Alotamide A, a novel neuropharmacological agent from the marine cyanobacterium Lyngbya bouillonii

Article abstract of DOI:10.1021/ol901438b

Alotamide A (1), a structurally Intriguing cyclic depsipeptide, was isolated from the marine mat-forming cyanobacterlum Lyngbya bouillonii collected In Papua New Guinea. It features three contiguous peptidic residues and an unsaturated heptaketide with oxidations and methylations unlike those found In any other marine cyanobacterial metabolite. Pure alotamide A (1) displays an unusual calcium influx activation profile In murine cerebrocortical neurons with an EC₅₀ of 4.18 uU.

Full text of DOI:10.1021/ol901438b

ORGANIC
LETTERS
2009
Vol. 11, No. 20
4704-4707
Alotamide A, a Novel
Neuropharmacological Agent from the
Marine Cyanobacterium Lyngbya
bouillonii
Irma E. Soria-Mercado,†,‡ Alban Pereira,† Zhengyu Cao,§ Thomas F. Murray,§ 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 92037, Facultad de Ciencias
Marinas, UniVersidad Auto´noma de Baja California, Ensenada, BC, 22830, Mexico,
and Department of Pharmacology, Creighton UniVersity School of Medicine,
Omaha, Nebraska 68178
wgerwick@ucsd.edu
Received June 27, 2009
ABSTRACT
Alotamide A (1), a structurally intriguing cyclic depsipeptide, was isolated from the marine mat-forming cyanobacterium Lyngbya bouillonii
collected in Papua New Guinea. It features three contiguous peptidic residues and an unsaturated heptaketide with oxidations and methylations
unlike those found in any other marine cyanobacterial metabolite. Pure alotamide A (1) displays an unusual calcium influx activation profile
in murine cerebrocortical neurons with an EC50 of 4.18 µM.
The secondary metabolites of marine cyanobacteria are
among the most structurally intriguing and biologically active
in the natural world.1 The majority of these compounds have
been reported from collections of a single species, Lyngbya
majuscula, accounting for nearly 185 chemical entities
reported to date.1,2 In turn, the less well explored species
L. bouillonii has also proven to be a rich source of new
natural product chemotypes, including linear tetrapeptides
(e.g., lyngbyapeptin),3a macrolides [e.g., lyngbyaloside and
(3) (a) Klein, D.; Braekman, J. C.; Daloze, D.; Hoffmann, L.; Castillo,
G.; Demoulin, V. Tetrahedron Lett. 1999, 40, 695–696. (b) Klein, D.;
Braekman, J. C.; Daloze, D.; Hoffmann, L.; Demoulin, V. J. Nat. Prod.
1997, 60, 1057–1059. (c) Klein, D.; Braekman, J. C.; Daloze, D.; Hoffmann,
L.; Demoulin, V. Tetrahedron Lett. 1996, 37, 7519–7520. (d) Klein, D.;
Braekman, J. C.; Daloze, D.; Hoffmann, L.; Castillo, G.; Demoulin, V. J.
Nat. Prod. 1999, 62, 934–936. (e) Tan, L. T.; Marquez, B. L.; Gerwick,
W. H. J. Nat. Prod. 2002, 65, 925–928. (f) Luesch, H.; Yoshida, W. Y.;
Moore, R. E.; Paul, V. J. Bioorg. Med. Chem. 2002, 10, 1973–1978. (g)
Gutierrez, M.; Suyama, T. L.; Engene, N.; Wingerd, J. S.; Matainaho, T.;
Gerwick, W. H. J. Nat. Prod. 2008, 71, 1099–1103. (h) Matthew, S.;
Schupp, P. J.; Luesch, H. J. Nat. Prod. 2008, 71, 1113–1116.
† University of California San Diego.
‡ Universidad Auto´noma de Baja California.
§ Creighton University School of Medicine.
(1) Tidgewell, K.; Clark, B. T.; Gerwick, W. H. The Natural Products
Chemistry of Cyanobacteria. In ComprehensiVe Natural Products Chemistry,
2nd ed.; Moore, B., Crews, P., Eds.; Elsevier Ltd.: Oxford, UK, 2009; in
press.
(2) (a) Gerwick, G. W.; Tan, L. T.; Sitachitta, N. Alkaloids Chem. Biol.
2001, 57, 75–184. (b) Tan, L T. Phytochemistry 2007, 68, 954–979. (c)
Van Wagoner, R. M.; Drummond, A. K.; Wright, J. L. AdV. Appl. Microbiol.
2007, 61, 89–217.
10.1021/ol901438b CCC: $40.75
Published on Web 09/15/2009
 2009 American Chemical Society

