Palmyramide a, a cyclic depsipeptide from a palmyra atoll collection of the marine cyanobacterium lyngbya majuscula

Article abstract of DOI:10.1021/np900428h

Bioassay-guided fractionation of the extract of a consortium of a marine cyanobacterium and a red alga (Rhodophyta) led to the discovery of a novel compound, palmyramide A, along with the known compounds curacin D and malyngamide C. The planar structure of palmyramide A. was determined by one- and two-dimensional NMR studies and mass spectrometry. Palmyramide A is a cyclic depsipeptide that features an unusual arrangement of three amino acids and three hydroxy acids; one of the hydroxy acids is the rare 2, 2-dimethyl-3- hydroxyhexanoic acid unit (Dmhha). The absolute configurations of the six residues were determined by Marfey's analysis, chiral HPLC analysis, and GC/MS analysis of the hydrolysate. Morphological and phylogenetic studies revealed the sample to be composed of a Lyngbya majuscula-Centroceras sp. association. MALDI-imaging analysis of the cultured L majuscula indicated that it was the true producer of this new depsipeptide. Pure palmyramide A showed sodium channel blocking activity in neuro-2a cells and cytotoxic activity in. H-460 human lung carcinoma cells.

Full text of DOI:10.1021/np900428h

J. Nat. Prod. 2010, 73, 393–398
393
Palmyramide A, a Cyclic Depsipeptide from a Palmyra Atoll Collection of the Marine
Cyanobacterium Lyngbya majuscula†
Masatoshi Taniguchi, Joshawna K. Nunnery, Niclas Engene, Eduardo Esquenazi, Tara Byrum, Pieter C. Dorrestein, 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
ReceiVed July 14, 2009
Bioassay-guided fractionation of the extract of a consortium of a marine cyanobacterium and a red alga (Rhodophyta)
led to the discovery of a novel compound, palmyramide A, along with the known compounds curacin D and malyngamide
C. The planar structure of palmyramide A was determined by one- and two-dimensional NMR studies and mass
spectrometry. Palmyramide A is a cyclic depsipeptide that features an unusual arrangement of three amino acids and
three hydroxy acids; one of the hydroxy acids is the rare 2,2-dimethyl-3-hydroxyhexanoic acid unit (Dmhha). The
absolute configurations of the six residues were determined by Marfey’s analysis, chiral HPLC analysis, and GC/MS
analysis of the hydrolysate. Morphological and phylogenetic studies revealed the sample to be composed of a Lyngbya
majuscula-Centroceras sp. association. MALDI-imaging analysis of the cultured L. majuscula indicated that it was
the true producer of this new depsipeptide. Pure palmyramide A showed sodium channel blocking activity in neuro-2a
cells and cytotoxic activity in H-460 human lung carcinoma cells.
Cyanobacteria are well known to produce a wide variety of
secondary metabolites displaying significant structural diversity and
biological activity.1,2 Because the production of secondary me-
tabolites depends on both the collection location and specific
species, our program has focused on collections from a wide variety
of tropical and subtropical locations including Panama, Papua New
Guinea, Curac¸ao, Jamaica, and Puerto Rico, and this has generally
been a productive approach.2 In this vein, we recently had the
opportunity to examine the unique marine flora and cyanobacteria
of Palmyra Atoll in the Northern Pacific Ocean, a remote Pacific
island with a unique biogeography.3 Bioassay-guided fractionation
of the extract of a cyanobacterial/red algal consortium led to the
discovery of a novel cyclic depsipeptide, palmyramide A (1), along
with the known compounds curacin D and malyngamide C.4,5 This
paper describes the isolation, structural determination including
Figure 1. Structure of palmyramide A (1).
stereostructure, biological activity, and identification of the produc-
ing organism of 1.
Analysis of 1- and 2D-NMR spectra including COSY, TOCSY,
HSQC, and HMBC led to the assignments of three amino acids
[valine (Val), N-methylvaline (N-MeVal), and proline (Pro)] and
three hydroxy acids (3-phenyllactic acid (Pla), lactic acid (Lac),
and 2,2-dimethyl-3-hydroxyhexanoic acid (Dmhha)) (Table 1). The
N-methyl group of N-MeVal was revealed by HMBC correlations
(H-19/C-15, H-15/C-19) and the chemical shifts of the NMe (δH
2.85, δC 30.5). In the Pla residue, the proton chemical shifts at δH
7.23-7.28 (monosubstituted benzene ring), δH 3.13 and 2.95
(methylene), and δH 5.02 (methine) were very similar to those
reported for phenylalanine. However, the carbon chemical shift of
the methine (δC 70.9) was consistent with that of an oxymethine,
indicating that this residue was not phenylalanine but rather
3-phenyllactic acid. The proton chemical shifts of the Lac residue
at δH 1.47 (methyl) and 4.81 (methine) are also very similar to
those reported for alanine, but once again the carbon chemical shift
of the methine (δC 69.0) indicated that this residue was an
R-hydroxy acid, namely, lactic acid. The identity of the Dmhha
residue was deduced through analysis of TOCSY, COSY, and
HMBC correlations as follows: the proton spin system from H-3
to H-6 was disclosed by TOCSY, and two singlet methyl proton
resonances H-7 (δH 1.21) and H-8 (δH 1.15) gave HMBC cross-
peaks to C-1, C-2, and C-3. Oxygenation at C-3 was once again
Results and Discussion
The crude extract of the consortium of cyanobacterium and red
alga collected at Palmyra Atoll was subjected to silica gel vacuum
liquid chromatography and assayed for sodium channel blocking
activity and cytotoxic activity. The bioactive fraction eluted with
100% EtOAc and was further purified by C18 SPE cartridge and
repetitive reversed-phase HPLC to yield palmyramide A (1) (Figure
1) as a colorless glassy solid.
