Natural products chemistry and taxonomy of the marine cyanobacterium Blennothrix cantharidosmum

Article abstract of DOI:10.1021/np800088a

A Papua New Guinea field collection of the marine cyanobacterium Blennothrix cantharidosmum was investigated for its cytotoxic constituents. Bioassay-guided isolation defined the cytotoxic components as the known compounds lyngbyastatins 1 and 3. However, six new acyl proline derivatives, tumonoic acids D-I, plus the known tumonoic acid A were also isolated. Their planar structures were defined from NMR and MS data, while their stereostructures followed from a series of chiral chromatographies, degradation sequences, and synthetic approaches. The new compounds were tested in an array of assays, but showed only modest antimalarial and inhibition of quorum sensing activities. Nevertheless, these are the first natural products to be reported from this genus, and this inspired a detailed morphologic and 16S rDNA-based phylogenetic analysis of the producing organism.

Full text of DOI:10.1021/np800088a

1530
J. Nat. Prod. 2008, 71, 1530–1537
Natural Products Chemistry and Taxonomy of the Marine Cyanobacterium Blennothrix
cantharidosmum
Benjamin R. Clark,† Niclas Engene,† Margaret E. Teasdale,‡ David C. Rowley,‡ Teatulohi Matainaho,§ Frederick A. Valeriote,
and William H. Gerwick*,†,|
Scripps Institution of Oceanography, UniVersity of California San Diego, 9500 Gilman DriVe, La Jolla, California 92093-0212, Department of
Biomedical and Pharmaceutical Sciences, College of Pharmacy, UniVersity of Rhode Island, Kingston, Rhode Island 02881, Discipline of
Pharmacology, School of Medicine and Health Sciences, UniVersity of Papua New Guinea, National Capital District, Papua New Guinea,
Henry Ford Health System, Detroit, Michigan 48202, and Skaggs School of Pharmacy and Pharmaceutical Sciences, UniVersity of California
San Diego, 9500 Gilman DriVe, La Jolla, California 92093-0212
ReceiVed February 7, 2008
A Papua New Guinea field collection of the marine cyanobacterium Blennothrix cantharidosmum was investigated for
its cytotoxic constituents. Bioassay-guided isolation defined the cytotoxic components as the known compounds
lyngbyastatins 1 and 3. However, six new acyl proline derivatives, tumonoic acids D-I, plus the known tumonoic acid
A were also isolated. Their planar structures were defined from NMR and MS data, while their stereostructures followed
from a series of chiral chromatographies, degradation sequences, and synthetic approaches. The new compounds were
tested in an array of assays, but showed only modest antimalarial and inhibition of quorum sensing activities. Nevertheless,
these are the first natural products to be reported from this genus, and this inspired a detailed morphologic and 16S
rDNA-based phylogenetic analysis of the producing organism.
Cyanobacteria are an ancient group of organisms well known
for their prolific production of natural products.1,2 Several genera,
such as Lyngbya and Microcystis, have been well investigated for
production of both dangerous toxins and drug leads.3,4 However,
there are numerous other cyanobacterial genera that have not been
studied in such detail. Recent advances in the molecular taxonomy
of cyanobacteria have allowed more rapid and precise identification
of cyanobacterial specimens and have the potential to assist in the
discovery of new natural products from some of these lesser-known
genera.
Blennothrix cantharidosmum Koma´rek (formerly Hydrocoleum
Gomont) is one of the most abundant species of mat-forming
benthic cyanobacteria in the tropics5 and an important contributor
to nitrogen fixation.6 Under bacteriological systems, Blennothrix
has been assigned as a subgenus of Microcoleus on the basis of
several common morphological features, most distinctively, the
presence of multiple trichomes within a single sheath.7 However,
recent phylogenetic studies have shown that Blennothrix species
are more closely related to the common planktonic genus
Trichodesmium.5,8 Despite their widespread occurrence, very few
studies have been carried out on the chemistry of Blennothrix, and
these studies have focused solely on their pigments.5,9 In this article,
we describe the first secondary metabolites reported from a
Blennothrix species and further investigate the taxonomy of this
important genus of marine cyanobacterium.
solid-phase extraction (SPE) and HPLC to yield the known
metabolites lyngbyastatins 110 and 311 as the active metabolites,
in addition to six new acylproline derivatives, the tumonoic acids
D-I (1-6), as well as the known compound tumonoic acid A (7).12
Structures of the tumonoic acids are given in Figure 1.
