Lipopeptides from the tropical marine cyanobacterium symploca sp.

Article abstract of DOI:10.1021/np401051z

A collection of the tropical marine cyanobacterium Symploca sp., collected near Kimbe Bay, Papua New Guinea, previously yielded several new metabolites including kimbeamides A-C, kimbelactone A, and tasihalide C. Investigations into a more polar cytotoxic fraction yielded three new lipopeptides, tasiamides C-E (1-3). The planar structures were deduced by 2D NMR spectroscopy and tandem mass spectrometry, and their absolute configurations were determined by a combination of Marfeys and chiral-phase GC-MS analysis. These new metabolites are similar to several previously isolated compounds, including tasiamide (4), grassystatins (5, 6), and symplocin A, all of which were isolated from similar filamentous marine cyanobacteria.

Full text of DOI:10.1021/np401051z

Article
pubs.acs.org/jnp
Lipopeptides from the Tropical Marine Cyanobacterium Symploca sp.
Emily Mevers,†,‡ F. P. Jake Haeckl,‡ Paul D. Boudreau,† Tara Byrum,† Pieter C. Dorrestein,‡,§
Frederick A. Valeriote,⊥ and William H. Gerwick*,†,§
†Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California San Diego, La
Jolla, California 92093, United States
‡Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, California 92093, United States
§Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, California 92093, United
States
⊥Division of Hematology and Oncology, Department of Internal Medicine, Henry Ford Hospital, Detroit, Michigan 48202, United
States
S
* Supporting Information
ABSTRACT: A collection of the tropical marine cyanobacte-
rium Symploca sp., collected near Kimbe Bay, Papua New
Guinea, previously yielded several new metabolites including
kimbeamides A−C, kimbelactone A, and tasihalide C.
Investigations into a more polar cytotoxic fraction yielded
three new lipopeptides, tasiamides C−E (1−3). The planar
structures were deduced by 2D NMR spectroscopy and
tandem mass spectrometry, and their absolute configurations
were determined by a combination of Marfey’s and chiral-
phase GC-MS analysis. These new metabolites are similar to
several previously isolated compounds, including tasiamide
(4), grassystatins (5, 6), and symplocin A, all of which were
isolated from similar filamentous marine cyanobacteria.
cytotoxicity and found to be inactive. Both the planar and
absolute configurations of these metabolites were determined
and have led to some intriguing insights into the biosynthetic
capability of this particular collection.
ecent sequencing efforts of marine cyanobacterial
Rgenomes have revealed their exceptional capacity to
produce a large diversity of intriguing secondary metabolites,
representing a range of distinct and unrelated biosynthetic
pathways.1,2 This has also been observed from chemical
investigations of such cyanobacterial species as Moorea
bouillonii and Moorea producens, formerly known as Lyngbya
bouillonii and Lyngbya majuscula, respectively.3 Both of these
latter organisms are known to produce a plethora of
metabolites, including the lyngbyabellins,4 apratoxins,5 laingo-
lide,6 lyngbyaloside,7 apramide A,8 and palau’imide9 from M.
bouillonii and lyngbyatoxin A,10 the jamaicamides,11 carmabin,12
various malyngamides,13 and barbamide14 from M. producens.
These examples of highly productive strains and improved
genomic insights into cyanobacterial biosynthetic capacity
demonstrate the utility of rigorously examining collections of
cyanobacteria for novel natural products, even when a given
strain has already been extensively investigated.
In the present investigation into a filamentous tuft-forming
cyanobacterium from Kimbe Bay, Papua New Guinea, which
already yielded kimbeamides A−C, kimbealactone A, and
tasihalide C,15 another chromatography fraction exhibited
strong cytotoxicity against several cancer cell lines and was
thus chosen for further evaluation. A subsequent NMR-guided
fractionation process yielded three new lipopeptides, tasiamides
C−E (1−3), two (C and D) of which were evaluated for
RESULTS AND DISCUSSION
■
Orange tufts of a tropical marine Symploca sp. were collected in
approximately 20 m of water near Kimbe Bay, Papua New
Guinea, in July 2007. The preserved collection was repetitively
Received: December 13, 2013
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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extracted (2:1 CH2Cl2/MeOH) and fractionated using normal-
phase vacuum liquid chromatography (VLC). Previously, a
middle polarity fraction (60% hexanes/40% EtOAc) of this
extract yielded several biologically active metabolites, including
kimbeamides A−C, kimbelactone A, and tasihalide C.15
Additionally, a relatively polar fraction eluting with 25%
MeOH/EtOAc exhibited cytotoxic activity against H-460
human lung cancer cells (81% toxicity at 3 μg/mL). Further
chromatography of this fraction using normal-phase solid-phase
extraction (SPE) and reversed-phase HPLC yielded 1.9 mg of
tasiamide C (1), 2.5 mg of tasiamide D (2), and 0.7 mg of
tasiamide E (3).
HR-ESITOFMS of 1 yielded an [M + Na]+ at m/z 839.4541,
giving a molecular formula of C41H64N6O11, with 13 degrees of
unsaturation. The IR spectrum featured absorptions indicative
of the presence of NH or OH protons and the presence of
amide or ester carbonyls (3371 and 1737 cm−1, respectively).
Further evidence of ester or amide carbonyls was present as
eight downfield signals in the 13C NMR spectrum (δC 167.8,
169.2, 169.9, 170.8, 172.1, 172.5, 173.7, and 175.7). Addition-
ally, the presence of a monosubstituted phenyl group was
evident from four downfield carbon signals, two of which were
composed of two carbons each as indicated by their relative
peak height (δC 126.4, 128.0 × 2, 129.2 × 2, and 136.4).
Further analysis of the 1H NMR spectrum revealed the
presence of three singlet methyls at shifts indicative of an O-
methyl (δH 3.67) and two N-methyl groups (δH 2.93 and 3.03),
along with two broad downfield signals suggestive of NH
protons (δH 6.69 and 6.78).
