard amino acid, L-threonine. Finally,
the hydroxy group at C-35 in the Dmetua portion of hoiamide B
(C-34 in hoiamide A) likely participates in a thioesterase-mediated
hydrolysis of the chain from the final PCP with coincident
lactonization, thereby forming the 26-membered macrocyclic ring.
Taxonomy of the Hoiamide-Producing Cyanobacterial
Strains. The two cyanobacteria that produced hoiamides B (2) and
C (3) were taxonomically compared with the hoiamide A (1)-
producing strain. Hoiamide A (1) was originally isolated from a
consortium of two different filamentous cyanobacteria identified
as Lyngbya majuscula (Harvey ex Gomont) and Phormidium gracile
(Meneghini ex Gomont) on the basis of morphology.8 This
cyanobacterial assemblage was predominantly composed of fine
Phormidium filaments entangled into thick pads that contained
embedded thicker, reddish Lyngbya filaments. As a result of these
entangled filaments, this Phormidium/Lyngbya consortium formed
extensive mats with cespitose short purple tufts. The hoiamide B
(2)-producing cyanobacterium, PNG06-64, was collected as dark
brownish-red mats covering the coral reefs, and its overall growth
morphology resembled that of the hoiamide A producer (Figure
7a). Interestingly, the hoiamide C producer possessed a distinctly
different thallus morphology compared with the hoiamide A and
B producers. The hoiamide C producer formed erect bundles of a
brownish-red purple color (Figure 7b).
7c,d). Embedded in the Phormidium of the hoiamide A and B
producers were also wider reddish filaments with disk-shaped cells
surrounded by distinct sheaths, which corresponded with the
Lyngbya morpho-type (Figure 7c inset).
The hoiamide B and C producers were phylogenetically analyzed
on the basis of their SSU (16S) rRNA genes to obtain a better
understanding of their evolutionary relationships and taxonomic
positions. The resulting phylogenies revealed that the Phormidium
morpho-types are related evolutionarily to various specimens of
Symploca, including the type-strain PCC 8002 and, thus, should
be reclassified as Symploca. The morphologically similar genera
Phormidium and Symploca both belong to the family Phormidiaceae
(Anagnostidis et Koma´rek, 1998) and are distinguished traditionally
by their thallus morphology. According to these systems, the
hoiamide A and B producers formed mats corresponding with the
definition of Phormidium, while the hoiamide C producer formed
erect bundles corresponding to the genus Symploca. However, our
phylogenetic analysis revealed that both the hoiamide B and C
producers belong to the genus Symploca, and it is suggested that
this traditional diacritical feature is taxonomically uninformative
and needs to be reconsidered. Along the same lines, the Lyngbya
morpho-type present in the mat producing hoiamide B was found
by 16S rRNA analysis to be closely related to the Oscillatoria type-
strain PCC 7515. Thus, while these two latter genera can have very
similar overall morphologies, our genetic analysis reveals the thicker
filaments in the mat to be Oscillatoria.
Microscopically, the three different hoiamide producers appeared
similar, with the vast majority of the biomass composed of fine
entangled filaments (7-10 µm) with isodiametric or slightly longer
cells. This description corresponds with the Phormidium morpho-
type previously described from the hoiamide A producer (Figure
Whether the biosynthesis of the hoiamides occurs in Oscillatoria
or Symploca can only be speculated upon at this point. The fact
that Symploca was the major component of all three samples and
that Oscillatoria was present only in the hoiamide A (1) and B (2)

1418 Journal of Natural Products, 2010, Vol. 73, No. 8
Choi et al.
Figure 7. Maximum-likelihood (PhyML) phylogenetic analysis of the hoiamide-producing cyanobacteria based on SSU (16S) rRNA nucleotide
sequences. The specimens are indicated as species, strain, and acc. nr in brackets. Specimens designated with an asterisk represent type-
strains obtained from Bergey’s Manual. The support values are indicated as boot-strap (PhyML) and posterior probability (MrBayes). The
scale bar is indicated at 0.04 expected nucleotide substitutions per site. Underwater field images of (A) the hoiamides A and B producer
and (B) the hoiamide C producer. Microscopic images of (C) hoiamides A and B and (D) hoiamide C producer.
Table 3. Biological Activities of Naturally Occurring
Hoiamides A-C (1-3) and Derivatives 4 and 5
inhibition of
activation of
VGSCa
Ca2+ oscillationsa
H-460b Neuro-2ac
compound (IC50 nM, 95% CI) (IC50 µM, 95% CI) (IC50 µM) (IC50 µM)
1
2
3
4
5
45.6 (30.3-68.6)
79.8 (29.5-215.5)
inactive
87.3 (19.8-385.0)
inactive
1.7 (0.7-4.2)
3.9 (1.2-13.0)
inactive
9.3 (1.0-90.0)
inactive
11.2
8.3
inactive
Inactive
inactive
2.1c
inactive
inactive
3.4d
inactive
a Mouse neocortical neurons. b Cytotoxicity to human lung
adenocarcinoma cells. c Cytotoxicity to mouse neuroblastoma cells.
d VGSC activation in mouse neuroblastoma cells.
