Dragonamides C and D, Linear Lipopeptides from the Marine Cyanobacterium Brown Lyngbya
polychroa
Sarath P. Gunasekera,? Cliff Ross,?,? Valerie J. Paul,*,? Susan Matthew,? and Hendrik Luesch?
Smithsonian Marine Station, 701 Seaway DriVe, Fort Pierce, Florida 34949, and Department of Medicinal Chemistry, UniVersity of Florida,
1600 SW Archer Road, GainesVille, Florida 32610
ReceiVed NoVember 27, 2007
Two new linear lipopeptides, 1 and 2, and a known compound, curacin D, have been isolated from a marine
cyanobacterium, brown Lyngbya polychroa, collected from Hollywood Beach, Fort Lauderdale, Florida. Their planar
structures were elucidated by 1D and 2D NMR techniques, and absolute configurations were assigned using chiral
HPLC. The new compounds were assigned the trivial names dragonamide C (1) and dragonamide D (2), as their peptide
moiety is related to previously reported dragonamides A and B.
Marine cyanobacteria, or blue-green algae, have proven to be a
rich source of novel biologically active secondary metabolites, in
particular small modified peptides. Several recent review articles
and related publications have shown that the genus Lyngbya has
yielded an impressive array of structurally diverse secondary
metabolites.1 As part of our ongoing efforts to discover cytotoxic
compounds produced by marine cyanobacteria around the south
Florida coast, we have collected a sample of brown Lyngbya
polychroa from Hollywood Beach, Fort Lauderdale, Florida. The
lipophilic extract of this cyanobacterium led us to identify two new
lipopeptides, dragonamides C (1) and D (2), and the antimitotic
agent curacin D, which was previously reported from the marine
cyanobacterium L. majuscula.2 Several linear and cyclic depsipep-
tides of 7-octynoic acid and linear depsipeptides of 7-octenoic acid
have been reported from L. majuscula.3?6 Structurally similar C8-
alkynoate units have been reported from Symploca laete-Viridis7
and marine mollusks.8 Dragonamides C (1) and D (2) represent
two new linear tetrapeptide-octynoates.3
The wet sample collected in October 2006 was stored frozen
until freeze-drying. The freeze-dried material was extracted first
with EtOAc-MeOH (1:1) and then with EtOH-H2O (1:1). The
EtOAc-MeOH-soluble fraction was partitioned between EtOAc
and H2O. The EtOAc-soluble fraction was further separated by
repeated column chromatography to give 94 mg of curacin D as a
pale yellow oil and a peptide-enriched fraction, which, after HPLC,
yielded purified dragonamide C (1) and dragonamide D (2).
Dragonamide C (1) was obtained as a colorless, amorphous
solid. HRESI/APCIMS supported the molecular formula of
C33H57N5O6 [(M + Na)+ m/z at 642.4223]. A strong IR
absorption at 1636 cm-1 implied the presence of amide func-
tionalities. The 1H and 13C NMR spectra indicated the presence
of a minor conformer (17% in CD3OD and CDCl3). However,
NMR analysis of the major conformer was not obstructed. The
1H NMR spectrum (Table 1) for 1 revealed the presence of four
singlets corresponding to N-methyl amide substituents (? 3.11,
3.05, 3.04, 2.97) and another low-field singlet for an O-methyl
substituent (? 3.66), eight high-field methyl doublets (? 0.96,
0.91, 0.90, 0.87, 0.85, 0.79, 0.77, 0.76), two high-field multiplets
(? 2.33 for 3H), and four low-field doublets for four R-protons
of amino acid residues (? 5.19, 5.18, 5.17, 4.65). In addition,
the 1H NMR spectrum indicated an olefinic singlet (? 5.33), three
methylene multiplets (? 2.70-2.15), and a characteristic triplet
for a terminal acetylenic proton [? 2.20 (J ) 2.7 Hz)].
Examination of the 13C NMR (Table 1) and multiplicity-edited
HSQC spectra revealed quaternary and methine 13C signals at ?