Figure 1. Select 2D NMR data for tasiamides C−E (1−3).
Analysis of the 2D NMR data (COSY, TOCSY, ROESY,
HSQC, and HMBC) enabled the assignment of eight COSY
spin systems consisting of five amino and two hydroxy acid
residues [proline methyl ester (Pro-Me ester), N-MePhe, Ala,
Ile, N-MeGln, and two 2-hydroxy-3-methylbutyric acids
(Hmba)], accounting for all 13 degrees of unsaturation and
thereby signifying an overall linear arrangement (Figure 1).
HMBC correlations from the NH and N-Me groups to the
carbonyl of the neighboring residues allowed for the assignment
of connections between N-MePhe and Ala (C-16 to C-17), N-
MeGln and Hmba-1 (C-31 to C-32), Ala and Ile (NH-1 to C-
20), and Ile and N-MeGln (N-H-2 to C-26), leading to three
partial structures, Pro-Me ester, N-MePhe−Ala−Ile−N-
MeGln−Hmba-1, and Hmba-2. Key ROESY correlations
revealed further connections between these fragments, one
between H-5a/b (δ 3.34/3.15) and H-8 (δ 5.52) connecting
Pro-Me ester to N-MePhe and the other between H-34 (δ
2.15) and H-38 (δ 4.09), making the final connection between
the two Hmba residues. Thus, tasiamide C was deduced to have
an overall linear structure consisting of Pro-Me ester−N-
MePhe−Ala−Ile−N-MeGln−Hmba-1−Hmba-2. This planar
constitution was supported by tandem mass spectroscopy
analysis (Figure 2).
The absolute configurations of several of the amino acids
(Pro, N-MePhe, Ala, and N-MeGln) were determined by
Marfey’s analysis. Authentic D and L standards for Pro, N-
MePhe, and Ala were each derivatized with D-(1-fluoro-2,4-
dinitrophenyl-5-D-alanine amide) (D-FDAA). Unfortunately,
authentic D-N-MeGlu was unavailable; therefore, chromato-
graphic standards were prepared by derivatizing L-N-MeGlu
with both D- and L-FDAA (upon acidic hydrolysis, N-MeGln
becomes N-MeGlu). Compound 1 was hydrolyzed and
derivatized with D-FDAA and analyzed by LC-MS in
comparison with the retention times of authentic standards.
From this analysis it was clear that three of the four amino acids
(Pro, Ala, and N-MeGln) were of the L configuration, while the
N-MePhe residue was of the D configuration (Supporting
Information).
The absolute configuration of the Ile residue was determined
by chiral-phase GC-MS comparison of N-Boc, O-Me-
derivatized authentic standards against the similarly derivatized
Ile residue released by acid hydrolysis of 1. The four protected
standards of L-Ile, L-allo-Ile, D-Ile, and D-allo-Ile were prepared
by first synthesizing the N-Boc-protected amino acids, followed
by methyl esterification of the carboxylic acid using diazo-
methane. From retention time comparison and co-injection
experiments it was clear that the Ile residue was of the D-allo
configuration (Supporting Information).
The absolute configuration of the final two Hmba stereo-
centers in 1 presented a challenge. Comparison of authentic
standards of S- and R-Hmba with the methylated hydrolysate
revealed that the natural product contained both S- and R-
Hmba residues. A similar situation has been previously reported
in closely related metabolites of this compound family.16
Following mild base treatment (1:1 0.5 N NaOH(aq)/MeOH),
only the terminal Hmba residue was released; this residue was
subsequently methyl esterified using diazomethane16 and, by
retention time comparison and co-injections with authentic
standards, was identified as the S configuration. The
penultimate Hmba must therefore have the R configuration.
In summary, the above experiments established that tasiamide
C (1) possessed a 2S, 8R, 18S, 21R, 22S, 27S, 33R, and 38S
absolute configuration.
HR-ESITOFMS of 2 yielded an [M + Na]+ at m/z 825.4371
1
for a molecular formula of C40H62N6O11. The IR and H and
13C NMR spectra were similar to those of 1; however, the
molecular formula indicated a reduction of 14 amu (e.g., a CH2
B
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Figure 2. Select low-resolution MS fragmentation cleavages for tasiamides C−E (1−3).
unit) relative to tasiamide C. Inspection of the 1H NMR
spectrum revealed the presence of only two singlet methyl
groups (e.g., one N-methyl at δH 3.02 and one O-methyl at δH
3.58) and three NH protons (δH 6.77, 6.89, and 6.98). From
the 2D NMR data, it was clear that the only modification
between 1 and 2 was the loss of the N-methyl on the Phe
residue, thus yielding a planar constitution of Pro-Me ester−
Phe−Ala−Ile−N-MeGln−Hmba-1−Hmba-2 for tasiamide D
(2). This assembly was corroborated by tandem mass
spectrometry analysis (Figure 2).
Further analysis of 1D and 2D NMR data (1H, 13C, COSY,
TOCSY, ROESY, HSQC, HMBC, and H2BC17) confirmed the
presence of seven residues, six amino acids, and one hydroxy
acid [Pro-Me ester, N-MePhe, Gly, Ile, N-MeGln, Leu, and 2-
hydroxy-4-methylpentanoic acid (H4mpa)18]. In a similar
fashion to 1, each of the connections between residues in 3
were revealed by key HMBC and ROESY correlations. HMBC
correlations from both N-methyl groups to the carbonyls on the
neighboring residues allowed for the assignment of connections
between N-MePhe to Gly (C-16 to C-17) and N-MeGln to Leu
(C-30 to C-31). Correspondingly, HMBC correlations from
amide protons to adjacent carbonyls allowed for the assignment
of connections between Ile and N-MeGln (NH-2 to C-25) and
between Leu and H4mpa (NH-3 to C-37), leading to three
partial structures, Pro-Me ester, N-MePhe−Gly, and Ile−N-
MeGln−Leu−H4mpa. Two ROESY correlations provided the
final connections between these fragments, one between H-5a/
b (δ 3.36/3.31) and H-8 (δ 5.55), connecting Pro-Me ester and
N-MePhe, and the other between H-18a/b (δ 4.05/3.81) and
H-21 (δ 1.82), making the final connection between Gly and
Ile. Thus, tasiamide E was deduced to have a planar linear
structure consisting of Pro-Me ester−N-MePhe−Gly−Ile−N-
MeGln−Leu−H4mpa, and this was supported by tandem mass
spectrometry analysis (Figure 2).