Figure 8. Concentration-response relationships for suppression of
spontaneous Ca2+ oscillations in neocortical neurons by various
hoiamide analogues.
producers suggest that Symploca is likely the origin of these unusual
metabolites. However, conflicting with this hypothesis is the
considerable evolutionary distance between the Symploca specimens
present in these three samples. In this regard, it is conceivable that
an Oscillatoria spp. was originally present in the hoiamide
C-producing collection, but that it was not observed due to the small
size of the retained voucher sample.
dependent on voltage-gated sodium channel mediated action
potentials.14 Although the mechanism(s) underlying hoiamide A
(1) and B (2) induced inhibition of calcium oscillations is presently
unknown, these natural products may disrupt the neurotransmission
that drives a neuronal network function. Interestingly, switching
from a Hiva residue in 1 to a Hmpa residue in 2 significantly
decreased cytotoxicity levels in the Neuro-2a neuroblastoma cell
line. The linear analogue hoiamide C (3) and the cyclic triacetylated
derivative (5), however, showed no significant pharmacological
activity in these assays, clearly suggesting that the macrocycle and
its hydrogen bond donors at C-3, C-13, and C-37 in 1 and 2 play
a key role in their interactions with molecular targets. This is also
true for analogue 4, which additionally shows that modifying the
alkyl side chain by esterification of the C-37 hydroxy group of
hoiamide A (1) decreases cytotoxicity significantly (no cytotoxicity
observed up to 27.0 µM) and thus allowed a low micromolar VGSC
activation to be measured for this derivative in mouse neuroblastoma
cells (IC50 3.3 µM). This result correlates with the VGSC activation
measured in mouse neocortical neurons (IC50 9.3 µM) and suggests
that the hoiamides may have more than one molecular target in
cells that separately involve suppression of Ca2+ oscillations and
VGSC activation.
Bioactivity of the Hoiamides. The biological properties of
hoiamides A-C (1-3) as well as two semisynthetic derivatives
(compounds 4 and 5) are summarized in Table 3. Both hoiamides
A (1) and B (2) stimulated sodium influx with EC50 values of 1.7
and 3.9 µM, respectively, in mouse neocortical neurons. Previously,
we have demonstrated that hoiamide A is a sodium channel
neurotoxin site 2 partial agonist.8 Given the structural similarity
between hoiamides A and B and comparable ability to stimulate
sodium influx, it is reasonable to conclude that hoiamide B is also
a site 2 sodium channel activator. Additionally, hoiamides A (1)
and B (2) were both found to potently suppress spontaneous calcium
oscillations in neocortical neurons with EC50 values of 45.6 and
79.8 nM, respectively (Figure 8). The effects of hoiamides A and
B on spontaneous calcium oscillations are therefore of greater
potency than their respective effects on sodium influx. This
inhibitory effect on spontaneous calcium oscillations is not related
to their ability to activate voltage-gated sodium channels, inasmuch
as sodium channel activators actually enhance calcium oscillation
amplitude and frequency in low concentrations and produce a
sustained elevation of cytoplasmic calcium concentration at higher
concentrations.13 Synchronized Ca2+ oscillations in neurons in
culture is considered to be a neuronal network phenomenon that is
Experimental Section
General Experimental Procedures. Optical rotations were measured
on a JASCO P-2000 polarimeter, UV spectra on a Beckman Coulter
DU800 spectrophotometer, and IR spectra on a Nicolet ThermoElectron

Neurotoxic Lipopeptides from Marine Cyanobacteria
Journal of Natural Products, 2010, Vol. 73, No. 8 1419
Nicolet IR100 FT-IR spectrometer using KBr plates. NMR spectra were
recorded with DMSO (δC 39.5, δH 2.50), pyridine (δC 150.3, δC 135.9,
δC 123.9, δH 8.73, δH 7.56, δH 7.21), or chloroform as internal standards
(δC 77.2, δH 7.26), on a Bruker 600 MHz spectrometer (600 and 150
polarity starting from 10% EtOAc in hexanes to 100% MeOH, to
produce nine fractions (A-I). The fraction eluting with 80% EtOAc
in hexanes (fraction F) was separated subsequently using RP HPLC
(Phenomenex Jupiter 10 µm C18, 250 × 10 mm, 85% MeOH-H2O at
3 mL/min, detection at 228, 254, and 280 nm) to give pure hoiamide
A (1, 21 mg, 2.8%) and hoiamide B (2, 8.9 mg, 1.2%).
1
MHz for H and 13C NMR, respectively), equipped with a 1.7 mm
MicroCryoProbe or a Varian 700 MHz spectrometer (700 and 175 MHz
for 1H and 13C NMR, respectively) with a 5 mm HCN cold probe. 15
N
Hoiamide B (2): pale yellow oil; [R]25 +5.0 (c 0.3, CHCl3); UV
D
NMR spectra were referenced to CH3NO2 from the observed dimension.
LR- and HRESIMS were obtained on a ThermoFinnigan LCQ
Advantage Max mass detector and Agilent 6200 ESI-TOF mass
spectrometer, respectively. HREIMS spectra were obtained also on a
ThermoFinnigan MAT900XL mass spectrometer. HPLC was carried
out using a Waters 515 pump system with a Waters 996 PDA detector.