High-resolution ESIMS of 1 gave [M + H]+ and [M + Na]+
peaks at m/z 672.3852 and 694.3669, respectively, indicating a
molecular formula of C36H53N3O9 and requiring 12 degrees of
unsaturation. The 1H NMR spectrum possessed five aromatic
protons at δH 7.23-7.28, an amide NH proton at δH 6.55, an
N-methyl group at δH 2.85, and two singlet methyl groups at δH
1.16 and 1.21. Correspondingly, the 13C NMR spectrum revealed
the presence of six ester/amide carbonyls and a monosubstituted
phenyl ring at δC 127.4, 128.6 (2C), 129.7 (2C), and 134.9,
accounting for 10 of the 12 degrees of unsaturation.
† Dedicated to the late Dr. John W. Daly of NIDDK, NIH, Bethesda,
Maryland, and to the late Dr. Richard E. Moore of the University of Hawaii
at Manoa for their pioneering work on bioactive natural products.
* To whom correspondence should be addressed. Tel: (858) 534-0578.
Fax: (858) 534-0529. E-mail: wgerwick@ucsd.edu.
10.1021/np900428h  2010 American Chemical Society and American Society of Pharmacognosy
Published on Web 10/19/2009

394 Journal of Natural Products, 2010, Vol. 73, No. 3
Taniguchi et al.
Table 1. NMR Spectroscopic Data (600 MHz, CDCl3) for Palmyramide A
residue
Dmhha
position
δC mult
δH (mult, J in Hz)
HMBC
1
2
3
174.9 qC
46.4 qC
5.57 (dd, 9.6, 1.8)
1.43 (m)
77.2 CH
31.6 CH2
C-1, C-2, C-4, C-5, C-7, C-8
C-3, C-5, C-6
4a
4b
1.52 (m)
C-5, C-6
5
6
7
8
9
10
11
12
13
NH
14
15
16
19.6 CH2
13.9 CH3
16.9 CH3
24.3 CH3
170.1 qC
58.3 CH
30.5 CH
17.8 CH3
18.3 CH3
1.36 (m)
0.92 (t, 7.2)
1.21 (s)
C-3, C-4, C-6
C-4, C-5
C-1, C-2, C-3, C-8
C-1, C-2, C-3, C-7
1.16 (s)
Val
4.46 (dd, 5.4, 3.0)
2.38 (dqq, 3.0, 7.2, 7.2)
0.89 (d, 7.2)
0.98 (d, 7.2)
6.55 (d, 5.4)
C-9, C-12, C-13
C-9, C-10, C-12, C-13
C-10, C-11, C-13
C-10, C-11, C-12
C-10, C-14
N -MeVal
169.7 qC
61.7 CH
27.3 CH
18.8 CH3
19.3 CH3
30.5 CH3
171.2 qC
56.7 CH
30.7 CH2
22.0 CH2
4.42 (d, 10.8)
2.13 (m)
0.70 (d, 6.6)
0.94 (d, 6.6)
2.85 (s)
C-14, C-16, C-17, C-18, C-19, C-20
C-14, C-15, C-17, C-18
C-15, C-16, C-18
C-15, C-16, C-17
C-15, C-20
17
18
19 (N-CH3)
20
21
Pro
3.22 (dd, 7.8, 3.0)
1.50 (m)
1.56 (m)
1.68 (m)
3.42 (ddd, 12.0, 6.6, 6.6)
3.57 (ddd, 12.0, 8.4, 4.8)
C-22, C-23
C-20, C-23, C-24
C-22, C-24
C-22, C-24
C-22, C-23, C-25
C-22, C-23
22
23a
23b
24a
24b
25
46.7 CH2
Pla
167.2 qC
70.9 CH
39.2 CH2
26
5.02 (dd, 12.0, 4.8)
2.95 (dd, 12.0, 4.8)
3.13 (dd, 12.0, 4.8)
C-25, C-27, C-28, C-34
C-25, C-26, C-28, C-29/33
C-25, C-26, C-28, C-29/33
27a
27b
28
29/33
30/32
31
34
35
36
134.9 qC
129.7 CH
128.6 CH
127.4 CH
168.3 qC
69.0 CH
7.23 (m)
7.28 (m)
7.25 (m)
C-27, C-31
C-28, C-30/32
C-29/33
Lac
4.81 (q, 6.6)
1.47 (d, 6.6)
C-34, C-36, C-1
C-34, C-35
16.7 CH3
inferred from its 13C NMR chemical shift at δC 77.2. The remaining
required degree of unsaturation suggested the overall monocyclic
nature of 1.