84.5 and 69.8, respectively, consistent with the terminal acety-
lenic group,9 and six putative carbonyl signals at ? 174.2, 173.6,
172.7, 172.6, 172.0, and 170.6. Examination of the HMBC data
(Table 1) connected four of these carbonyl signals (? 174.2,
172.7, 172.6, and 170.6) to the four N-methyl amide groups in
the molecule. Corroborated by HMBC data (Table 1), the
putative carbonyl at ? 173.6 belonged to a conjugated enol
methyl ether group along with the methine 13C NMR signals at
? 92.2.10 These data accounted for the eight degrees of
unsaturation inherent in the molecular formula. Analysis of the
2D DQF COSY and edited HSQC spectra revealed the presence
of four isopropyl spin systems for four N-Me valine residues,
and the order of these residues was assigned by analysis of the
HMBC data (Table 1). The presence of a free amide group at
* To whom correspondence should be addressed. Tel.: (772) 472-0982.
Fax: (772) 461-8154. E-mail: paul@si.edu.
? Smithsonian Marine Station.
? University of Florida.
? Current address: Department of Biology, University of North Florida,
Jacksonville, FL 32224.
J. Nat. Prod. 2008, 71, 887?890 887
10.1021/np0706769 CCC: $40.75 ? 2008 American Chemical Society and American Society of Pharmacognosy
Published on Web 04/05/2008
the C-terminal end was evident from the presence of two
exchangeable singlets (? 5.02 and 6.02 in CDCl3), which are
mutually coupled and showed long-range correlations to the
C-terminal amide carbonyl group (? 171.5 in CDCl3, Table 1).
Further analysis of the 2D DQF COSY and edited HSQC spectra
indicated that the terminal acetylenic proton (H-37) and the three
sequentially coupled methylene groups (H2-33 to H2-35) con-
stitute a H33 to H37 spin system, as evidenced by a long-range
coupling between H2-35 and H-37 (J ) 2.7 Hz). The HMBC
spectrum exhibited three-bond coupling between H2-35 (? 2.15)/
terminal C-37 (? 69.8) and methylene C-33 (? 32.3), H2-34 (?
1.74)/C-36 acetylenic qC (? 84.5) and C-32 olefinic qC (? 173.6),
H2-33 (? 2.70, 2.66)/C-35 (? 18.9) and olefinic C-31 (? 92.2),
H3-38 (O-Me, ? 3.66)/C-32 olefinic qC (? 173.6), H-31 (? 5.33)/
C-33 (? 32.3), and two-bond coupling between H-31 (? 5.33)/
C-32 (? 173.6) and C-30 amide carbonyl group (? 172.0).
Combination of the above data established the connectivity
between C-30 and C-38 and thus confirmed the structure of the
terminal fatty acid moiety as 3-methoxy-2-en-7-octynoic acid.
Further, the HMBC spectrum exhibited three-bond coupling
between H3-29 (N-Me Val-4, ? 3.05)/C-30 amide carbonyl signal
(? 172.0) and thus established the connectivity between the N-Me
Val-4 moiety and the terminal acid group in the molecule. The
stereochemistry of the C-31 double bond was assigned on the
basis of correlations seen in the NOESY spectrum. Strong
correlation between H-31 and O-Me-38, but not between H-31
and H2-33, assigned an E geometry to the C-31 double bond.
Dragonamide D (2) was obtained as a colorless solid. HRESI/
APCIMS supported the molecular formula of C32H55N5O6 [(M
+ Na)+ m/z at 628.4067]. A strong IR absorption at 1631 cm-1
indicated the presence of amide functionalities. The 1H and 13C
NMR spectra indicated the presence of a minor conformer (8%
in CD3OD and 12% CDCl3). The 1H NMR spectrum of the major
conformer (Table 1) was similar to the 1H NMR spectrum for
1, including the presence of four tertiary N-methyl amides,
methyl doublets suggestive of valine isopropyl groups, and the
characteristic terminal acetylenic signal; however, an O-methyl
singlet (? 3.66) was absent (Table 1). In addition, the 1H NMR
spectrum for 2 showed a low-field methylene AB quartet (? 3.85,
3.67, J ) -16.5 Hz) instead of the olefinic proton and methoxy
singlet observed in dragonamide C (1). The presence of a
carbonyl signal at ? 205.5 in addition to the five amide carbonyl
peaks in the 13C NMR spectrum for 2 and the above 1H NMR
data suggested a possible keto?enol relationship between
compounds 1 and 2. HMBC analysis (Table 1) established the
connectivity between C-30 and C-37 and thus established the
structure of the terminal fatty acid moiety as 3-keto-7-octynoic
acid. Further 2D DQF COSY, and HMBC analyses confirmed
the connectivity of all amino acid residues in the molecule.