Cyanobacterial Collections and Taxonomic Identification. The
hoiamide B (2)-producing cyanobacterium (collection code: PNG-4-
28-06-1) was collected by scuba at a depth of 15-18 m at the Gallows
Reef of Papua New Guinea, in April 2006 (150°44.878′ E, 10°15.612′
S). The hoiamide C (3)-producing cyanobacterium (collection code:
PNG-5-19-05-7) was collected by scuba on a 10 m deep reef wall near
Pigeon Island, Papua New Guinea, in May 2005 (152°20.266′ E,
4°16.0.63′ S). Morphological characterization was performed using an
Olympus IX51 epifluorescent microscope (100×) equipped with an
Olympus U-CMAD3 camera. Taxonomic identification of cyanobac-
terial specimens was performed in accordance with current phycological
systems.15
Polymerase Chain Reaction (PCR) and Cloning. Approximately
50 mg of algal biomass was cleaned and pretreated using TE (10 mM
Tris; 0.1 M EDTA; 0.5% SDS; 20 µg/mL RNase)/lysozyme (1 mg/
mL) at 37 °C for 30 min followed by incubation with proteinase K
(0.5 mg/mL) at 50 °C for 1 h. Genomic DNA was extracted using the
Wizard Genomic DNA purification kit (Promega Inc., Madison, WI)
following the manufacturer’s specifications. DNA concentration and
purity were measured on a DU 800 spectrophotometer (Beckman
Coulter). The 16S rRNA genes were PCR-amplified from isolated DNA
using the modified lineage-specific primers, 106F 5′-CGGACGGGT-
GAGTAACGCGTGA-3′ and 1509R 5′-GGCTACCTTGTTACGACTT-
3′/1445R 5′-GGTAACGACTTCGGGCGTG-3′. The PCR reaction
volumes were 25 µL containing 0.5 µL (∼50 ng) of DNA, 2.5 µL of
10 × 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 H2O. 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 50 °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 manufacturer’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.
HM072001-HM072003.
Phylogenetic Inferences. All gene sequences were aligned using
MUSCLE v4.016 and refined using the SSU secondary structures model
for Escherichia coli J01695.17 Best-fitting nucleotide substitution models
optimized by maximum likelihood were selected using corrected
Akaike/Bayesian Information Criterion (AIC/BIC) in ModelTest 3.7.18
The evolutionary histories of the cyanobacterial genes were inferred
using maximum likelihood (ML) and Bayesian inference algorithms.
The maximum likelihood inference was performed using PhyML
v2.4.4.19 The analysis was run using the GTR+I+G model (selected
by AIC and BIC) assuming a heterogeneous substitution rate and
gamma substitution of variable sites (proportion of invariable sites
(pINV) ) 0.529, shape parameter (R) ) 0.453, number of rate
categories ) 4). Bootstrap resampling was performed on 500 replicates.
Bayesian analysis was conducted using MrBayes 3.1.20 The Bayesian
inference was performed using the GTR+I+G substitution model
(pINV ) 0.450, R ) 0.449, number of rate categories ) 4) with Markov
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 were
being sampled with a frequency of every 100 generations. The MCMC
convergence was detected by AWTY.21
(MeCN) λmax 250 nm (log ε 3.86); IR (KBr) νmax 3386, 2966, 2933,
1742, 1672 cm-1; 1H, 13C, and 2D NMR data, see Table 1; HRESIMS
m/z [M + H]+ 940.4584 (calcd for C45H73N5O10S3 940.4598).
Acid Hydrolysis and Marfey’s Analysis.22 Hoiamide B (2, 150
µg) was treated with 150 µL of 6 N HCl at 110 °C for 30 min. The
reaction product was obtained by lyophilization, and the residue was
dissolved in 150 µL of water. An aliquot (100 µL) of the resuspended
residue was transferred into a 1.5 mL glass vial and dried. The dried
hydrolysate was dissolved in 100 µL of 1 M sodium bicarbonate, and
then 25 µL of 1% L-FDLA (1-fluoro-2,4-dinitrophenyl-5-L-leucine
amide) was added in acetone. The solution was vortexed and incubated
at 40 °C for 60 min. The reaction was quenched by the addition of 50
µL of 2 N HCl, and then the reaction mixture was diluted with 100 µL
of MeOH and 10 µL of the solution was analyzed by LC-ESIMS.
The reaction products from advanced Marfey’s method were
separated on a RP HPLC column (Phenomenex Jupiter 5 µm C18
column, 4.6 × 250 mm, 5.0 µm) with a stepped gradient elution of
0.1% TFA in water (eluent A) and 100% MeCN (eluent B). Gradient
program: 0-5 min, B, 30%; 5-25 min, B, 30-70%; 25-30 min, B,
70%; 30-35 min, B, 100%; flow rate, 500 µL/min. The column
temperature was kept at 30 °C. The amino acids, derivatized with
advanced Marfey’s reagents, were detected using ESIMS. The retention
times of authentic amino acid L-FDLA derivatives were L-Thr (19.63
min), L-allo-Thr (20.45 min), D-allo-Thr (21.45 min), and D-Thr (23.05
min).