The absolute configurations of the residues in 3 were
determined as described above for compounds 1 and 2.
Analysis of the Marfey’s derivatized hydrolysate with the
retention times of authentic standards on LC-MS revealed that
three of the four amino acids (Pro, Leu, and N-MeGln) were of
the L configuration, while the N-MePhe residue was of the D
configuration. As for the chiral GC-MS analysis of the Ile
residue, retention time comparison and co-injections confirmed
that it was also of the L configuration. The absolute
configuration of the H4mpa was determined using chiral-
phase GC-MS, both comparing retention time and co-
injections with authentic standards, thus confirming its S
configuration and establishing the absolute configuration of
tasiamide E (3) as 2S, 8R, 20S, 21S, 26S, 32S, and 38S.
Tasiamides C−E (1−3) are of close structural relation to
several families of known metabolites, the grassystatins,
tasiamides, and symplocin A. In this regard, 1−3 are structurally
most similar to tasiamide (4), with 3 varying only in the
The absolute configurations of the residues in compound 2
were determined in an identical fashion to that described above
for compound 1. Analysis of the retention times of the Marfey’s
derivatized hydrolysate and authentic standards by LC-MS
revealed that three of the four amino acids (Pro, Ala, and N-
MeGln) were of the L configuration and that the Phe residue
was of the D configuration. Chiral-phase GC-MS analysis of the
Ile residue, N-Boc and O-methylated as with 1, showed by
retention time comparison and co-injections that this residue
was of the L configuration. Chiral-phase GC-MS analysis of the
methylated hydrolysate of 2 exhibited peaks matching both S-
and R-Hmba. Analysis of the methylated mild base hydrolysate
confirmed that, as with 1, the terminal Hmba was of S
configuration, and so the penultimate Hmba was of R
configuration, establishing the absolute configuration of
tasiamide D (2) as 2S, 8R, 17S, 20S, 21S, 26S, 32R, and 37S.
HR-ESITOFMS of 3 yielded an [M + Na]+ at m/z 852.4842
in agreement with a molecular formula of C42H67N7O10,
1
requiring 13 degrees of unsaturation. The IR and H and 13C
NMR spectra again featured similarities to 1 and 2; however,
the molecular weight was 13 and 27 amu greater than 1 and 2,
respectively, and thus could not be readily attributed to a single
modification. Closer inspection of the 1H NMR spectrum
revealed the presence of three singlet methyls (δH 2.98, 2.99,
and 3.72), similar to 1, and three amide protons (δH 6.78, 6.82,
and 7.01), as seen in 2, suggesting that one of the hydroxy acids
was replaced by an amino acid. Further evidence of this was
seen by comparison of the 13C NMR spectra of 1 and 3; in
tasiamide C there were two α-carbons with shifts indicative of
hydroxy acids at δC 74.6 and 76.4, whereas there was only one
such peak in tasiamide E at δC 71.1.
C
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Table 1. NMR Data for Tasiamide C (1) in CDCl3
δH (J in
Article
δH (J in
a
c
a
b
b
c
b
b
residue
position
δC , type
Hz)
HMBC
COSY
3a, 3b
residue
position
22
δC , type
Hz)
HMBC
COSY
Pro-Me
Ester
1
172.1, C
36.1, CH
1.91, m
21, 23, 24, 21, 25
25
2
59.5, CH
4.37, dd
1, 3
1, 2, 4
23a
23b
24
24.7, CH2 1.30, m
25
21, 22, 24 23a, 24
23a, 23b
15.4, CH3 0.74, d (6.8) 21, 22, 23 22
6.78, d (7.5) 26 21
23b, 24
(7.6, 7.5)
1.02, m
3a
3b
4a
28.7, CH2 2.16, m
1.86, m
2, 3b, 4a
2, 3a, 4b
11.1, CH3 0.78, t (7.4) 22, 23
1, 2, 4, 5
5
25
25.2, CH2 1.92, m
3a, 4b,
5a, 5b
NH-2
26
N-MeGlu
169.2, C
56.7, CH
4b
5a
5b
1.75, m
47.1, CH2 3.34, m
3.15, m
3, 5
3
3b, 4a,
5a, 5b
27
4.93, dd
(8.0, 7.0)
26, 28, 29, 28a, 28b
32
4a, 4b,
5b
28a
28b
29a
24.5, CH2 2.35, m
26, 27, 29, 27, 28b
30
3, 4
1
4a, 4b,
5a
1.86, m
26, 27, 29, 27, 28a,
30
29a
6
7
8
52.3, CH3 3.67, s
167.8, C
32.7, CH2 2.26, m
27, 28a, 28, 28b,
30 29b
N-MePhe
55.9, CH
5.52, dd
(9.8, 6.1)
7, 9, 16
9a, 9b
8, 9b
8, 9a
29b
30
2.10, m
173.7, C
30 29a
9a
9b
34.7, CH2 3.14, m
2.87, dd
7, 8, 10,
11, 15
NH2a
NH2b
31
6.62, bs
6.46, bs
8, 10, 11,
15
(14.3, 9.7)
31.2, CH3 3.03, s
170.8, C
27, 32
10
136.4, C
Hmba-1
Hmba-2
32
11/15
129.2, CH
7.10, m
7.15, m
7.11, m
9, 10, 12,
14
12, 14
33
76.5, CH
4.85, d (7.9) 32, 34, 35, 34
36, 37
12/14
128.0, CH
10, 11, 13, 11, 13,
15
34
30.0, CH
2.15, m
33, 35, 36 33, 35,
36
15
13
16
17
18
126.4, CH
12, 14
8, 17
12, 14
35
36
37
38
39
18.3, CH3 0.99, d (6.7) 33, 34, 36 34
18.2, CH3 0.93, d (7.0) 33, 34, 35 34
175.7, C
30.5, CH3 2.93, s
172.5, C
Ala
Ile
45.1, CH
4.67, dq
(7.4, 7.3)
17, 19, 20 19
74.6, CH
31.7, CH
4.09, d (3.7) 37, 39
39
2.07, m
38, 40, 41 38, 40,
41
19
17.4, CH3 0.76, d (7.1) 17, 18
6.69, d (8.6) 20
18
18
NH-1
20
40
41
18.9, CH3 0.97, d (6.9) 39, 41
39
39
169.9, C
16.2, CH3 0.83, d (6.9) 39, 40
a
b
c
600 MHz for 1H NMR. 500 MHz for HMBC and COSY. 125 MHz
21
58.0, CH
4.14, dd
(8.2, 7.3)
20, 22, 23, 22
25
for 13C NMR.