lyngbouilloside (2) among others],3b-e as well as a group
of exceptionally active cyclic depsipeptides (apratoxins
A-E).3f-h As part of our assay-based screening program
for new neuroactive compounds from cyanobacteria,4 we
found that the extract of a Papua New Guinea collection
of L. bouillonii exhibited potent activation of calcium
influx in mouse cerebrocortical neurons. A number of
critical biological processes such as muscle contraction,
neurotransmission, hormone secretion, enzyme regulation,
and cell membrane permeability are modulated by calcium
ion concentrations in cells.5 For example, glutamate-
mediated intracellular Ca2+ overload is known to contrib-
ute to neuronal death in several human pathological
conditions (hypoxia-ischemia, hypoglycemia trauma, epi-
lepsy)6,7 and possibly neurodegenerative disorders such as
Alzheimer’s, Huntington’s, and motor neuron disease.8,9 The
profile of Ca2+ influx induced by this L. bouillonii crude
extract was unique and stimulated an assay-guided isola-
tion of the active constituent and ensuing structure
elucidation. The result was the discovery of alotamide A
(1), a structurally intriguing cyclic depsipeptide of mixed
polyketide/nonribosomal peptide biosynthetic origin.
Samples of L. bouillonii were collected by scuba in Milne
Bay near the town of Alotau, Papua New Guinea. The
organic extract (CH2Cl2/methanol 2:1, 311.7 mg) was
subjected to silica gel vacuum column chromatography
(stepwise gradient hexanes/EtOAc/MeOH) to produce nine
fractions (A-I). Neurotoxic fraction F was subjected to a
combination of further bioassays and 1H NMR-guided
fractionation, comprised of silica gel column chromatogra-
phies and reversed-phased HPLC to afford pure alotamide
molecular formula C32H49O5N3S and ten degrees of unsat-
uration. As described below, extensive analysis of 1 by 2D
NMR, including HSQC, HMBC, COSY, and NOESY,
confirmed the presence of a peptidic C1-C14 fragment as
well as a larger polyketide C15-C32 section (Figure 2). A
Figure 2. Partial structures of alotamide A (1) derived from analysis
of 2D NMR data and their assembly by HMBC correlations.
deshielded doublet R-amino proton at δ 4.95 (H2, δC 61.7)
was coupled to a multiplet at δ 2.19 which in turn showed
correlations to two methyl groups at δ 1.03 and 0.74,
consistent with the amino acid valine, and confirmed by
HMBC (Table 1). An HMBC from H2 to an N-methyl carbon
resonance at δ 31.4 (C6) suggested this to be N-methylvaline.
The H2 resonance was coupled with two carbonyls at δ 169.7
(C1) and δ 168.9 (C7); HMBC between the N-methyl at δ
2.84 (H36) and carbonyl C7 indicated this resonance was
associated with an adjacent residue. HMBC connection also
was observed between C7 and a methine at δ 4.80 (H8)
which in turn was coupled to a deshielded methylene at δ
3.41/3.98 (H9a/b; C9 δ35.0). HMBC correlations between
H9a and carbon resonances at δ 76.5 (C8) and 173.2 (C10),
combined with the unique chemical shifts of C7-C10,
identified this to be a cysteine-derived thiazoline ring. A final
R-amino methine resonance at δ 4.36 (H11, δC 60.6) showed
HMBC to C10, providing linkage to the third residue. COSY
connections were observed from the H11 methine to an
adjacent series of methylenes (H12-H14). H11 also showed
HMBC to all three of these methylene carbons and, on the
basis of this connectivity and chemical shifts, defined the
amino acid proline. Thus, the tripeptide component was
found to be composed of N-methylvaline (C1-C6), a
cysteine-derived thiazolene ring (C7-C9), and a proline
residue (C10-C14) and accounted for 5 of the 10 unsatura-
tions present in alotamide (1).
A (2.8 mg, 0.9%) (1) [[R]25 -1.9 (c 0.0158, CH2Cl2)]
D
(Figure 1), accompanied by the previously reported metabo-
lite lyngbouilloside (3.2 mg, 1.0%) (2).3e
Figure 1. Metabolites isolated from a collection of L. bouillonii
collected from near Alotau, Papua New Guinea, in 2003.
HRESIMS of 1 yielded an [M + H]+ peak at m/z 588.3477
(calcd for C32H50O5N3S, 588.3471), consistent with the
A weak HMBC correlation between the H12b proton and
carbonyl resonance at δ 172.1 (C15), together with strong
NOE cross peaks between the methine at δ 4.36 (H11) and
methyl at δ 1.83 (H17) and between H17 and the H14
methylene provided a linkage with the next section of
alotamide A. An allylically coupled (J ) 1.2 Hz) olefinic
proton (H18) and olefinic methyl group (H317) both showed
HMBC correlations to the C15 carbonyl peak and, on the
(4) Pereira, A.; Cao, Z.; Murray, T. F. Gerwick, W. H. Chem. Biol.
2009, 16, 893-906.
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Org. Lett., Vol. 11, No. 20, 2009
.
4705