The molecular formula of tumonoic acid D (1) was determined
to be C16H29NO3 by HRESIMS (m/z 284.2211, [M + H]+).
Analysis of 1H NMR data (Table 1) revealed downfield
resonances (δH 4.64, d; 3.62, ddd; 3.51, ddd) characteristic of a
proline residue, while the large methylene envelope (δH 1.24-1.28)
and methyl signals (δH 1.15, d; 0.88, t) suggested a branched
fatty acid. HMBC correlations from the methyl doublet to
carbons at 179.9, 38.1, and 34.1 ppm confirmed its location at
C-2. 2D NMR analysis (Figure 2) enabled the assignment of all
protons and carbons in the proline residue and the terminal
regions of the fatty acid moiety. The three remaining carbons
(δC 29.2, 29.4, 29.6) were assigned as the methylene carbons in
the central portion of the fatty acid, which was consistent with
the observed molecular formula. However, no HMBC correla-
tions were observed linking the proline and fatty acid portions
of 1, but on the basis of the downfield shifts of H-2′ and H-5′,
it was clear that the proline nitrogen was part of an amide group,
leading to the structure shown.
Tumonoic acid E (2) (C18H31NO4, m/z 326.2321, [M + H]+)
possessed more complex 1H and 13C NMR spectra than 1. While
resonances consistent with a proline residue were present, it was
clear that the fatty acid moiety of 2 was more highly function-
alized. The presence of a downfield doublet (δH 5.23, d), along
with two sp2 carbon resonances (δC 137.0, 132.3), suggested
the presence of a double bond. Also observed were resonances
consistent with an oxygenated methine (δH 4.15, d; δC 80.6)
and two additional methyls. A broad IR stretch at 3381 cm-1
confirmed the presence of a free hydroxy group. COSY and
HMBC correlations (Figure 2) situated the oxygenated methine
at H-3, while HMBC correlations from the three methyl groups
(δH 1.05, d; δC 14.3, δH 1.63, br s; δC 10.9, and δH 0.95, d; δC
20.9) confirmed their positions at C-2, C-4, and C-6, respectively.
Likewise, HMBC correlations situated the vinylic proton at H-5,
while ROESY correlations between H-5 and H-3 confirmed the
E-geometry of the double bond. The H-2 pentet (δH 2.82, dq, J
) 6.8, 6.5) of the fatty acid moiety was characteristic for an
Results and Discussion
Natural Products Isolation and Structure Elucidation. The
strain was collected near Duke of York Island in Papua New Guinea
in 2005. The specimen was extracted with CH2Cl2-MeOH and
fractionated by silica vacuum liquid chromatography. Initial assays
revealed activity against an H-460 cell line in several of the polar
fractions, which were subsequently fractionated by reversed-phase
* To whom correspondence should be addressed. Tel: (858) 534-0578.
Fax: (858) 534-0529. E-mail: wgerwick@ucsd.edu.
† Scripps Institution of Oceanography.
‡ University of Rhode Island.
§ University of Papua New Guinea.
Henry Ford Health System.
| University of California San Diego.
10.1021/np800088a CCC: $40.75
2008 American Chemical Society and American Society of Pharmacognosy
Published on Web 08/13/2008

Natural Products Chemistry of Blennothrix cantharidosmum
Journal of Natural Products, 2008, Vol. 71, No. 9 1531
Figure 1. Structures of tumonoic acids D-I (1-6) and A (7) and epi-tumonoic acid D (8). Configurations for the fatty acid portions of 2-6
are relative only.