terminal residue, while compounds 1 and 2 possess other
relatively simple modifications. In 2008, Li and co-workers
revised the original configuration of the N-MeGln residue in 4
from L to D based on the comparison of analytical data (13C
NMR and specific rotation) of the natural product with four
stereoisomers obtained from a total synthesis (containing both
L and D stereoisomers of N-MeGln and Leu; Table 2).19
However, the original assignments for all of the residues in 4
are consistent with that of 3, and a comparison of 13C shifts
revealed no significant differences between 3 and 4, along with
the four additional synthetic stereoisomers (A−D, Table 2)
(Supporting Information).20 Furthermore, the opposite specific
rotation signs for tasiamides C and D suggest a structural
solely on these data. This also suggests that the misassignment
in tasiamide (4) may involve residues other than just the N-
MeGln, and thus, a broader investigation into the configuration
of 4 will be necessary to clarify its correct absolute
configuration.
It is interesting to note that there appears to be considerable
biosynthetic flexibility in this family of metabolites. Insights are
thus provided by comparing the amino or hydroxy acid residues
in each member of this family (Table 2). The first three
residues (A, B, and C) starting from the carboxy terminus of
these molecules are rather well conserved, with each analogue
containing a Pro-Me ester followed by the incorporation of D-
Phe, which is N-methylated in all but one analogue, and then
the incorporation of an Ala, Aba (2-aminobutyric acid), or Gly
as the third residue. Residues E, H, and J are also conserved
residues (statine, Hmba, and an N,N-dimethylated amino acid,
respectively), but they occur only in a subset of the natural
products. Residues F and I are slightly less conserved, with
three different polar amino acids (N-MeGln, Asn, and Ser)
incorporated into residue F and four different hydroxy acids
(Hmpa, H4mpa, Hpa, and H3mpa)18 in residue I. Also
observed are variations in the absolute configurations of these
residues. For example, residues D and J in eight of nine
metabolites incorporate an L residue, while a single metabolite
has a D-allo residue at this position. At residue F all of the
variance between these two metabolites (3, [α]25 −22.2; 4,
D
[α]21 +15.0). Therefore, a more detailed analysis of the 13C
D
NMR data for tasiamide and the four synthetic analogues was
conducted using DP4 probability calculations and revealed that
the carbon data alone are insufficient to deduce the correct
configuration in tasiamide (4).21 The probability of the original
experimental data matching each analogue was calculated as
1.3% for analogue A (4), 56.0% for analogue B, 3.0% for
analogue C, and 39.7% for analogue D. Because analogues B
and D have the same sign and magnitude of specific optical
rotation and are indistinguishable by DP4 calculations, it is not
possible to unequivocally assign the configuration of 4 based
D
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Table 2. Residue Sequence Comparisons of the Tasiamide/Grassystatin/Symplocin Superfamily of Metabolites
d
d
compound
A
B
C
D
E
F
G
H
I
J
[α]D
grassystatin A (5)16
L-OMe-
Pro
D-NMe-
Phe
L-Ala
L-Thr
(S,S)-
Sta
L-Asn
L-Leu
D-
Hmba
L-Hmba
L-N,N-diMe- −4.4 (c 0.08,
Val MeOH)
grassystatin B (6)16
grassystatin C16
symplocin A23
L-OMe-
D-NMe- L-Aba
L-Thr
L-Ile
L-Val
Ile
(S,S)-
L-Asn
L-Leu
L-Leu
L-Tyr
Leu
D-
L-Hmba
L-N,N-diMe- −5.0 (c 0.1,
Pro
Phe
Sta
Hmba
Val
MeOH)
L-OMe-
Pro
D-NMe-
Gly
Gly
Gly
Ala
(S,S)-
Sta
L-NMe-
Gln
D-allo-
H3mpa
−21.9 (c 0.04,
Phe
MeOH)
L-OMe-
Pro
D-NMe-
(R,S)-
Sta
L-Ser
D-
Hmba
D-N,N-
diMe-Ile
+16 (c 2.18,
MeOH)
Phe
a
tasiamide (4)20
OMe-
Pro
NMe-
NMe-
Gln
Hmba
Hpi
+15 (c 0.4,
CHCl3)
Phe
a
tasiamide B24
OMe-
Pro
NMe-
Phe
Leu
Ahppa
NMe-
Gln
Val
−28 (c 0.4,
CHCl3)
tasiamide C (1)
tasiamide D (2)
tasiamide E (3)
L-OMe-
D-NMe-
L-Ala
L-Ala
Gly
Gly
Gly
Gly
Gly
D-allo-
L-NMe-
D-
L-Hmba
L-Hmba
L-H4mpa
L-Hmba
L-Hmba
L-Hmba
L-Hmba
−37 (c 1.17,
Pro
Phe
Ile
Gln
Hmba
CHCl3)
L-OMe-
Pro
D-Phe
L-Ile
L-Ile
L-Ile
L-Ile
L-Ile
L-Ile
L-NMe-
Gln
D-
Hmba
−85 (c 1.67,
CHCl3)
L-OMe-
Pro
D-NMe-
Phe
L-NMe-
Gln
L-Leu
L-Leu
L-Leu
−22 (c 0.53,
CHCl3)
tasiamide synthetic analogue L-OMe-
D-NMe-
Phe
L-NMe-
Gln
−12.6 (c 0.4,
b
A (4)19
Pro
CHCl3)
tasiamide synthetic analogue L-OMe-
D-NMe-
Phe
D-NMe-
Gln
+15 (c 0.4,
CHCl3)
c
B19
tasiamide synthetic analogue L-OMe-
C19
Pro
tasiamide synthetic analogue L-OMe-
D19
Pro
Pro
D-NMe-
Phe
L-NMe-
Gln
D
−15.6 (c 0.4,
-Leu
CHCl3)
D-NMe-
Phe
D-NMe-
Gln
D-Leu
+18.5 (c 0.4,
CHCl3)
a
Stereochemical assignments for tasiamide and tasiamide B intentionally omitted due to unresolved stereochemical questions (see discussion).