The sequencing of these residues in 1 was accomplished by
interpretation of HMBC correlations (Table 1). Correlations from
the NH proton or N-methyl protons to the neighboring carbonyl
carbons were observed between Val/N-MeVal and N-MeVal/Pro.
Correlations from methine or methylene protons to carbonyl carbons
of the neighboring amino or hydroxy acids were also observed
between Pro/Pla, Pla/Lac, and Lac/Dmhha. Thus, the planar
structure of 1 was determined as shown in Figure 1.
The absolute configurations of the three amino acids in palym-
ramide A (1) were determined by HPLC analysis of the acid
hydrolysate derivatized with Marfey’s reagent [NR-(2,4-dinitro-5-
fluorophenyl)-L-valinamide: DFVA].6 LC/MS analysis of the
derivatives eluting by a linear gradient MeCN/H2O (0.1% HCOOH)
revealed the presence of D-N-MeVal and L-Pro, whereas the
configuration of the Val residue could not be determined because
of the overlap between the peaks of L-Val and Marfey’s reagent.
After exploring a variety of HPLC conditions, LC/MS analysis using
a linear gradient of MeOH/H2O (0.1% HCOOH) and a Merck
LiChrospher 100 RP-18 column enabled separation of the peaks
of L-Val and the Marfey’s reagent and, thus, allowed determination
of the Val residue in 1 to be L. The absolute configurations of Pla
and Lac were determined as L by chiral HPLC analysis. The
absolute configuration of the ꢀ-hydroxy group of Dmhha was
deduced by chromatographic comparisons with authentic S- and
R-Dmhha, which were stereoselectively synthesized via a tertiary
aldol reaction (Figure 2).7 The absolute configurations of the
intermediates 4a and 4b formed in these syntheses were confirmed
Figure 2. Stereoselective synthesis of S- and R-Dmhha. (a) 1.
n-BuLi, THF, -78 °C. 2. Isobutyryl chloride. (b) 1. LDA, THF,
-78 °C. 2. Ti(O-i-Pr)3Cl. 3. Butanal. (c) 1. 30% aqueous H2O2. 2.
LiOH/H2O, THF/H2O (4:1).
by the Mosher ester method8 and optical rotations. Chiral GC/MS
analysis of the Dmhha methyl ester liberated from 1 by acid
hydrolysis revealed only the presence of R-Dmhha. Thus, the
absolute configuration of palmyramide A (1) was determined as
shown in Figure 1.
As noted, palmyramide A (1) was isolated from an environmental
sample composed of a consortium of a cyanobacterium and a red
alga (Figure 3A and B) and obtained from two different populations
collected from different sampling sites at Palmyra Atoll. Morpho-
logical and phylogenetic studies revealed that both samples were
of similar composition and composed of Lyngbya majuscula Harvey

Cyclic Depsipeptide from Lyngbya majuscula
Journal of Natural Products, 2010, Vol. 73, No. 3 395
Figure 3. MALDI-TOF imaging of the palmyramide A (1)-producing cyanobacterium Lyngbya majuscula. (A) Photograph of the consortium
of Lyngbya majuscula and Centroceras sp. at the time of collection. (B) Photomicrograph of the consortium of Lyngbya majuscula (a) and
Centroceras sp. (b). (C) Epifluorescent image of the cultured Lyngbya majuscula at 590 nm (c, orange) and MALDI image of m/z 672 ()
palmyramide A, 1, d, green).
ex Gomont (Cyanophyta) and Centroceras sp. (Rhodophyta). The
L. majuscula appeared as blackish tufts approximately 10 cm in
height (Figure 3A). Microscopically, the Lyngbya filaments were
long (>1 cm), straight or slightly waved, cylindrical, and 40.2 (
3.4 µm (n ) 3) wide. The trichomes were enclosed with distinct
visible, colorless sheaths. The cells were disk-shaped (39.1 ( 2.8
µm wide and 3.5 ( 0.9 µm long; n ) 30) with no or only very
small constrictions at the cross-walls. The terminal cells were
rounded and noncapitated, and lacked calyptras. Taxonomic
identifications and evolutionary histories were inferred for both the
L. majuscula and Centroceras sp. components of this consortium
using the conserved 16S/18S (SSU) rRNA genes and the more
variable RNA polymerase γ-subunit (rpoC1) gene. Furthermore,
both genetic markers were obtained from the consortium obtained
at both sampling sites and confirmed that these were the same two
organisms in each case. An identical 16S rRNA gene (1373 base
pairs or ∼95% of the gene coverage; GenBank Acc. No. GQ231522)
and rpoC1 gene (866 base pairs; GenBank Acc. No. GQ231521)
were obtained from the L. majuscula of both populations. The
evolutionary history was inferred from the 16S rRNA gene using
the Minimum Evolution and MrBayes algorithms, which resulted
in conserved tree topologies and similar bootstrap support. These
collections of L. majuscula claded tightly (<1% sequence diver-
gence) with other Pacific strains of Lyngbya within the “marine
Lyngbya lineage” (Figure 4). The 18S rRNA genes (206 bp;
GenBank Acc. No. GQ246180) obtained from the two collections
of Centroceras sp. were also identical in sequence.
human lung carcinoma cells, palmyramide A (1) showed modest
cytotoxic effects with an IC50 value of 39.7 µM.