Preparation and Chiral Analysis of 2-Hydroxy-3-methylpen-
tanoic Acid (Hmpa).9 L-Ile (20 mg) was dissolved in 5 mL of cold (0
°C) 0.2 N HClO4, and then 2 mL of NaNO2(aq) was added with rapid
stirring. The reaction mixture was stored at room temperature until
evolution of N2 subsided (1 h). The solution was boiled for 3 min,
cooled to room temperature, and then saturated with NaCl. The mixture
was extracted three times with Et2O, and the Et2O layer was then dried
under N2 (g) to give 17.3 mg of the oily 2S,3S-Hmpa. Correspondingly,
2R,3R-Hmpa (16.2 mg), 2S,3R-Hmpa (13.1 mg), and 2R,3S-Hmpa (15.6
mg) were synthesized with the same procedure from D-Ile, L-allo-Ile,
and D-allo-Ile, repectively. Each authentic stereoisomer of Hmpa was
dissolved in 2 mM CuSO4(aq) with retention times measured by chiral
HPLC (Phenomenex, Chirex-D-penicillamine, 4.6 × 50 mm, 0.8 mL/
min, 87.5% 2 mM CuSO4(aq) in MeCN). The retention time of the
Hmpa residue in acid hydrolysate of 2 matched with 2S,3S-Hmpa (24.1
min; 2S,3R-Hmpa, 20.9 min; 2R,3S-Hmpa, 31.6 min; 2R,3R-Hmpa, 37.0
min).
Ozonolysis, Oxidation, Acid Hydrolysis, and Chiral HPLC. A
portion (800 µg) of 2 was dissolved in 2 mL of CH2Cl2 at room
temperature, and O3 was bubbled through the sample for 15 min. The
pale blue solution was dried under N2(g), resuspended in 200 µL of
mixed oxidation solution (H2O2-HCOOH, 1:2), incubated at 70 °C
for 20 min, and then dried under N2(g). The products were resuspended
in 200 µL of 6 N HCl and reacted at 110 °C for 2 h. The acid
hydrolysates were dried under N2 (g), dissolved in 2 mM CuSO4(aq),
and injected over chiral HPLC (Phenomenex, Chirex-D-penicillamine,
4.6 × 50 mm, 0.3 mL/min, 85% 2 mM CuSO4(aq) in MeCN). Synthetic
standards of 2S-methyl cysteic acid (MeCysA) and 2R-MeCysA were
prepared by Pattenden’s method.23 The retention time of products
resulting from the acid hydrolysate of 2 matched the synthetic
2S-MeCysA standard (9.8 min; 2R-MeCysA, 11.0 min).
Preparation of MTPA Ester of Hoiamide B. Duplicate samples
of compound 2 (1 mg) were dried and dissolved in 1 mL of anhydrous
pyridine, and a catalytic amount of DMAP (dimethylaminopyridine)
was added. Separately and into each vial, 15 µL of R-MTPA chloride
and 15 µL of S-MTPA chloride were added. The reaction vials were
stored at 40 °C for 72 h with stirring. The acylation products were
purified using RP HPLC (Phenomenex Jupiter 5 µm C18, 4.6 × 250
mm, 85% MeOH-H2O at 1 mL/min). The m/z values of the two
diastereomeric MTPA derivatives of compound 2 were observed by
ESIMS, and the 1H NMR spectrum was assigned by 2D NMR
experiments including TOCSY, HSQC, and HMBC.
Isolation of Hoiamide B (2). The cyanobacterial tissue (141 g, dry
wt) was extracted repetitively with 2:1 CH2Cl2-MeOH to afford 900
mg of crude extract. A portion of the extract (761 mg) was fractionated
by silica gel VLC with a stepwise gradient solvent system of increasing

1420 Journal of Natural Products, 2010, Vol. 73, No. 8
37-S-MTPA Ester of the 2,3-Dehydro Derivative of Hoiamide B
Choi et al.
NMR (600 MHz, pyridine-d5) δ 8.30 (1H, s, H-20), 7.60 (1H, d, J )
10.1 Hz, NH-4), 7.42 (1H, d, J ) 3.6 Hz, OH), 5.95 (1H, s, OH), 5.38
(1H, s, OH), 4.45 (1H, d, J ) 9.0 Hz, H-3), 4.43 (1H, ddd, J ) 11.4,
3.8, 2.4 Hz, H-23), 4.41 (1H, dd, J ) 10.8, 1.5 Hz, H-25), 4.19 (1H,
d, J ) 11.4 Hz, H-12a), 4.17 (1H, m, H-36a), 4.15 (1H, dd, J ) 9.6,
9.6 Hz, H-4), 4.15 (1H, d, J ) 11.4 Hz, H-16a), 4.13 (1H, m, H-36b),
3.93 (1H, dd, J ) 6.6, 4.9 Hz, H-27), 3.54 (1H, d, J ) 10.8 Hz, H-16b),
3.51 (1H, dd, J ) 15.6, 1.8 Hz, H-22a), 3.35 (3H, s, H-35), 3.33 (1H,
d, J ) 11.4 Hz, H-12b), 3.16 (1H, dd, J ) 15.0, 10.2 Hz, H-22b), 2.92
(1H, dddd, J ) 9.6, 7.2, 7.2, 7.2 Hz, H-2), 2.52 (1H, m, H-24), 2.05
(1H, m, H-26), 2.04 (3H, s, H-17), 2.02 (1H, m, H-5), 1.87 (1H, quintet,
J ) 6.0 Hz, H-28), 1.78 (1H, m, H-6a), 1.77 (3H, s, H-13), 1.59 (1H,
m, H-29a), 1.39 (1H, m, H-6b), 1.38 (2H, m, H-30), 1.37 (1H, m,
H-29b), 1.34 (3H, d, J ) 7.2 Hz, H-9), 1.20 (3H, d, J ) 6.6 Hz, H-33),
1.15 (3H, d, J ) 6.0 Hz, H-32), 1.14 (3H, t, J ) 7.2 Hz, H-37), 0.97
(3H, d, J ) 6.6 Hz, H-8), 0.95 (3H, d, J ) 7.2 Hz, H-34), 0.93 (3H,
t, J ) 7.8 Hz, H-7), 0.89 (3H, t, J ) 7.2 Hz, H-31); 13C NMR (125
MHz, pyridine-d5) δ 178.9 (C-14), 176.4 (C-1), 174.8 (C-10), 170.4
(C-21), 163.6 (C-18), 148.4 (C-19), 122.1 (C-20), 85.7 (C-11), 85.0
(C-15), 81.6 (C-23), 76.4 (C-27), 71.62 (C-3), 71.57 (C-25), 60.5 (C-
36), 56.8 (C-35), 54.1 (C-4), 45.3 (C-2), 43.0 (C-16), 41.9 (C-12), 37.9
(C-26), 37.3 (C-24), 37.2 (C-29), 36.7 (C-5), 35.5 (C-28), 34.2 (C-
22), 27.0 (C-17), 26.4 (C-13), 26.3 (C-6), 20.7 (C-30), 16.0 (C-8), 14.8
(C-31), 14.6 (C-9), 14.5 (C-37), 14.2 (C-32), 11.5 (C-7), 10.41 (C-
33), 10.38 (C-34); HRESIMS m/z [M + H]+ 771.3860 (calcd for
C37H63N4O7S3, 771.3859).