b
c
d
Matches original configuration proposed by Williams et al.20 Proposed revised structure of tasiamide according to Ma et al.19 Abbreviations
developed based on IUPAC nomenclature of each residue.18
amino acids were originally deduced as L; however, in tasiamide
and tasiamide B these residues were reassigned as D based on
total synthesis. Although non-ribosomal peptide synthetase
(NRPS)-derived peptides can show variations in the incorpo-
rated amino acid, it is difficult to imagine a single biosynthetic
pathway that would be capable of producing all of these
metabolites, especially those of varying absolute configurations,
which usually requires an epimerase.22
Both the grassystatins and symplocin A were reported to
possess exceptional inhibitory activity toward cathepsin E
(grassystatin A: IC50 = 886 pM, symplocin A: IC50 = 300
pM);16,23 however, this activity is likely due to the presence of a
statine residue, which is absent in the tasiamides. Additionally,
tasiamide and tasiamide B both showed moderate cytotoxicity
against KB cells, with IC50 values of 0.48 and 0.8 μM,
respectively.19,24 Due to limited isolated quantities of tasiamide
E (3), only tasiamides C (1) and D (2) were evaluated for their
cytotoxicity against the HCT-116 colon cancer cell line and
were found to be inactive (tasiamide C, IC50 > 25 μM;
tasiamide D, IC50 ≈ 25 μM).
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured on a JASCO P-2000 polarimeter, UV spectra on a Beckman
Coulter DU-800 spectrophotometer, and IR spectra on a Nicolet IR-
100 FT-IR spectrophotometer using KBr plates. NMR spectra were
recorded with chloroform as an internal standard (δC 77.0, δH 7.26 for
CHCl3) on a Varian Unity 500 MHz spectrometer (500 and 125 MHz
for 1H and 13C NMR, respectively), a Varian Unity 300 MHz
spectrometer (300 and 75 MHz for 1H and 13C NMR, respectively), a
Varian VNMRS (Varian NMR System) 500 MHz spectrometer
equipped with a cold probe (500 and 125 MHz for 1H and 13C NMR),
and a Bruker 600 MHz spectrometer equipped with a 1.7 mm
MicroCyroProbe (600 and 150 MHz for 1H and 13C NMR). LR- and
HR-ESIMS data were obtained on ThermoFinnigan LCQ Advantage
Max and Thermo Scientific LTQ Orbitrap-XL mass spectrometers,
respectively. Tandem mass spectroscopy experiments were run with a
Biversa Nanomate (Advion Biosystems) electrospray source for a
Finnigan LTQ-FTICR-MS instrument (Thermo-Electron Corpora-
tion) running Tune Plus software version 1.0. HPLC was carried out
using a Waters 515 pump system with a Waters 996 PDA detector.
GC-MS was conducted with a Thermo Electron Corp. DSQ/TRACE-
GC-Ultra GCMS system. All solvents were either distilled or of HPLC
quality. Acid hydrolysis was performed using a Biotage (Initiator)
microwave reactor equipped with high-pressure vessels.
Cyanobacterial Collection. The tasiamides C−E-producing
cyanobacterium (collection code: PNG 07/14/07-6) was collected
by hand on reefs 20 m deep from approximately eight nearby sites
located in Kimbe Bay off the north coast of New Britain, Papua New
Guinea (S 5°26.292′, E 150°40.813′). Field notes identified the
puffballs as a consortium of Schizothrix sp. with a minor amount of
Lyngbya sp. present. The collected samples were stored in 1:1 EtOH/
seawater in the field, and the supernatant was decanted and discarded
before shipment. As was previously reported, a portion of the
cyanobacterial biomass was used for 16S rRNA sequencing, which
revealed it claded with “tropical marine Symploca” (acc. no.