Palmyramide A (1) is a novel cyclic depsipeptide that features
an unusual arrangement of three amino acids and three hydroxy
acids. Among them, Dmhha is the most unusual hydroxy acid and
has previously been reported only in guineamides E and F, two
metabolites isolated from Papua New Guinea collections of Lyngbya
majuscula.13 Since environmental samples of marine organisms are
almost always collected as a consortium, as mixtures, or with
symbionts, it is necessary to determine the true source of interesting
secondary metabolites by other approaches. The npMALDI-I
method is well suited to directly observing the physical location of
metabolites in intact marine organisms or assemblages.10 In this
case, palmyramide A (1) was isolated from a consortium of two
organisms, a cyanobacterium and a red alga; however, analysis by
npMALDI-I showed that L. majuscula is the true producer. Indeed,
the cyanobacterium L. majuscula is one of the most prolific
producers of natural products, with more than 185 reported
compounds to date.2 In this study, our investigation of L. majuscula
from a new geographical region was rewarded by the discovery of
a new sodium channel blocking and cytotoxic cyclic lipopeptide,
palmyramide A (1), and suggests that a continued investigation of
this organism from new locations will be productive in the discovery
of novel natural compounds with pharmaceutical potential.
Experimental Section
General Experimental Procedures. Optical rotations were measured
with a JASCO P-2000 polarimeter. IR and UV spectra were recorded
with a Nicolet IR-100 FT-IR and Beckman-Coulter DU800 spectro-
photometer, respectively. NMR spectra were recorded on Varian Inova
500 and Bruker Avance III DRX600 with the solvent CDCl3 (δH at
7.26, δC at 77.0) used as an internal standard. High-resolution mass
spectra were obtained on a Thermo Scientific LTQ-XL Orbitrap mass
spectrometer. LC/MS analysis was carried out on a Finnigan LCQ
advantage mass spectrometer with a Finnigan Surveyor HPLC system.
HPLC was performed using a Waters 515 pump and a Waters 996
photodiode array spectrometer.
Collection. The consortium of a cyanobacterium and a red alga was
collected by hand from shallow water (<1 m) at two different sites in
the western lagoon south of Strawn Island, Palmyra Atoll, USA, in
August 2008 (GPS coordinates: 05′53.04 N, 162′05.161 W and
05′52.172 N, 162′05.047 W). The environmental samples were stored
in EtOH/H2O (1:1) at -20 °C, while the genetic materials were
preserved in RNA stabilization solution at -20 °C (RNAlater, Ambion
Inc.). Voucher specimens are available from WHG as collection
numbers PAL 8/17/08-2 and PAL 8/15/08-1.
Extraction and Isolation. Approximately 417.1 g (dry weight) of
the consortium was extracted repeatedly with CH2Cl2/MeOH (2:1) to
yield 7.1 g of crude extract. A portion of the extract (1.5 g) was
subjected to silica gel vacuum liquid chromatography (VLC, hexanes/
EtOAc/MeOH) to produce nine chemically distinct fractions. The
fraction eluted with EtOAc was subjected to C18 SPE (MeOH/H2O,
stepwise) and repetitive reversed-phase HPLC (Phenomenex Jupiter
C18, 10 × 250 mm, 70% MeCN, 3 mL/min) to obtain 6.1 mg of
In order to determine which organism was responsible for
production of palmyramide A (1), a natural product MALDI-TOF-
imaging (npMALDI-I) approach was applied to the environmental
samples stored in either EtOH/H2O (1:1) at -20 °C or RNA
stabilization solution (RNAlater) at -20 °C.9,10 In principal, the
npMALDI-I approach can visualize the spatial distribution of
secondary metabolites in intact tissues of marine organisms.
However, in this case the molecular weight of 1 was not detected
in any of the L. majuscula or Centroceras sp. samples. We reasoned
that this surprising result might be due to the instability of the
palmyramide in the preserved biological samples. However, we
were able to adapt into laboratory culture the cyanobacterial
component of this consortium, and npMALDI-I of fresh Lyngbya
filaments possessed a robust signal for a compound with the
molecular weight of 1, indicating that L. majuscula is the producer
of this new depsipeptide (Figure 3C).
Pure palmyramide A (1) was assayed for sodium channel
blocking and cancer cell cytotoxic activities (presumably due the
presence of one N-methyl amide in 1, it occurs as a 95:5 mixture
of major and minor conformers by 1H NMR; see Supporting
Information).11,12 In the sodium channel blocking assay, 1 inhibited
the veratridine- and ouabain-induced sodium overload and resulting
cytotoxicity in neuro2a cells, presumably by blocking the voltage-
gated sodium channel with an IC50 value of 17.2 µM. In H-460

396 Journal of Natural Products, 2010, Vol. 73, No. 3
Taniguchi et al.
Figure 4. Phylogenetic tree of the cyanobacterial order Oscillatoriales based on the 16S rRNA gene and placement of this field collection
of Lyngbya majuscula.
palmyramide A (1, 20.1 min) and 2.0 mg of malyngamide C (14.1
min). The fraction eluting with 40% EtOAc in hexanes was purified
by silica gel column chromatography [hexane/EtOAc (6:1)] to yield
28.6 mg of curacin D.