Neocortical Neuron Culture. Primary cultures of neocortical
neurons were obtained from embryonic day 16 Swiss-Webster mice.
Briefly, pregnant mice were euthanized by CO2 asphyxiation, and
embryos were removed under sterile conditions. Neocortices were
collected, stripped of meninges, minced by trituration with a Pasteur
pipet, and treated with trypsin for 25 min at 37 °C. The cells were
then dissociated by two successive trituration and sedimentation steps
in soybean trypsin inhibitor and DNase containing isolation buffer,
centrifuged and resuspended in Eagle’s minimal essential medium with
Earle’s salt (MEM), and supplemented with 1 mM L-glutamine, 10%
fetal bovine serum, 10% horse serum, 100 IU/mL penicillin, and 0.10
mg/mL streptomycin, pH 7.4. Cells were plated onto poly-L-lysine-
coated 96-well (9 mm) clear-bottomed black-well culture plates (Costar)
at a density of 1.5 × 105 cells/well. Cells were then incubated at 37 °C
in a 5% CO2 and 95% humidity atmosphere. Cytosine arabinoside (10
µM) was added to the culture medium on day 2 after plating to prevent
proliferation of nonneuronal cells. The culture media was changed on
days 5 and 7 using a serum-free growth medium containing Neurobasal
Medium supplemented with B-27, 100 IU/mL penicillin, 0.10 mg/mL
streptomycin, and 0.2 mM L-glutamine. Neocortical cultures were used
in experiments between 8 and 13 days in Vitro (DIV). All animal use
protocols were approved by the Institutional Animal Care and Use
Committee (IACUC) at Creighton University.
1
(6): pale yellow, amorphous solid; H NMR (CDCl3, 600 MHz) δH
8.70 (1H, br s, NH-2), 7.79 (1H, s, H-30), 6.90 (1H, q, J ) 7.1 Hz,
H-3), 6.77 (1H, d, J ) 10.1 Hz, NH-14), 5.09 (1H, d, J ) 4.1 Hz,
H-6), 4.96 (1H, d, J ) 9.3 Hz, H-37), 4.79 (1H, d, J ) 10.2 Hz, H-35),
3.97 (1H, dd, J ) 8.2, 8.0 Hz, H-13), 3.86 (1H, br s, OH-13), 3.82
(1H, d, J ) 11.6 Hz, H-22a), 3.79 (1H, d, J ) 10.5 Hz, H-33), 3.74
(1H, dd, J ) 9.5, 8.7 Hz, H-14), 3.69 (1H, d, J ) 11.4 Hz, H-26a),
3.32 (1H, d, J ) 11.4 Hz, H-26b), 3.25 (1H, d, J ) 11.6 Hz, H-22b),
3.17 (3H, s, H-45), 2.78 (1H, d, J ) 15.4 Hz, H-32a), 2.70 (1H, dd, J
) 15.3, 10.2 Hz, H-32b), 2.48 (1H, dq, J ) 7.1, 7.1 Hz, H-12), 2.32
(1H, ddq, J ) 10.4, 3.7, 6.9 Hz, H-34), 2.11 (1H, m, H-7), 2.08 (1H,
m, H-36), 1.84 (3H, s, H-27), 1.76 (3H, d, J ) 7.1 Hz, H-4), 1.74 (1H,
m, H-38), 1.68 (1H, m, H-8a), 1.55 (3H, s, H-23), 1.44 (1H, m, H-15),
1.40 (1H, m, H-16a), 1.31 (3H, d, J ) 7.1 Hz, H-19), 1.30 (2H, m,
H-39), 1.30 (2H, m, H-40), 1.26 (1H, m, H-8b), 1.07 (1H, m, H-16b),
1.02 (3H, d, J ) 6.9 Hz, H-10), 0.97 (3H, d, J ) 7.1 Hz, H-43), 0.89
(3H, t, J ) 7.2 Hz, H-9), 0.88 (3H, d, J ) 6.9 Hz, H-18), 0.85 (3H, t,
J ) 7.1 Hz, H-41), 0.82 (3H, d, J ) 6.8 Hz, H-42), 0.77 (3H, t, J )
7.4 Hz, H-17), 0.72 (3H, d, J ) 6.9 Hz, H-44); LRESIMS m/z 1138.57
[M + H]+, 1160.58 [M + Na]+.