JQ388599).15
CONCLUSION
■
The new metabolites tasiamides C−E (1−3) were isolated
from a Kimbe Bay, Papua New Guinea, collection of the
tropical marine cyanobacterium Symploca sp. These metabolites
add to the growing family of structurally homologous natural
products [tasiamide (4), grassystatins (5, 6), and symplocin A]
that have all been isolated from similar tuft-forming
cyanobacteria. This particular Symploca sp. is a prolific producer
of secondary metabolites, as several structurally diverse natural
products were isolated from this collection including
kimbeamides A−C, kimbelactone, tasihalide C, and the three
tasiamides reported herein.15
E
dx.doi.org/10.1021/np401051z | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Extraction and Isolation. The cyanobacterial biomass (101.7 g
dry wt) was extracted with 2:1 CH2Cl2/MeOH to afford 1.8 g of dried
extract. A portion of the extract was fractionated by silica gel VLC
using a stepwise gradient solvent system of increasing polarity starting
from 100% hexanes to 100% MeOH (nine fractions, A−I). The
fraction eluting with 25% MeOH/75% EtOAc (fraction H) was
separated further using RP HPLC [4 μ Synergi Hydro, isocratic 65%
MeCN/35% H2O] to yield pure tasiamide C (1, 1.9 mg), tasiamide D
(2, 2.5 mg), and tasiamide E (3, 0.7 mg).
authentic D-NMeGlu was not available, L-NMeGlu standard was
derivatized with both the L-FDAA and D-FDAA. The NMeGlu residue
was analyzed by RP HPLC using a Kinetex 5 μ C18 100A column (4.6
× 100 Å mm). The HPLC condition began with 5% MeOH/95% H2O
acidified with 0.1% FA(aq) followed by a gradient profile to 45%
MeOH/55% H2O acidified with 0.1% FA(aq) over 125 min at a flow
of 0.4 mL/min, monitoring from 200 to 600 nm. The retention times
of the derivatives of the authentic amino acids when analyzing for 1
were D-Ala (64.8 min), L-Ala (71.1 min), D-Pro (66.6 min), L-Pro (69.4
min), D-NMePhe (78.4 min), L-NMePhe (79.7 min), L-NMeGlu
reacted with L-FDAA (93.3 min), and L-NMeGlu reacted with D-FDAA
(91.9 min); the derivatives of the hydrolysate product of 1 gave peaks
with retention times of 71.5, 69.8, 78.3, and 91.8 min, according to L-
Ala, L-Pro, D-NMePhe, and L-NMeGlu, respectively. The retention
times of the derivatives of the authentic amino acids when analyzing
for 2 were D-Ala (59.3 min), L-Ala (63.6 min), D-Pro (60.4 min), L-Pro
(62.6 min), D-Phe (78.7 min), L-Phe (83.8 min), L-NMeGlu reacted
with L-FDAA (93.3 min), and L-NMeGlu reacted with D-FDAA (91.9
min); the derivatives of the hydrolysate product of 2 gave peaks with
retention times of 64.0, 62.6, 83.8, and 91.7 min, according to L-Ala, L-
Pro, D-Phe, and L-Glu. The retention times of the derivatives of the
authentic amino acids when analyzing for 3 were D-Leu (68.4 min), L-
Leu (85.8 min), D-Pro (60.4 min), L-Pro (62.6 min), D-NMePhe (78.6
min), L-NMePhe (79.2 min), L-NMeGlu reacted with L-FDAA (107.6
min), and L-NMeGlu reacted with D-FDAA (103.9 min); the
derivatives of the hydrolysate product of 3 gave peaks with retention
times of 85.7, 62.2, 78.2, and 104.5 min, according to L-Leu, L-Pro, D-
NMePhe, and L-NMeGlu.
Preparation and GC-MS Analysis of Isoleucine (Ile) in
Tasiamides C−E (1−3). An aliquot (∼0.1 mg) of the above
hydrolysate product was dissolved in 100 μL of H2O and then treated
with 0.26 mg (0.43 μmol) of NaHCO3 followed by 0.94 mg (0.43
μmol) of di-tert-butyl dicarbonate. After 16 h at room temperature
(rt), the reaction mixture was quenched by 500 μL of 5% KHSO4 and
back extracted with 3 × 1 mL of EtOAc, and the combined organic
layers were dried over MgSO4. The reaction product was then treated
with freshly prepared diazomethane in diethyl ether. Each of the four
Ile standards were prepared in a similar fashion.
The derivatized hydrolysate product and standards were analyzed
by chiral-phase GC-MS using a Chirasil-Val (Agilent Technologies
J&W Scientific, 30 m × 0.25 mm) under the following conditions: the
initial oven temperature was 40 °C, kept for 2 min, followed by a ramp
from 40 to 75 °C at a rate of 10 °C/min, kept for 5 min, followed by
another ramp to 110 °C, at a rate of 0.5 °C/min, followed by a final
ramp to 200 °C, at a rate of 25 °C/min, kept for 2 min. The retention
times for the four authentic standards when analyzing 1 were D-allo-Ile
(49.6 min), L-allo-Ile (49.9), D-Ile (51.5 min), and L-Ile (51.7 min); the
derivatized hydrolysis product of 1 yielded a peak at 49.6 min,
according to D-allo-Ile. The retention times for the four authentic
standards when analyzing 2 and 3 were D-allo-Ile (47.7 min), L-allo-Ile
(48.2), D-Ile (50.1 min), and L-Ile (50.5 min); the derivatized
hydrolysis products of 2 and 3 each yielded a peak at 50.6 min,
according to L-Ile.
Tasiamide C (1): white, amorphous solid; [α]25 −37 (c 1.2,
D
CHCl3); UV (MeOH) λmax (log ε) 211.0 (3.84); IR (neat) νmax 3371,
2965, 2933, 2877, 1737, 1644, 1521, 1453, 1263, 1201, 1178, 1098,
1
1031 cm−1; H NMR (600 MHz, CDCl3) and 13C NMR (125 MHz,
CDCl3), see Table 1; HRESIMS m/z 839.4541 [M + Na]+ (calcd for
C41H64N6O11Na, 839.4525).