Chiral HPLC Analysis. Palmyramide A (0.5 mg) in 0.5 mL of 6 N
HCl was heated in a sealed tube at 110 °C for 17 h, and the reaction mixture
extracted with EtOAc. The organic layer was dried under N2, and the
residue dissolved in 200 µL of MeOH. The hydrolysate was analyzed by
chiral HPLC on a Phenomenex Chirex 3126 (4.6 × 250 mm) at a flow
rate of 0.7 mL/min with UV detection at 254 nm. The isocratic solvent
system of 2.0 mM CuSO4 in H2O was used to determine the configuration
of L-Lac (L 16.4 min, D 20.3 min). The isocratic solvent system of 2.0
mM CuSO4 in H2O/2-propanol (85:15) was used to determine the
configuration of L-Pla (L 53.2 min, D 74.1 min).
(S)-4-Benzyl-3-isobutyryl-5,5-dimethyloxazolidin-2-one (3a). Ox-
azolidinone 2a (102 mg, 0.497 mmol) was dissolved in dry THF (1.3
mL) and treated with n-BuLi (1.42 M in hexanes, 350 µL, 0.497 mmol,
1 equiv) dropwise at -78 °C. After 1 h, isobutyryl chloride (130 µL,
1.242 mmol, 2.5 equiv) in THF (0.8 mL) was added at -78 °C. The
reaction went to completion within 1 h, as monitored by TLC, and
was quenched with H2O (2.1 mL). The mixture was extracted with
Et2O (4 × 4 mL), dried over MgSO4, and concentrated in Vacuo. The
product was purified by flash chromatography on silica gel 60 (Et2O/
hexanes, 1:19), yielding the title compound as a nearly colorless oil
(132.5 mg, 97% yield): [R]D -34.0 (c 0.87, CHCl3); 1H NMR (CDCl3)
δ 1.15 (3H, d), 1.17 (3H, d), 1.36 (3H, s), 1.38 (3H, s), 2.89 (1H, dd),
3.09 (1H, dd), 3.76 (1H, m), 4.51 (1H, dd), 7.21-7.32 (5H, m); HRMS
m/z 275.1516 [M]+ (calcd for C16H21NO3, 275.1516).
Palmyramide A (1): colorless glass; [R]D +19.6 (c 0.25, CHCl3);
UV (MeOH) λmax (log ꢀ) 203 (4.27); IR (neat) νmax 3409, 2965, 2876,
1
1741, 1660, 1504, 1454, 1387, 1261, 1197, 1146, 1100 cm-1; H and
13C NMR, see Table 1; HRESIMS m/z 672.3852 [M + H]+ (calcd for
C36H54N3O9, 672.3855).
Marfey’s Analysis. Palmyramide A (0.5 mg) in 0.5 mL of 6 N HCl
was heated in a sealed tube at 110 °C for 17 h, and the reaction mixture
extracted with EtOAc. The aqueous layer was dried under N2, and the
residue dissolved in 400 µL of H2O. Subsequently, 200 µL of this
solution was treated with 40 µL of 1% DFVA solution in acetone and
8.5 µL of 1 M NaHCO3 and heated at 90 °C for 5 min. After cooling
to room temperature, 20 µL of 1 N HCl was added and the solution
was analyzed by LC/MS on a Merck LiChrospher 100 RP-18 (4 ×
125 mm) at a flow rate of 0.8 mL/min using a linear gradient from
MeCN/H2O (30:70, 0.1% HCOOH) to MeCN/H2O (70:30, 0.1%
HCOOH) over 60 min. The DFVA derivatives from the acid hydrolysate
were identified by comparing the retention times with those of authentic
standards, as follows: D-N-MeVal 20.9 min (L-N-MeVal 15.8 min) and
L-Pro 8.0 min (D-Pro 10.6 min). The configuration of Val could not be
determined in the above solvent system because L-Val and the Marfey’s
reagent coeluted. However, employing a linear gradient from MeOH/
H2O (30:70, 0.1% HCOOH) to MeOH/H2O (70:30, 0.1% HCOOH)
over 60 min cleanly resolved these peaks and identified the configuration
of Val in 1 to be L (L 34.1 min, D 51.5 min).
(R)-4-Benzyl-3-isobutyryl-5,5-dimethyloxazolidin-2-one (3b). Ox-
azolidinone 3b was prepared in the same way as oxazolidinone 3a from
the D-phenylalanine-derived oxazolidinone 2b (81% yield, due to a
1
partial, accidental loss of product): [R]D +33.6 (c 1.08, CHCl3); H

Cyclic Depsipeptide from Lyngbya majuscula
Journal of Natural Products, 2010, Vol. 73, No. 3 397
NMR (CDCl3) δ 1.16 (3H, d), 1.17 (3H, d), 1.36 (3H, s), 1.38 (3H, s),
2.89 (1H, dd), 3.09 (1H, dd), 3.76 (1H, m), 4.51 (1H, dd), 7.21-7.32
(5H, m); HRMS m/z 275.1517 [M]+ (calcd for C16H21NO3, 275.1516).