Table 1. NMR Data of Alotamide A (1) in CDCl3
a
carbon
δC
δH, multiplicity (J Hz)b
type
COSY
HMBC (1H to 13C)
NOESY
1
2
3
4
5
6
7
8
169.7
61.7
25.7
17.3
20.1
31.4
168.9
76.5
35.0
4.95 d (10.8)
2.19 m
0.74 d (6.6)
1.03 d (6.6)
2.84 s
CH
CH
CH3
CH3
N CH3
3
1, 3, 4, 5, 6, 7
1, 2, 4, 5
2, 3, 5
2, 3, 4
2, 7
4, 5
4, 5
2, 3
2, 3
2, 4, 5
3
3
8, 18, 23, 24
4.80 ddd (1.8, 8.4, 12.6)
3.41 dd (9.0, 11.4)
3.98 dd (11.4, 12.6)
CH
CH2
9a, 9b
8, 9b
8, 9a
7, 9, 10
8, 10
7, 8
6, 18, 20, 24
20
9a
9b
10
11
12a
12b
13a
13b
14
15
16
17
18
19
20
21a
21b
22
23
24
25
26
27
28
29a
29b
30
31
32
173.2
60.6
30.9
4.36 d (7.2)
2.07 m
2.10 m
1.81 m
1.92 m
CH
CH2
12a
11, 13a
13a
12a, 12b, 14
14
13a, 13b
10, 12, 13, 14
10, 13, 14
10, 11, 15
11, 12
17, 20
21.5
CH2
CH2
45.2
172.1
131.1
14.4
133.5
30.1
3.52 m
12, 13
1.83 d (1.2)
5.34 dd (1.2, 9.0)
2.67 m
1.09 d (7.8)
2.23 m
CH3
CH
CH
CH3
CH2
18
17, 19
18, 20, 21a, 21b
19
19, 22
19, 22
21a, 21b, 23
22, 24
15, 16, 18, 19
15, 17, 19, 21
11, 14, 20
6, 8, 21a
19.4
38.1
18, 19, 21
19
8, 9a, 11, 17, 22
18, 23
2.30 m
128.0
129.9
126.9
134.4
15.5
46.5
67.9
43.0
5.63 ddd (3.6, 10.8, 14.8)
6.23 ddd (1.2, 10.8, 14.8)
5.68 d (10.8)
CH
CH
CH
21, 24
21, 24, 25
22, 23, 26, 27
20
6, 21a, 26
6, 8, 27
23
1.72 s
2.25 m
5.57 ddt (3.3, 10.2, 10.2)
1.58 ddd (3.0, 9.6, 14.2)
1.65 ddd (3.0, 9.3, 14.6)
3.15 m
CH3
CH2
CH
24, 25, 27
24, 25, 26, 28, 29
1
30
27, 28, 31
29, 32
29, 30
30
23, 28
24, 29a
26
27, 31
31
28
27, 29b
30
28
29a, 31
30
CH2
73.2
19.0
56.4
CH
CH3
OCH3
1.11 d (6.0)
3.29 s
29a, 29b
a Recorded at 150 MHz. b Recorded at 600 MHz.
basis of a 9.0 Hz coupling of H18 to a high-field methine
proton at δ 2.67, defined an R,ꢀ-unsaturated amide. Despite
H18 apparently having an unusually high-field shift for the
ꢀ-proton of an R,ꢀ-unsaturated amide, this was supported
by comparisons with synthetic compounds which mimic this
section of alotamide.10 H19 was coupled to a doublet methyl
group at δ 1.09 and to both protons of a diasterotopic
methylene (H221). HMBC from the doublet methyl to C18,
C19, and C21 confirmed these positional assignments. The
H221 protons were in turn coupled to an olefinic proton at δ
5.63 which was part of a conjugated diene (sequential
couplings to δ 6.23 and 5.68). By HMBC a second olefinic
methyl group (H326) was placed at the distal end of this
diene. Moreover, HMBC from H326 as well as H24 to a
methylene at higher field (δ 2.25) placed it as the other
terminating substituent of the diene. The remainder of the
polyketide section was deduced from sequential COSY
correlations between δ 2.25, a methine on an oxygen bearing
carbon, a high-field methylene, a second oxygen-bearing
carbon, and a terminating doublet methyl group (δ 1.11).
HMBC located a methoxy group at the latter oxygenated
site, whereas the deshielded nature of H28 was consistent
with an ester substituent (δH 5.57, δC 67.9). A key HMBC
from H28 to C1 located this position as the site of
macrolactonization to the tripeptide section of the molecule
(Figure 2). Thus, an unusual polyketide fragment, 11-
hydroxy-13-methoxy-2,4,9-trimethyltetradeca-2,6,8-trieno-
ic acid, completed the macrocyclic planar structure of
alotamide A (1).
Compound 1 was ozonolyzed (25 °C, 10 min), followed
by oxidative workup (H2O2-HCO2H) and hydrolysis with
6 M HCl (110 °C, 18 h), and then analyzed by chiral HPLC,
revealing the presence of N-methyl-L-(S)-valine, L-(S)-
proline, and D-(S)-cysteic acid. Additional stereochemical
(10) Jew, S. S.; Terashima, S.; Koga, K. Tetrahedron 1979, 35, 2337–
2343.
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Org. Lett., Vol. 11, No. 20, 2009