Tasiamide D (2): white, amorphous solid; [α]25 −85 (c 1.6,
D
CHCl3); UV (MeOH) λmax (log ε) 220.0 (3.86) nm; IR (neat) νmax
3318, 2965, 2931, 1739, 1648, 1526, 1453, 1203, 1033 cm−1; 1H NMR
(500 MHz, CDCl3) δ 7.17 (2H, m), 7.13 (1H, m), 7.09 (2H, m), 6.98
(1H, d, J = 6.8), 6.89 (1H, d, J = 7.7), 6.77 (1H, d, J = 6.7), 5.98 (1H,
bs), 5.62 (1H, bs), 5.08 (1H, dd, J = 7.6, 6.7), 4.85 (1H, d, J = 7.5),
4.76 (1H, q, J = 6.6), 4.43 (1H, dq, J = 8.0, 6.8), 4.13 (1H, m), 4.12
(1H, m), 4.11 (1H, m), 3.58 (3H, s), 3.47 (1H, dd, J = 8.7, 5.1), 3.02
(3H, s), 2.94 (2H, m), 2.68 (1H, m), 2.48 (1H, m), 2.32 (1H, m), 2.25
(1H, m), 2.14 (1H, m), 2.07 (1H, m), 1.97 (1H, m), 1.81 (1H, m),
1.78 (2H, m), 1.35 (2H, m), 1.26 (3H, d, J = 7.2), 1.07 (2H, m), 0.99
(3H, d, J = 6.1), 0.97 (3H, d, J = 6.2), 0.93 (3H, d, J = 7.0), 0.82 (3H,
t, J = 6.8), 0.82 (6H, d, J = 6.3); 13C NMR (75 MHz, CDCl3) δ 175.8,
174.6, 172.2, 171.8, 170.7, 170.4, 136.2, 129.1, 128.5, 127.0, 76.7, 75.1,
59.4, 59.1, 57.1, 52.5, 52.2, 48.8, 46.9, 39.0, 35.9, 33.0, 32.1, 31.1, 29.9,
29.0, 24.7, 24.6, 24.0, 18.7, 18.4, 18.1, 16.1, 15.7, and 11.5; HRESIMS
m/z 825.4371 [M + Na]+ (calcd for C40H62N6O11Na, 825.4369).
Tasiamide E (3): white, amorphous solid; [α]25 −22 (c 0.5,
D
CHCl3); UV (MeOH) λmax (log ε) 207.0 (4.10) nm; IR (neat) νmax
3329, 2926, 2958, 1743, 1645, 1521, 1454, 1281, 1177 cm−1; 1H NMR
(600 MHz, CDCl3) δ 7.26 (2H, m), 7.22 (2H, m), 7.20 (1H, m), 7.01
(1H, d, J = 9.3), 6.82 (1H, d, J = 9.4), 6.78 (1H, d, J = 4.4), 5.61 (1H,
bs), 5.55 (1H, t, J = 7.4), 5.25 (1H, bs), 5.06 (1H, t, J = 7.4), 4.95 (1H,
q, J = 7.9), 4.39 (1H, dd, J = 8.1, 5.2), 4.29 (1H, dd, J = 8.8, 6.3), 4.09
(1H, dd, J = 9.1, 4.5), 4.05 (1H, dd, J = 17.7, 3.3), 3.81 (1H, dd, J =
17.7, 3.3), 3.72 (3H, s), 3.36 (1H, m), 3.31 (1H, m), 3.27 (1H, dd, J =
13.6, 8.2), 2.99 (3H, s), 2.98 (3H, s), 2.82 (1H, dd, J = 13.6, 6.9), 2.31
(1H, m), 2.24 (1H, m), 2.18 (1H, m), 2.12 (1H, m), 1.99 (1H, m),
1.93 (1H, m), 1.87 (2H, m), 1.86 (1H, m), 1.82 (1H, m), 1.80 (1H,
m), 1.60 (2H, m), 1.54 (2H, m), 1.43 (1H, m), 1.11 (1H, m), 0.96
(6H, d, J = 6.6), 0.95 (3H, d, J = 6.4), 0.92 (3H, d, J = 6.4), 0.88 (3H,
d, J = 7.2), 0.87 (3H, t, J = 7.7); 13C NMR (125 MHz, CDCl3) δ
175.2, 174.4, 174.0, 172.5, 171.5, 169.7, 167.8, 136.8, 129.4, 128.4,
126.8, 71.1, 58.9, 57.6, 56.9, 56.2, 52.3, 46.8, 43.1, 41.2, 37.2, 35.1,
31.9, 31.0, 29.7, 28.8, 25.0, 24.8, 24.7, 24.6, 23.4, 23.0, 22.7, 22.3, 21.4,
15.6, 11.3; HRESIMS m/z 852.4842 [M + Na]+ (calcd for
C42H67N7O10Na, 852.4842).
Acid Hydrolysis and Marfey’s Analysis of Tasiamides C−E
(1−3). Tasiamides C−E (1−3, 0.2 mg) were treated separately with
400 μL of 6 N HCl in a microwave reactor at 160 °C for 5 min. An
aliquot of the reaction product was dissolved in 500 μL of a 1 mg/mL
solution of D-FDAA (1-fluoro-2,4-dinitrophenyl-5-D-alanine amide) in
acetone followed by the addition of 20 μL of 1 N NaHCO3. The
solution was maintained at 40 °C for 1 h, at which time the reaction
was quenched by the addition of 40 μL of 1 N HCl. The reaction
mixture was then dried down under N2(g) and resuspended in 200 μL
of 50% H2O/50% MeCN, and 10 μL of the solution was analyzed by
LC-ESIMS.
The Marfey’s derivatives of the hydrolysate and standards reacted
with D-FDAA (Ala, Phe, N-MePhe, Pro, and Leu) were analyzed by RP
HPLC using a Phenomenex Luna 5 μm C18 column (4.6 × 250 mm).