37-R-MTPA Ester of the 2,3-Dehydro Derivative of Hoiamide
1
B (7): pale yellow, amorphous solid; H NMR (CDCl3, 600 MHz) δH
8.72 (1H, br s, NH-2), 7.78 (1H, s, H-30), 6.86 (1H, q, J ) 7.0 Hz,
H-3), 6.79 (1H, d, J ) 10.2 Hz, NH-14), 5.08 (1H, d, J ) 4.1 Hz,
H-6), 4.95 (1H, dd, J ) 9.6, 2.0 Hz, H-37), 4.75 (1H, d, J ) 10.4 Hz,
H-35), 3.96 (1H, dd, J ) 7.7, 7.7 Hz, H-13), 3.92 (1H, br s, OH-13),
3.83 (1H, d, J ) 11.6 Hz, H-22a), 3.77 (1H, d, J ) 10.1 Hz, H-33),
3.75 (1H, dd, J ) 9.5, 8.7 Hz, H-14), 3.68 (1H, d, J ) 11.3 Hz, H-26a),
3.33 (1H, d, J ) 11.4 Hz, H-26b), 3.24 (1H, d, J ) 11.6 Hz, H-22b),
3.17 (3H, s, H-45), 2.78 (1H, d, J ) 15.2 Hz, H-32a), 2.68 (1H, dd, J
) 15.4, 10.3 Hz, H-32b), 2.48 (1H, dq, J ) 7.1, 7.1 Hz, H-12), 2.30
(1H, ddq, J ) 10.5, 3.7, 6.9 Hz, H-34), 2.11 (1H, m, H-7), 2.08 (1H,
m, H-36), 1.83 (3H, s, H-27), 1.77 (1H, m, H-38), 1.75 (3H, d, J )
7.1 Hz, H-4), 1.68 (1H, m, H-8a), 1.55 (3H, s, H-23), 1.49 (1H, m,
H-15), 1.43 (1H, m, H-16a), 1.32 (2H, m, H-39), 1.30 (2H, m, H-40),
1.30 (3H, d, J ) 7.1 Hz, H-19), 1.25 (1H, m, H-8b), 1.08 (1H, m,
H-16b), 1.02 (3H, d, J ) 6.9 Hz, H-10), 0.94 (3H, d, J ) 7.1 Hz,
H-43), 0.92 (3H, d, J ) 6.6 Hz, H-18), 0.91 (3H, t, J ) 7.5 Hz, H-9),
0.86 (3H, d, J ) 6.9 Hz, H-42), 0.83 (3H, t, J ) 7.1 Hz, H-41), 0.80
(3H, t, J ) 7.4 Hz, H-17), 0.71 (3H, d, J ) 6.9 Hz, H-44); LRESIMS
m/z 1138.56 [M + H]+, 1160.47 [M + Na]+.
Isolation of Hoiamide C (3). The cyanobacterial filaments (ap-
proximately 81 g, dry wt) were extracted repeatedly with
CH2Cl2-MeOH (2:1) to afford 1.42 g of crude extract. A portion of
the extract (1.19 g) was fractionated by silica gel VLC with a stepped
gradient elution of hexanes, EtOAc, and MeOH. The bioactive fraction
F (79.2 mg) was subjected to RP HPLC (Phenomenex Jupiter 10 µm
C18, 10 × 250 mm, 65% MeCN-H2O at 3 mL/min, detection at 228,
254, and 280 nm) to yield 2.9 mg of hoiamide C (3).
Intracellular Ca2+ Monitoring. Neocortical neurons grown in 96-
well plates were used for [Ca2+]i measurements at 12-13 DIV. Briefly,
the growth medium was removed and replaced with dye loading buffer
(50 µL/well) containing 4 µM fluo-3 and 0.04% pluronic acid F-127
in Locke’s buffer (8.6 mM Hepes, 5.6 mM KCl, 154 mM NaCl, 5.6
mM glucose, 1.0 mM MgCl2, 2.3 mM CaCl2, 0.0001 mM glycine, pH
7.4). After 1 h incubation in dye loading buffer, the neurons were
washed four times in fresh Locke’s buffer (200 µL/well) using an
automated cell washer (BioTek instrument, Inc., Winooski, VT) and
transferred to a FlexStation II (Molecular Devices, Sunnyvale, CA).
The final volume of Locke’s buffer in each well was 150 µL. Cells
were excited at 485 nm, and Ca2+-bound Fluo-3 emission was detected
at 535 nm. Fluorescence readings were taken once every 1.5 s for 60 s
to establish the baseline, and then 50 µL of hoiamide analogue solution
(4×) was added to each well from the compound plate at the rate of
52 µL/s, yielding a final volume of 200 µL/well.