analysis of 1 was limited by the amount of available
compound at this point in the structure elucidation (<0.1 mg);
thus, only the double bond/proline amide bond geometries
were determined (Figure 3). The C16-C17 olefin was
are observed (heterocyclization, N-methylation). Cyclization
of alotamide A to a 21-membered ring likely occurs
concurrent with offloading from the final NRPS module.14
Alotamide A (1) displayed a unique profile when tested
in murine cerebrocortical neurons. Specifically, it produced
both a concentration-dependent elevation of intracellular
calcium concentration as well as an increase in the frequency
of spontaneous calcium oscillations in these cells (EC50 4.18
µM) (Figure 4 and Supporting Information). Although the
Figure 3. Proline amide bond and double-bond geometry assign-
ments for the C11-C32 section of alotamide A (1).
determined as E from NOE correlations between the H317
and H320 as well as between H18 and one of the methylene
protons at H21 (Table 1). A trans-disubstituted olefin
coupling constant of 14.8 Hz was observed between H22
and H23. An NOE between the H26 methyl singlet and H23
vinylic proton was complemented by an NOE between H24
and H27, thus defining the C24-C25 olefin as E. This
assignment was supported by the upfield chemical shift in
C26 (δ 15.5) and comparison with related diene-containing
model systems.9,10 These data established the configuration
of these three double bonds as 16E, 22E, and 24E. The cis
conformation for the D-proline amide bond was determined
by the diagnostic difference in chemical shifts between the
proline ꢀ and γ carbons.11,12 This assignment was reinforced
by NOE data which showed the proximity of the H11
methine and H17 methyl group.
From a biosynthetic perspective, alotamide A (1) appears
to derive from integration of PKS (e.g., loading plus six PKS
modules) and NRPS pathways (e.g., three NRPS modules).
There are four methylations of the polyketide, three of which
likely involve SAM as a methyl source (the C30 OCH3, C17
and C20 CCH3 groups). The C26 methyl resides at a
predicted carbonyl site in the nascent polyketide and thus
likely involves the ꢀ-branch mechanism using an HMGCoA
synthase casette of enzymes.13 In the peptide section,
modifications typical of cyanobacterial secondary metabolites
Figure 4. Concentration-response profile for alotamide A-induced
elevation in the frequency of spontaneous Ca2+ oscillations in mouse
cerebrocortical neurons. Depicted is a three-parameter logistic fit
to the frequency data that yielded an alotamide A EC50 value of
4.18 µM (95% confidence intervals 2.32-7.54 µM).
molecular target for this activity is presently unknown, a
preliminary pharmacological evaluation has excluded volt-
age-gated sodium and calcium channels as potential sites of
action. Additional possible mechanisms of action include
allosteric enhancement of glutamate receptor function or
antagonism of voltage-gated-potassium channels. The latter
remains an intriguing possibility inasmuch as we have
observed similar profiles on spontaneous calcium oscillations
in cerebrocortical neurons with potassium channel antago-
nists. It will therefore be of considerable interest to further
characterize the site of action and full pharmacological
consequences in mammalian neurons of this new type of
cyanobacterial neurotoxin.
Acknowledgment. We thank the NIH (NS053398) and
the Consejo Nacional de Ciencia y Tecnolog´ıa (CoNaCyT,
Republic of Mexico for a sabbatical fellowship to I.S.M.)
for support. We also thank O. Vining for extract preparation,
N. Engene for 16S rRNA identification, and A. Jansma for
NMR assistance.
(11) Tan, L. T.; Williamson, R. T.; Gerwick, W. H.; Watts, K. S.;
McGough, K.; Jacobs, R. J. Org. Chem. 2000, 65, 419–425
.
Supporting Information Available: Experimental details,
full NMR data of alotamide A (1), and calcium oscillation
data. This material is available free of charge via the Internet
at http://pubs.acs.org.
(12) Siemion, I. Z.; Wieland, T.; Pook, K. H. Angew. Chem., Int. Ed.
1975, 14, 702–703
.
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Hakansson, K.; Sherman, D. H.; Smith, J. L. J. Biol. Chem. 2007, 282,
35954–35963
.
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.
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