Table 1. NMR Data (CDCl3) for Tumonoic Acids D-F (1-3)a
tumonoic acid D (1)
tumonoic acid E (2)
tumonoic acid F (3)
δC
δH
δC
δH
δC
δH
fatty acid
1
2
3
4
5
6
7
8
179.9
38.1
34.1
27.5
29.6
29.4
29.2
31.8
22.6
14.1
178.2
41.3
80.6
132.3
137.0
32.1
37.1
28.8
22.7
14.1
175.6
40.0
77.5
33.9
33.6
27.3
29.7
29.2
29.5
31.8
22.6
14.1
13.7
12.9
2.59 dq (6.8, 6.8)
1.69 m, 1.43 m
1.26 m
1.25 m
1.25 m
1.25 m
1.24 m
1.28 m
0.88 t (6.8)
2.82 dq (6.8, 6.5)
4.15 d (6.5)
2.96 dq (9.6, 6.8)
5.21 dd (9.6, 1.5)
1.75 br tq (5.9, 6.7)
1.31 m
1.25 m
1.25 m
1.25 m
1.25 m
1.25 m
1.28 m
5.23 d (9.3)
2.38 m
1.33 m, 1.21 m
1.22 m
1.29 m
0.87 t (7.2)
9
10
11
12
2-Me
4-Me
6-Me
OAc
0.88 t (6.9)
1.14 d (6.8)
0.91 d (6.8)
16.8
1.15 d (6.8)
14.3
10.9
20.9
1.05 d (6.8)
1.63 br s
0.95 d (6.6)
169.9
20.8
1.99 s
proline
1′
2′
3′
171.4
60.5
26.6
n/ob
60.2
27.3
172.8
59.7
27.8
4.64 d (7.6)
2.56 m 1.94 m
4.62 d (9.5)
2.51 m 2.03 m
4.55 dd (8.1, 2.5)
2.41 ddd (2.5, 6.1, 10.0)
2.00 m
4′
5′
24.8
48.0
2.03 m
3.62 ddd (9.5, 7.8, 3.2)
3.51 ddd (9.8, 9.5, 7.1)
24.8
48.0
2.04 m
3.71 ddd (9.2, 7.6, 7.6)
3.60 ddd (9.2, 7.0, 3.6)
24.6
47.7
2.10 m
3.64 dd (8.4, 5.1)
a
1H NMR spectra were recorded at 500 MHz, and 13C spectra at 75 MHz. b Not observed.
anti-configuration of the 2-Me and 3-OH substituents.12,13 The
relative configuration at C-6 was not assigned.
and derivatization with Mosher’s acid chloride (both R and S).
However, this proved unsuccessful. Prior research has suggested
that reduction of the C-1 amide to an alcohol is necessary before
successful esterification can occur.12 Given the small amount
of material available, further attempts were not made.
The molecular formula of tumonoic acid G (4) was determined
to be C25H43NO7 (m/z 470.3106, [M + H]+) by HRESIMS. While
1H and 13C NMR analyses revealed the presence of the same
proline and 2-methyldecanoyl moieties as tumonoic acid D (1),
the spectra were significantly more complex, with additional
downfield methine and methyl resonances being the most
distinctive new elements. COSY analysis established the presence
of a spin system incorporating two methines (δH 5.05, d; δH
2.05, m), two methyls (δH 0.91, t; δH 0.96, d), and a diaste-
reotopic methylene unit (δH 1.44, m; δH 1.28, m), consistent
Tumonoic acid F (3) (C21H37NO5, m/z 384.2736, [M + H]+)
also possessed a complex fatty acid moiety, two secondary
methyls (δH 1.14, d and 0.91, d), and an oxygenated methine
proton (δH 5.21, d) shifted further downfield than that of 2. In
combination with the presence of a deshielded methyl group (δH
1.99, s; δC 20.8) and an additional carbonyl resonance (δC 169.9),
this suggested the presence of an O-acetyl group at C-3, which
was confirmed by 2D NMR analysis (Figure 2). HMBC
correlations also positioned the secondary methyls at C-2 and
C-4, respectively. As for 2, the H-2 pentet of the fatty acid
moiety suggested an anti-arrangement for the 2-Me and 3-OAc
moieties. In an attempt to determine the absolute configuration
at C-3, a small sample of 3 was subjected to base hydrolysis

1532 Journal of Natural Products, 2008, Vol. 71, No. 9
Clark et al.