Hoiamide C (3): colorless oil; [R]23 +16 (c 0.2, CHCl3); CD λ
D
295 nm (∆ε -0.21), λ 280 nm (∆ε -0.11), λ 245 nm (∆ε -0.37), λ
220 nm (∆ε +2.42); UV (MeCN) λmax 249 nm (log ε 3.66); IR (neat)
1
νmax 3389, 2925, 2853, 1731, 1656, 1520, 1182, 1084, 735 cm-1; H
and 13C NMR data, see Table 2; HREIMS m/z [M]+ 770.3743 (calcd
for C37H62N4O7S3, 770.3775).
Preparation of Hoiamide C (3) from Hoiamide A (1). Hoiamide
A (10.1 mg, 0.011 mmol) was dissolved in a mixture of dioxane-H2O
2:1 (3 mL) and treated with LiOH monohydrate (10.0 mg, 0.21 mmol)
at 25 °C. The mixture was stirred at room temperature until TLC (70%
EtOAc in hexanes) showed the absence of starting material (1 h). The
solvent of mixture was then removed under reduced pressure, and the
resulting residue was redissolved in EtOH (15 mL), treated with 12 N
HCl (5 µL, 0.06 mmol) at 25 °C, and stirred at the same temperature
until TLC showed the appearance of a new product (72 h). At this
point, the crude reaction was concentrated to dryness, reconstituted in
H2O, and extracted with EtOAc (3 × 20 mL). The organic layer was
dried (Na2SO4) and filtered, and upon solvent removal under vacuum,
the resulting residue was purified via silica gel column chromatography
(70% EtOAc in hexanes) to yield pure hoaimide C (3) (1.3 mg, 16%)
as a colorless oil: [R]23D +32 (c 0.6, CHCl3); CD λ 295 nm (∆ε -0.35),
λ 280 nm (∆ε -0.15), λ 260 nm (∆ε +0.13), 245 nm (∆ε -0.11), λ
220 nm (∆ε +3.98); UV (MeCN) λmax 250 nm (log ε 3.84); IR (neat)
Intracellular Sodium Concentration ([Na+]i) Measurement. The
neocortical neurons cultured in 96-well plates (DIV 8-13) were washed
four times with Locke’s solution using an automated cell washer
(Bioteck Instrument Inc.). The background fluorescence of each well
was measured and averaged prior to dye loading. Cells were then
incubated for 1 h at 37 °C with dye loading buffer (50 µL/well)
containing 10 µM SBFI-AM and 0.02% Pluronic F-127. After 1 h
incubation in dye loading medium, cells were washed five times with
Locke’s buffer, leaving a final volume of 150 µL in each well. The
plate was then transferred to the plate chamber of a FlexStation II
1
νmax 3366, 2963, 2927, 1731, 1655, 1516, 1179, 1083, 671 cm-1; H

Neurotoxic Lipopeptides from Marine Cyanobacteria
Journal of Natural Products, 2010, Vol. 73, No. 8 1421
W. H. J. Am. Chem. Soc. 2000, 122, 12041–12042. (e) Orjala, J.;
Nagle, D. G.; Hsu, V.; Gerwick, W. H. J. Am. Chem. Soc. 1995, 117,
8281–8282. (f) Luesch, H.; Yoshida, W. Y.; Moore, R. E.; Paul, V. J.;
Corbett, T. H. J. Am. Chem. Soc. 2001, 123, 5418–5423. (g) Luesch,
H.; Chanda, S. K.; Raya, R. M.; DeJesus, P. D.; Orth, A. P.; Walker,
J. R.; Izpisu´a Belmonte, J. C.; Schultz, P. G. Nat. Chem. Biol. 2006,
2, 158–167.
(Molecular Devices, Sunnyvale, CA). Cells were excited at 340 and
380 nm, and Na+-bound SBFI emission was detected at 505 nm.
Fluorescence readings were taken once every 5 s for 60 s to establish
the baseline, and then 50 µL of hoiamide analogue containing solution
(4×) was added to each well from the compound plate at a rate of 52
µL/s, yielding a final volume of 200 µL/well.
Data Analysis. Time-response and concentration-response graphs
were generated using Graphpad Prism software (Graphpad Software
Inc., San Diego, CA). The EC50 values were determined by nonlinear
regression analysis using a logistic equation.
(5) Jones, A. C.; Gu, L.; Sorrels, C. M.; Sherman, D. H.; Gerwick, W. H.
Curr. Opin. Chem. Biol. 2009, 13, 216–223.
(6) Clare, J. J.; Tate, S. N.; Nobbs, M.; Romanos, M. A. Drug DiscoVery
Today 2000, 5, 506–520.
(7) (a) Catterall, W. A.; Cestele, S.; Yarov-Yarovoy, V.; Yu, F. H.; Konoki,
K.; Scheuer, T. Toxicon 2007, 49, 124–141. (b) Denac, H.; Mevissen,
M.; Scholtysik, G. Naunyn-Schmiedebergs Arch. Pharmacol. 2000,
362, 453–479. (c) Taylor, C. P.; Meldrum, B. S. Trends Pharmacol.
Sci. 1995, 16, 309–316. (d) Cestele, S.; Catterall, W. A. Biochimie
2000, 82, 883–892.