Another batch of the same brown Lyngbya polychroa was
Table 1. NMR Spectroscopic Data (600 MHz, CD3OD) for Dragonamides C (1) and D (2)
dragonamide C (1) dragonamide D (2)
unit position ?C mult. ?H (J in Hz) HMBCa ?C mult ?H (J in Hz) HMBCa
NH2b 1 N 5.02, s 2b N 5.28, s
6.06, s 6.05, s
N-Me Val-1 2 174.2, qC 174.2, qC
2b 171.5, qC
3 63.1, CH 4.65, d (10.2) 2, 5, 6, 7, 8 63.1, CH 4.65, d (10.9) 2, 5, 8
4 N
5 27.4, CH 2.21, m 6, 7 27.4, CH 2.21, m 2
6 18.8, CH3 0.76, d (6.5) 3 18.8, CH3 0.76, d (6.9) 3
7 19.7, CH3 0.96, d (6.5) 3 19.7, CH3 0.96, d (6.9) 3
8 31.4, CH3 3.11, s 3, 9 31.4, CH3 3.11, s 3, 9
N-Me Val-2 9 172.6 qC 172.7, qC
10 59.7, CH 5.19, d (10.8) 9, 13, 14, 15 59.7, CH 5.19, d (10.9) 9, 12, 15
11 N N
12 28.8, CH 2.33, m 28.8, CH 2.34, m 9
13 18.4, CH3 0.79, d (6.4) 10 18.5, CH3 0.78, d (6.9) 10
14 19.6, CH3 0.91, d (6.4) 10 19.6, CH3 0.91, d (6.5) 10
15 32.1, CH3 3.04, s 10, 16 31.2, CH3 3.04, s 10, 16
N-Me Val-3 16 172.7, qC 172.1, qC
17 59.7, CH 5.17, d (10.2) 16, 19, 22 59.9, CH 5.18, d (10.9) 16, 19, 22
18 N N
19 28.5, CH 2.33, m 28.5, CH 2.35, m 16
20 18.6, CH3 0.85, d (6.7) 17 18.4, CH3 0.87, d (6.7) 17
21 18.6, CH3 0.77, d (6.7) 17 18.4, CH3 0.82, d (6.8) 17
22 31.6, CH3 2.97, s 17, 23 31.1, CH3 3.04, s 17, 23
N-Me Val-4 23 170.6, qC 171.8, qC
24 59.9, CH 5.18, d (10.8) 23, 27, 28, 29 60.0, CH 5.13, d (10.9) 23, 26, 29
25 N N
26 28.5, CH 2.33, m 28.8, CH 2.31, m
27 19.9, CH3 0.87, d (6.4) 24 19.9, CH3 0.87, d (6.5) 24
28 20.0, CH3 0.90, d (6.5) 24 20.0, CH3 0.88, d (6.5) 24
29 31.3, CH3 3.05, s 24, 30 31.5, CH3 2.88, s 24, 30
MeO-Oya-2-ene 30 172.0, qC 170.5, qC
31 92.2, CH 5.33, s 30, 32, 33 49.6, CH2 3.85, d (-16.5) 30, 32
3.67, d (-16.5)
32 173.6, qC 205.5, qC
33 32.3, CH2 2.70, m 31, 32, 34, 35 42.4, CH2 2.69, m 32, 34, 35
2.66, m 31, 32, 34, 35
34 27.9, CH2 1.74, m 32, 33, 35, 36 23.4, CH2 1.75, m 32, 33, 35, 36
35 18.9, CH2 2.15, dt (2.7, 7.5) 33, 34, 36, 37 18.3, CH2 2.20, dt (2.7, 7.5) 33, 34, 36, 37
36 84.5, qC 84.1, qC
37 69.8, CH 2.20, t (2.7) 70.3, CH 2.24, t (2.7)
38 55.9, CH3 3.66, s 31, 32
a HMBC correlations, optimized for 2/3JCH ) 8 Hz, are from proton(s) stated to the indicated carbon. b Data recorded in CDCl3.