(S)-4-Benzyl-3-(3-hydroxy-2,2-dimethylhexanoyl)-5,5-dimethylox-
azolidin-2-one (4a). Oxazolidinone 3a (47.6 mg, 0.173 mmol) was
dissolved in dry THF (2 mL) and added dropwise to a solution of LDA
(1.5 equiv) in dry THF (1.76 mL) at -78 °C [prepared by addition of
1.35 M n-BuLi (192 µL, 0.259 mmol, 1.5 equiv) to a solution of
diisopropylamine (39 µL, 0.277 mmol, 1.6 equiv) in dry THF at -78
°C, the solution was warmed to 0 °C for 15 min, then cooled to -78
°C]. After 30 min, a solution of chlorotriisopropoxy titanium IV (1 M
in THF, 692 µL, 0.691 mmol, 4 equiv) was added dropwise, and the
solution was warmed to -40 °C. After 1 h, the solution was cooled to
-78 °C, butyraldehyde (46.5 µL, 0.519 mmol, 3 equiv) in THF (2
mL) was added dropwise, and the solution was warmed to -40 °C.
After 3 h, the reaction was quenched with saturated NH4Cl (4 mL)
and stirred with Celite until warmed to room temperature. The filtrate
was extracted with EtOAc (4 × 4 mL), washed with brine (4 mL),
dried over Na2SO4, and concentrated in Vacuo. The product was purified
by flash chromatography on silica gel 60 (DCM/hexanes/MeCN, 49.5:
49.5:1), yielding the title compound as a nearly colorless oil (32.3 mg,
chloride (0.015 mL, 0.078 mmol), and DMAP (2.0 mg, 0.017 mmol).
The mixture was stirred at room temperature overnight, followed by
purification on a silica gel column to yield the S-MTPA ester 6a (5.7
mg, yield 62.5%). The same procedure for the synthesis of 6a was
used for the synthesis of the R-MTPA ester 6b (2.2 mg, yield 29.6%).
The absolute configuration of oxymethine carbon of 4a was determined
to be S by ∆δ values for the protons adjacent to the MTPA esters
moieties.
1
6a: H NMR (CDCl3) δ 0.83 (3H, t), 1.14 (2H, m), 1.30 (3H, s),
1.32 (3H, s), 1.34 (3H, s), 1.42 (3H, s), 1.50 (1H, m), 1.61 (1H, m),
2.85 (1H, dd), 3.12 (1H, dd), 3.54 (3H, s), 4.42 (1H, dd), 6.15 (1H,
dd), 7.20-7.56 (10H, m); HRMS m/z 586.2389 [M + Na]+ (calcd for
C30H36 F3NO6Na, 586.2387)
1
6b: H NMR (CDCl3) δ 0.88 (3H, t), 1.26 (2H, m), 1.28 (3H, s),
1.30 (3H, s), 1.33 (3H, s), 1.38 (3H, s), 1.48 (1H, m), 1.56 (1H, m),
2.82 (1H, dd), 3.04 (1H, dd), 3.55 (3H, s), 4.44 (1H, dd), 6.36 (1H,
dd), 7.20-7.56 (10H, m); HRMS m/z 586.2385 [M + Na]+ (calcd for
C30H36 F3NO6Na, 586.2387).
Morphological Characterization. Taxonomic identification of the
cyanobacterium was performed in accordance with bacteriological
systems14 and current phycological systems.15 Morphological charac-
terizations were performed using an Olympus IX51 epifluorescent
microscope (100× objective) equipped with an Olympus U-CMAD3
camera. Size measurements were calculated as an average with standard
deviation of 10 neighboring cells from three different filaments of a
population.
1
54% yield): [R]D -33.0 (c 0.48, CHCl3); H NMR (CDCl3) δ 0.94
(3H, t), 1.28 (2H, m), 1.33 (3H, s), 1.34 (3H, s), 1.35 (3H, s), 1.37
(3H, s), 1.60 (2H, m), 2.39 (1H, brd), 2.90 (1H, dd), 3.10 (1H, dd),
4.11 (1H, m), 4.56 (1H, dd), 7.20 - 7.35 (5H, m); HRMS m/z 370.1992
[M + Na]+ (calcd for C20H29NO4Na, 370.1989).
DNA Extraction, PCR-Amplification, and Cloning. DNA was
extracted from approximately 40 mg of cleaned cyanobacterial filaments
using the Wizard Genomic DNA purification kit (Promega Inc.)
following the manufacturer’s specifications. The isolated DNA was
further purified using a Genomic-tip 20/G kit (Qiagen Inc.). DNA
concentration and purity were measured on a DU 800 spectrophotometer
(Beckman Coulter Inc.). The 16S rRNA genes were PCR-amplified
using the cyanobacterial-specific primers 106F and 1509R,16 the rpoC1
genes using the degenerate primers LrpoC1-F (5′-CYTGYTTNC-
CYTCDATDATRTC-3′) and LrpoC1-R (5′-YTNAARCCNGARATG-
GAYGG-3′), and the 18S rRNA gene using the universal primers U1F
and U1R, as previously described.17 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 of dNTP mix (25 mM each of dATP, dTTP,
dGTP, and dCTP), 0.5 µL of each primer (10 µM), 0.5 µL of PfuUltra
IV fusion HS DNA polymerase, and 20.5 µL of dH2O. 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
analyzed on a (1%) agarose gel in SB buffer and visualized by EtBr
staining. PCR products were subcloned using the Zero Blunt TOPO
PCR cloning kit (Invitrogen) into the pCR-Blunt IV TOPO vector and
then transformed into TOPO cells and cultured on LB-kanamycin plates.