Acknowledgment. We thank A. C. Jones and T. L. Simmons for
assistance with collection of the different hoiamide-producing cyano-
bacterial samples from Papua New Guinea, as well as A. Jansma for
assistance with the Bruker 600 MHz TCI cryoprobe spectrometer
(supported in part by the Skaggs School of Pharmacy and Pharmaceuti-
cal Sciences, UCSD). Support of chemical and pharmacological aspects
of the work was provided by NIH NS053398. 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.
(8) (a) Pereira, A.; Cao, Z.; Murray, T. F.; Gerwick, W. H. Chem. Biol.
2009, 16, 893–906. (b) Pereira, A.; Cao, Z.; Murray, T. F.; Gerwick,
W. H. Chem. Biol. 2009, 16, 1208.
(9) Mamer, O. A. Methods Enzymol. 2000, 324, 3–10.
(10) Grindberg, R. V.; Shuman, C. F.; Sorrels, C. M.; Wingerd, J.; Gerwick,
W. H. In Modern Alkaloids, Structure, Isolation, Synthesis and Biology;
Fattorusso, E., Taglialatela-Scafati, O., Eds.; Wiley-VCH Verlang
GmbH: Weinheim, Germany, 2008; pp 139-170.
(11) Namikoshi, M.; Murakami, T.; Fujiwara, T.; Nagai, H.; Niki, T.;
Harigaya, E.; Watanabe, M. F.; Oda, T.; Yamada, J.; Tsujimura, S.
Chem. Res. Toxicol. 2004, 17, 1692–1696.
(12) Ramaswamy, A. V.; Sorrels, C. M.; Gerwick, W. H. J. Nat. Prod.
2007, 70, 1977–1986.
(13) Dravid, S. M.; Baden, D. G.; Murray, T. F. J. Neurochem. 2004, 89,
739–749.
Supporting Information Available: LR- and HRESIMS, one-
dimensional (1H and 13C) and two-dimensional (COSY, TOCSY,
HSQC, HMBC, ROESY, HETLOC, and HSQMBC) NMR spectra of
2, 3, 6, and 7, LC-ESIMS profile and CD spectra of natural and
semisynthetic 3, and bioassay results of 1 and 2. This material is
available free of charge via the Internet at http://pubs.acs.org.
References and Notes
(14) Dravid, S. M.; Murray, T. F. Brain Res. 2004, 1006, 8–17.
(15) Koma´rek, J.; Anagnostidis, K. In Su¨ꢀwasserflora Von Mitteleuropa;
Bu¨del, B.; Ga¨rtner, G.; Krienitz, L.; Schagerl, M., Eds.; Gustav Fischer:
Jena, 2005; 19/2, pp 483-606.
(1) Tidgewell, K.; Clark, B. T.; Gerwick, W. H. In ComprehensiVe Natural
Products Chemistry, 2nd ed.; Moore, B.; Crews, P., Eds.; Elsevier:
Oxford, UK, 2010; pp 141-188.
(2) (a) Fitch, C. P.; Bishop, L. M.; Boyd, W. L.; Gortner, R. A.; Rogers,
C. F.; Tilden, J. E. Cornell Vet. 1934, 24, 30–39. (b) Walsh, P.;
Fleming, L.; Solo-Gabriele, H.; Gerwick, W. H., Eds. Oceans and
Human Health, Risks and Remedies from the Sea; Elsevier Press:
Oxford, UK, 2008.
(16) Edgar, R. C. BMC Bioinf. 2004, 32, 1792–1797.
(17) 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.
(18) Posada, D.; Crandall, K. A. Bioinformatics 1998, 14, 817–818.
(19) Guindon, S.; Gascuel, O. Syst. Biol. 2003, 52, 696–704.
(20) Ronquist, F.; Huelsenbeck, J. P. Bioinformatics 2003, 12, 1572–1574.
(21) Nylander, J. A. A.; Wilgenbusch, J. C.; Warren, D. L.; Swofford, D. L.
Bioinformatics 2008, 15, 581–583.
(22) Fujii, K.; Ikai, Y.; Oka, H.; Suzuki, M.; Harada, K. Anal. Chem. 1997,
69, 5146–5151.
(23) Pattenden, G.; Thom, S. M.; Jones, M. F. Tetrahedron 1993, 49,
2131–2138.
(3) Marner, F.-J.; Moore, R. E.; Hirotsu, K.; Clardy, J. J. Org. Chem.
1977, 42, 2815–2819.
(4) (a) Gerwick, W. H.; Proteau, P. J.; Nagle, D. G.; Hamel, E.; Blokhin,
A.; Slate, D. J. Org. Chem. 1994, 59, 1243–1245. (b) Blokhin, A. V.;
Yoo, H. D.; Geralds, R. S.; Nagle, D. G.; Gerwick, W. H.; Hamel, E.
Mol. Pharmacol. 1995, 48, 523–531. (c) Marquez, B. L.; Watts, K. S.;
Yokochi, A.; Roberts, M. A.; Verdier-Pinard, P.; Jimenez, J. I.; Hamel,
E.; Scheuer, P. J.; Gerwick, W. H. J. Nat. Prod. 2002, 65, 866–871.
(d) Wu, M.; Okino, T.; Nogle, L. M.; Marquez, B. L.; Williamson,
R. T.; Sitachitta, N.; Berman, F. W.; Murray, T. F.; McGough, K.;
Jacobs, R.; Colsen, K.; Asano, T.; Yokokawa, F.; Shioiri, T.; Gerwick,
NP100468N