888 Journal of Natural Products, 2008, Vol. 71, No. 5 Notes
extracted with EtOAc. 1H NMR analysis of this extract in CDCl3
indicated the presence of the methoxy and other N-Me signals
corresponding to dragonamide C (1). Further, we have noticed
that dragonamide C (1) in CDCl3 was stable for a long period
of time, suggesting that dragonamide D (2) is probably not an
acid-catalyzed artifact formed during MeOH extractions.
The configuration of the N-Me Val residues in 1 and 2 was
assigned by chiral HPLC analysis, comparing the amino acid content
in the acid hydrolysates with N-Me D- and L-valine standards. Their
retention times established an L-configuration for all N-Me Val
residues in 1 and 2.3
1H and 13C NMR spectroscopic data of our isolated curacin D
were identical to those data reported2 for curacin D, previously
isolated from the cyanobacterium Lyngbya majuscula collected from
St. Croix in the U.S. Virgin Islands. The observed specific rotation
value for our isolated curacin D ([R]D +34) closely matched that
reported2 in the literature for curacin D ([R]D +33), indicating that
both compounds have the same absolute configuration, although
not all stereocenters are determined yet. These data confirmed the
structure of the isolate as curacin D. No other reported curacins
were found in this collection.
Dragonamides C (1) and D (2) were tested for biological
activity in cancer cell viability assays. Compounds 1 and 2
showed weak activity, with GI50 values of 56 and 59 ?M against
U2OS osteosarcoma cells, 22 and 32 ?M against HT29 colon
adenocarcinoma cells, and 49 and 51 ?M against IMR-32
neuroblastoma cells, respectively. These data are similar to
cytotoxicity data reported for dragonamides A and B against
other cell lines.3
Experimental Section
General Experimental Procedures. Optical rotations were recorded
on a Jasco DIP-370 digital polarimeter. IR spectra were obtained on a
Bruker Vector 22 FT-IR spectrometer. NMR data were collected on a
JEOL ECA-600 spectrometer operating at 600.17 MHz for 1H and 150.9
MHz for 13C. The edited-gHSQC experiments were optimized for JCH
) 140 Hz, and the gHMBC spectra were optimized for 2/3JCH ) 8 Hz.
1H NMR chemical shifts (referenced to residual CHCl3 observed at ?
7.24 and residual CH3OH observed at ? 3.30) were assigned using a
combination of data from 2D DQF COSY and gHMQC experiments.
Similarly, 13C NMR chemical shifts (referenced to CDCl3 observed at
? 77.0 and CD3OD observed at ? 49.0) were assigned on the basis of
multiplicity-edited HSQC experiments. The LRESIMS was obtained
on a Finnigan LTQ LC-MS with an electrospray ionization detector.
The HRMS data were obtained using an Agilent LC-TOF mass
spectrometer equipped with an APCI/ESI multimode ion source detector
at the Mass Spectrometer Facility at the University of California,
Riverside, CA.
Collection, Extraction, and Isolation. The sample of brown
Lyngbya polychroa cyanobacterium was collected in October 2006
from Hollywood Beach, Fort Lauderdale, FL. The sample was
identified by one of us (V.J.P.), and a voucher specimen is
maintained at the Smithsonian Marine Station, Fort Pierce, FL. This
specimen displayed an average cell width of 36.9 ?m, cell length
of 12.6 ?m, and sheath width of 2.8 ?m. The freeze-dried material
(65 g) was first extracted with EtOAc-MeOH (1:1) and then with
EtOH-H2O (1:1). Concentration of the extracts by rotary evapora-
tion at 45 ?C under reduced pressure furnished 6.8 g (10.5% yield)
of the organic extract and 7.5 g of a polar extract (11.6% yield).
The EtOAc-MeOH-soluble fraction was partitioned between EtOAc
and H2O. The H2O-soluble fraction was further partitioned between
n-BuOH and H2O. Concentration of these extracts furnished 1.7 g
of an EtOAc-soluble fraction, 0.8 g of an n-BuOH-soluble fraction,
and 3.9 g of a H2O-soluble material. The EtOAc-soluble fraction
(1.7 g) was chromatographed on a column of Si gel. The column
was prepared in a mixture of hexanes-EtOAc (1:1) and eluted with
a hexanes-EtOAc-MeOH step gradient system to give 10 fractions.