Plasmid DNA was isolated using the QIAprep Spin miniprep kit
(Qiagen) and sequenced with pCR-Blunt IV TOPO vector specific
primers M13F and M13R. Sequencing of the 16S rRNA genes’ middle
regions was improved using the internal primers 359F and 781R.16
Gene sequences were analyzed for anomalies using the Pintail software
with the cutoff size set at >600 bp before submission to GenBank/
EMBL/DDBJ.18 The gene sequences are available in the GenBank/
EMBL/DDBJ databases under accession numbers L. majuscula 16S
rRNA gene (GQ231522), L. majuscula rpoC1 (GQ231521), and the
Centroceras sp. 18S rRNA gene (GQ246180).
(R)-4-Benzyl-3-(3-hydroxy-2,2-dimethylhexanoyl)-5,5-dimethy-
loxazolidin-2-one (4b). This was prepared in the same way as 4a from
3b (35% yield): [R]D +32.4 (c 0.69, CHCl3); 1H NMR (CDCl3) δ 0.94
(3H, t), 1.28 (2H, m), 1.33 (3H, s), 1.34 (3H, s), 1.35 (3H, s), 1.37
(3H, s), 1.60 (2H, m), 2.39 (1H, brd), 2.90 (1H, dd), 3.10 (1H, dd),
4.11 (1H, m), 4.56 (1H, dd), 7.20-7.35 (5H, m); HRMS m/z 370.1991
[M + Na]+ (calcd for C20H29NO4Na, 370.1989).
(S)-3-Hydroxy-2,2-dimethylhexanoic Acid (5a). A hydrogen per-
oxide solution (30%, 13 µL, 0.115 mmol, 3.6 equiv) and lithium
hydroxide monohydrate (2.1 mg, 0.051 mmol, 1.6 equiv), dissolved in
H2O (61 µL), were added successively to a solution of 4a (11 mg,
0.031 mmol) in 4:1 THF/H2O (275 µL) at 0 °C. After stirring 1 h, 20
min at 0 °C, sodium sulfite (16.6 mg, 0.132 mmol, 4.2 equiv) was
added. THF was removed in Vacuo, and the residual aqueous solution
was partitioned between CH2Cl2 (3 × 1 mL) and H2O. The combined
aqueous layers were acidified to pH 1 with 1 N HCl. The aqueous
layer was extracted with Et2O (3 × 1 mL), dried over MgSO4, filtered,
and concentrated in Vacuo, yielding the title compound as a colorless
oil (3.8 mg, 75% yield): [R]D -35.5 (c 0.24, CHCl3); 1H NMR (CDCl3)
δ 0.95 (3H, t), 1.20 (3H, s), 1.25 (3H, s), 1.25-1.70 (4H, m), 3.66
(1H, dd); HRMS m/z 183.0995 [M + Na]+ (calcd for C8H16O3Na,
183.0992).
(R)-3-Hydroxy-2,2-dimethylhexanoic acid (5b). This was prepared
in the same way as 5a from 4b (69% yield): [R]D +26.5 (c 0.28,
1
CHCl3); H NMR (CDCl3) δ 0.95 (3H, t), 1.20 (3H, s), 1.25 (3H, s),
1.25-1.70 (4H, m), 3.66 (1H, dd); HRMS m/z 183.0994 [M + Na]+
(calcd for C8H16O3Na, 183.0992).
Chiral GC/MS Analysis. Palmyramide A (0.5 mg) in 0.5 mL of 6
N HCl was heated in a sealed tube at 110 °C for 17 h, and the reaction
mixture extracted with EtOAc. The organic layer was dried under N2,
and half of the residue was dissolved in 600 µL of 1:1 Et2O/MeOH
and treated with diazomethane for 25 min. Solvent and excess
diazomethane were removed with N2 gas, and the residue was
resuspended in CH2Cl2. Standards of the R-Dhhma and S-Dhhma methyl
esters were prepared similarly. Capillary GC/MS analysis was conducted
using a Cyclosil B column (Agilent Technologies J&W Scientific, 30 m
× 0.25 mm) under the following conditions: the initial oven temperature
was 40 °C, followed by an immediate ramp from 40 to 90 °C at a rate
of 5 °C/min, and held at 90 °C for 55 min. The Dhhma methyl ester
derived from 1 eluted at 42.00 min, while the R-Dhhma methyl ester
eluted at 41.90 min. The S-Dhhma methyl ester eluted at 39.82 min.
Co-injection of the Dhhma methyl ester derived from compound 1 with
the R-Dhhma methyl ester yielded a single peak at 41.95 min, while
co-injection with the S-Dhhma methyl ester yielded a peak at 39.87
min and a peak at 42.00 min, confirming that the ꢀ-ester linkage of
the Dhhma residue of 1 is R.