The HPLC conditions began with 10% MeCN/90% H2O acidified
with 0.1% formic acid (FA) (aqueous) followed by a gradient profile to
50% MeCN/50% H2O acidified with 0.1% FA(aq) over 85 min at a
flow of 0.4 mL/min, monitoring from 200 to 600 nm. Because
Base Hydrolysis to Determine Configuration of Hmba Units
in Tasiamides C and D (1 and 2). Tasiamides C (1) and D (2)
(0.15 mg) were treated separately with 150 μL of 1:1 0.5 N
NaOH(aq)/MeOH (1:1) solution at room temperature for 72 h.16
The reaction mixture was neutralized by the addition of 40 μL of 1 N
HCl(aq) and back extracted with 3 × 1 mL of EtOAc, and the
combined organic layers were dried over MgSO4. The product was
then treated with freshly prepared diazomethane in diethyl ether for 5
min at rt. The derivatized acid hydrolysate was analyzed by chiral-
phase GC-MS using a Cyclosil B column (Agilent Technologies J&W
Scientific, 30 m × 0.25 mm) under the following conditions: the initial
oven temp was 35 °C, kept for 15 min, followed by a ramp to 60 °C, at
a rate of 1.5 °C, followed by a ramp to 170 °C, at a rate of 5 °C/min,
kept for 5 min. The retention times for the authentic standards were
R-Hmba (34.9 min) and S-Hmba (35.8 min); the derivatized
hydrolysis product for 1 and 2 each exhibited a single peak at 35.8
and 35.9 min, respectively, according to S-Hmba.
F
dx.doi.org/10.1021/np401051z | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
GC-MS Analysis of H4mpa in Tasiamide E (3). The derivatized
hydrolysate product used in the Ile analysis from 3 was also used in the
H4mpa analysis. Authentic standards were synthesized following a
previously published method.25 The derivatized hydrolysis product
and authentic standards were analyzed by chiral-phase GC-MS using a
Cyclosil B column under the following conditions: the initial oven
temp was 40 °C, kept for 15 min, followed by a ramp to 90 °C, at a
rate of 1.5 °C/min, followed by a ramp to 200 °C, at a rate of 10 °C/
min, kept for 5 min. The retention times for the authentic standards
were R-H4mpa (43.2 min) and S-H4mpa (43.8 min); the derivatized
hydrolysis product yielded a peak at 43.7 min, consistent with S-
H4mpa.
2008, 71, 1113−1116. (d) Tidgewell, K.; Engene, N.; Byrum, T.;
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ASSOCIATED CONTENT
* Supporting Information
■
S
(11) Edwards, D. J.; Marquez, B. L.; Nogle, L. M.; McPhail, K.;
Goeger, D. E.; Roberts, M. A.; Gerwick, W. H. Chem. Biol. 2004, 11,
817−833.
1H NMR, 13C NMR, COSY, ROESY, TOCSY, HSQC, HMBC,
and H2BC (only for 3) spectra in CDCl3 for tasiamides C−E
(1−3) and complete NMR data tables for tasiamides D and E
(2 and 3). LC-MS and GC-MS chromatograms for chiral-phase
analyses, MS fragmentation data for tasiamides C−E (1−3),
and descriptions of both the H-460 and HCT-116 cytotoxicity
assays. This material is available free of charge via the Internet
at http://pubs.acs.org.
(12) Hooper, G. J.; Orjala, J.; Schatzman, R. C.; Gerwick, W. H. J.
Nat. Prod. 1998, 61, 529−533.
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(15) (a) Nunnery, J. K.; Engene, N.; Byrum, T.; Cao, Z.; Jabba, S. V.;
Pereira, A. R.; Matainaho, T.; Murray, T. F.; Gerwick, W. H. J. Org.
Chem. 2012, 77, 4198−4208. (b) Nunnery, J. K. Ph.D. Dissertation,
University of California San Diego, 2012.
AUTHOR INFORMATION
Corresponding Author
*Tel: 858-534-0578. Fax: 858-534-0576. E-mail: wgerwick@
ucsd.edu.
■
(16) Kwan, J. C.; Eksioglu, E. A.; Liu, C.; Paul, V. J.; Luesch, H. J.
Med. Chem. 2009, 52, 5732−5747.
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2005, 127, 6154−6155.
(18) The hydroxy acid abbreviations were developed from their
corresponding IUPAC names as follows [IUPAC name (abbreviation),
common name]: 2-hydroxypropanoic acid (Hpa), lactic acid and
sarcolactic acid; 2-hydroxy-3-methylbutyric acid (Hmba), valic acid
and 2-hydroxyisovaleric acid (Hiva); 2-hydroxy-3-methylpentanoic
acid (H3mpa), isoleucic acid and 2-hydroxy-3-methylvaleric acid
(Hmva); 2-hydroxy-4-methylpentanoic acid (H4mpa), leucic acid and
2-hydroxyisocaproic acid (Hica). Although several of these acids have
common names, none of these are structurally descriptive of the
residue and thus could give rise to confusion.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the government of Papua New Guinea for permission
to collect the cyanobacterial specimens, J. Nunnery for initial
purification work, E. Theodorakis for use of the microwave
reactor, and the UCSD mass spectrometry facility for their
analytical services. The 500 MHz NMR 13C XSens cold probe
was supported by NSF CHE-0741968. We also acknowledge
The Growth Regulation & Oncogenesis Training Grant NIH/
NCI (T32A009523-24) for a fellowship to E.M., and NIH
Grants (CA100851 and GM107550) for support of the
research.
(19) Ma, Z.; Song, N.; Li, C.; Zhang, W.; Wang, P.; Li, Y. J. Pept. Sci.
2008, 14, 1139−1147.
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(22) von Dohren, H.; Dieckmann, R.; Pavela-Vrancic, M. Chem. Biol.
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