Fraction 2, which eluted with hexanes-EtOAc (1:1), was rechro-
matographed on a column of Si gel using hexanes followed by 10%
EtOAc-hexanes to give three subfractions. Subfractions 2 and 3
were combined and rechromatographed on a column of Si gel with
hexanes followed by 5% EtOAc-hexanes to give 94 mg of curacin
D (yield, 0.14% dry wt) as a pale yellow oil. Fraction 6, which
eluted with 50% EtOAc-hexanes, was further separated by reversed-
phase HPLC (semipreparative, 5 ?m, RP-18) using a 20% H2O
-MeOH mixture to give 25 mg of dragonamide C (1, yield, 0.038%
dry wt) and 4 mg of dragonamide D (2, yield, 0.006% dry wt).
Dragonamide C (1): colorless, amorphous solid; [R]25D -226 (c
1.66, MeOH); IR (KBr film) ?max 2924, 2940, 1685, 1636, 1458, 1399,
1257, 1238, 1000 cm-1; 1H and 13C NMR data, see Table 1, assignments
were made by interpretation of 2D DQF COSY, HSQC, and HMBC
data; HRESI/APCIMS m/z 642.4223 [M + Na]+ (calcd for
C33H57N5O6Na, 642.4207).
Dragonamide D (2): colorless solid; [R]25D -250 (c 0.26, MeOH);
IR (KBr film) ?max 2925, 2940, 2930, 1690, 1631, 1467, 1420, 1388,
301, 1259, 1098 cm-1; 1H and 13C NMR data, see Table 1, assignments
were made by interpretation of 2D-DQF-COSY, HSQC, and HMBC
data; HRESI/APCIMS m/z 628.4067 [M + Na]+ (calcd for
C32H55N5O6Na, 628.4050).
Curacin D: pale yellow oil; [R]25D +34 (c 0.17, CHCl3) [lit.2 [R]D
+33 (c 0.14, CHCl3)]; 1H and 13C NMR data are identical with those
reported2 in the literature; LRESIMS (positive ion) m/z 260.4 [M +
H]+.
Absolute Configurations of the Peptide Portions of Compounds
1 and 2. Each of compounds 1 and 2 (0.1 mg) was dissolved in 0.3
mL of 6 N HCl and heated in a sealed tube at 115 ?C for 18 h. The
product mixtures were dried, and each hydrolysate was reconstituted
in 0.2 mL of H2O and analyzed by chiral HPLC, comparing the
retention times with those of authentic standards [Phenomenex
Chirex (D) penicillamine, 4.6 ? 250 mm, 5 ?m]; solvent 2.0 mM
CuSO4-MeCN (95:5); detection 254 nm. The retention times (tR
min) for authentic standards were N-Me-L-Val (12.8) and N-Me-D-
Val (17.5). The hydrolysates of 1 and 2 showed peaks at 12.8 min,
but not at 17.5 min, indicating the presence of only N-Me-L-Val in
both compounds.3
Cell Viability Assays. Cells were plated in 96-well plates (U2OS,
5000 cells; HT29, 10 000 cells; IMR-32, 30 000 cells) and 24 h later
treated with various concentrations of dragonamides C and D or solvent
control (1% EtOH). After 48 h of incubation, cell viability was
measured using MTT according to manufacturer?s instructions (Prome-
ga).
Acknowledgment. This article was developed under the auspices
of the Florida Sea Grant College Program with support from NOAA,
Office of Sea Grant, U.S. Department of Commerce, Grant No.
NA06OAR4170014. We thank the Harbor Branch Oceanographic
Institution spectroscopy facility for 600 MHz NMR spectrometer time,
low-resolution MS, and optical rotation measurements. We also thank
K. Arthur for providing the cell dimensions of the specimen. The high-
resolution mass spectrometric analyses were performed by the UCR
mass spectrometer facility, Department Chemistry, University of
California at Riverside. This is contribution number 725 from the
Smithsonian Marine Station at Fort Pierce.
Supporting Information Available: 1H and 13C NMR spectra in
CD3OD for dragonamides C (1) and D (2) and 2D-NOESY spectrum
in CD3OD for dragonamide C (1). 1H and 13C NMR spectra of curacin
D in C6D6. This material is available free of charge via the Internet at
http://pubs.acs.org.
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NP0706769
890 Journal of Natural Products, 2008, Vol. 71, No. 5 Notes