Phylogenetic Analysis. The gene sequences were aligned in
ClustalW XXL in MEGA 4.0 with standard gap opening and extension
penalties without gaps.19 The evolutionary histories of the cyanobac-
terial 16S rRNA genes were inferred using the Minimum Evolution
(ME) algorithm in MEGA 4.0 as well as the Bayesian (MrBayes
algorithm) method using TOPALi v2.5.20 All algorithms were per-
formed with 1000 bootstrap replicates. The evolutionary distances
(pairwise sequence divergence) were computed using the Maximum
Composite Likelihood method. The ME tree was searched using the
Close-Neighbor-Interchange (CNI) algorithm at a search level of 1.21
All positions containing gaps and missing data were eliminated from
the data set (complete deletion option) for a total of 855 bp.
Culture. Specimens for culturing were isolated under microscopy
and cleaned by running individual filaments through 1% agar SWBG-
11 plates. The isolated specimens were cultured in SWBG-11 medium
Mosher’s Method. To a solution of 4a (5.4 mg, 0.016 mmol) in
CH2Cl2 (1 mL) were added Et3N (0.010 mL, 0.078 mmol), R-MTPA

398 Journal of Natural Products, 2010, Vol. 73, No. 3
Taniguchi et al.
at 28 °C with 35 g/L Instant Ocean (Aquarium Systems Inc.). The
cultures were kept in a 16 h light/8 h dark cycle with a light intensity
of ∼7 µmol photon/s/m2 provided by 40 W cool white fluorescent lights.
Filament Sample Preparation. Filaments were removed from the
parent culture using small, sterile, blunt-tip tweezers and transferred
to a Petri dish containing a small amount of distilled H2O in order to
remove excess salt and media; this process was repeated. Using the
same tweezers, the filament was carefully removed and laid horizontally
on a Bruker MSP 96 stainless steel microflex target plate. Excess liquid
on the surface was carefully blotted using the corner of a Kimwipe.
The plate and filament were allowed to dry at room temperature until
visibly dry.
104 cells/mL in 180 µL of RPMI medium with FBS. After 16 h, the
test compounds were dissolved in DMSO and diluted into medium
without FBS and then added to the wells. After 48 h, cell viability was
determined by the MTT assay.
Acknowledgment. We thank R. C. Coates for help with the
collection of the organisms, J. Smith for identification of the red alga,
Y. Su for accurate mass measurement, and T. L. Suyama and A. Pereira
for helpful discussions. Financial support for this work was provided
by NS053398.
1
Supporting Information Available: H NMR, 13C NMR, COSY,
Image Acquisition. After the filament had dried on the target plate,
a photograph (Nikon Coolpix, 3 megapixel) of the plate was taken to
use as spatial teach reference for the Bruker Microflex MALDI
instrument. Next, epifluorescent images of the length of the filament
were acquired at 4× magnification using an Olympus IX511 microscope
with an excitation filter of 590 nm. The resulting images were tiled
together using Photoshop CS to create a single, epifluorescent image
of the filament.
MALDI Matrix Deposition. After image acquistion, a MALDI
matrix composed of 35 mg/mL R-cyanohydroxycinnamic acid, 35 mg/
mL DHB, 75% MeCN, and 0.2% TFA was coated onto the MALDI
MSP 96 plate using a Paasche airbrush (www.paascheairbrush.com)
and repeated side-to-side strokes until an even, thin crystalline layer
occluded the background of the plate. The target plate containing the
sample and matrix was placed in an empty Petri dish until analysis.
MALDI MS and Imaging. The target plate containing the sample
was inserted into a Microflex Bruker Daltonics mass spectrometer
outfitted with the Compass 1.2 software suite (consists of FlexImaging
2.0, FlexControl 3.0, and FlexAnalysis 3.0). The sample was run in
positive mode, with 80 µm raster intervals in X and Y and 20-25%
absolute laser power. Briefly, a photomicrograph of the sample to be
imaged by mass spectrometry was loaded onto the Fleximaging
command window. Three teach points were selected in order to align
the background image with the sample target plate. After the target
plate calibration was complete, the AutoXecute command was used to
run the sample. The settings under the FlexControl panel were used as
previously described.10
Image Visualization and Analysis. Using the Bruker FlexImaging
2.0 software, a window containing the m/z values corresponding to the
palmyramide A (1) isotope cluster was selected and assigned a bright
green color. It was clear that the spatial distribution of the specified
mass window colocalized precisely with the filament contained in the
teach image. These visual results were exported and overlaid on the
epifluorescent images of the filament acquired before matrix application
using Photoshop CS.
Biological Activity. Sodium channel blocking activity was measured
as previously described.11 Neuro2a mouse neuroblastoma cells were
seeded in 96-well plates at 3.0 × 105 cells/mL in 200 µL of RPMI
medium with FBS. After 16 h, the test compounds were dissolved in
DMSO and diluted into medium without FBS and then added to the
wells. A solution of 10 mM ouabain and 1 mM veratridine was applied
to wells to cause sodium overload cytotoxicity. After 24 h, cell viability
was determined by the MTT assay and sodium channel blocking activity
was calculated as percentage of recovery from the toxicity of ouabain
and veratridine.
TOCSY, HSQC, and HMBC spectra in CDCl3 for palmyramide A (1).
This material is available free of charge via the Internet at http://
pubs.acs.org.
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Cytotoxicity was measured in NCI H-460 human lung tumor cells
using the MTT assay.12 Cells were seeded in 96-well plates at 3.3 ×
NP900428H