Figure 2. Elucidation of planar structures of tumonoic acids D-I (1-6). COSY correlations are indicated by bolded bonds, while key
HMBC correlations are designated by arrows (proton to carbon).
Table 2. NMR Data (CDCl3) for Tumonoic Acids G-I (4-6)a
tumonoic acid G (4)
tumonoic acid H (5)
tumonoic acid I (6)
δC
δH
δC
δH
δC
δH
fatty acid
1
2
3
4
5
6
7
8
176.1
37.8
33.8
27.3
29.1
29.3
29.7
31.8
22.6
14.1
16.7
175.9
37.7
33.8
27.4
29.5
29.3
29.7
31.8
22.6
14.0
16.8
175.9
35.2
41.2
30.1
36.1
29.0
22.9
14.4
2.56 dq (6.7, 6.7)
1.64 m 1.37 m
1.31 m
1.27 m
1.27 m
1.27 m
1.24 m
1.28 m
0.87 t (6.8)
1.11 d (6.7)
2.55 tq (6.8, 6.7)
1.64 m, 1.36 m
1.30 m
1.26 m
1.26 m
1.26 m
1.26 m
1.28 m
0.89 t (6.7)
1.10 d (6.7)
2.67 dq (6.8, 6.8)
1.64 ddd (13.5, 6.8, 6.8) 1.19 m
1.48 m
1.32 m, 1.02 m
1.31 m, 1.21 m
1.28 m
0.88 t (6.7)
9
10
2-Me
4-Me
proline
1′
2′
3′
4′
5′
16.9
20.1
1.08 d (6.7)
0.88 d (6.8)
172.3
59.1
29.5
25.0
47.1
172.2
59.2
29.2
25.0
47.1
172.1
59.2
29.2
24.9
47.0
4.50 dd (7.7, 4.5)
2.23 m, 2.05 m
2.18 m, 2.02 m
3.69 ddd (9.8, 7.4, 6.0)
3.60 ddd (9.8, 6.5, 6.5)
4.51 dd (8.1, 4.9)
2.24 m, 2.08 m
2.18 m, 2.01 m
3.70 ddd (9.9, 7.1, 6.0)
3.59 ddd (9.9, 9.6, 6.7)
4.52 dd (7.3, 4.5)
2.21 m, 2.11 m
2.20 m, 2.02 m
3.68 ddd (9.4, 7.3, 6.8)
3.61 ddd (9.4, 7.1, 6.9)
hydroxy acid 1
1′′
2′′
3′′
4′′
5′′
3′′-Me
169
168.9
77.2
29.8
18.6
169.5
75.0
36.6
25.8
11.6
14.2
75.5
36.6
25.8
11.5
14.2
5.05 d (3.3)
2.05 m
1.44 m, 1.28 m
0.91 t (7.4)
0.96 d (6.8)
5.06 d (4.1)
2.33 m
1.00 d (7.0)
5.20 d (3.3)
2.05 m
1.41 m, 1.25 m
0.92 t (7.3)
0.95 d (7.0)
17.0
0.98 d (6.8)
hydroxy acid 2
1′′′
2′′′
3′′′
4′′′
5′′′
3′′′-Me
n/ob
71.1
16.7
n/ob
79.0
30.2
18.5
n/ob
77.2
36.4
24.5
11.4
15.2
5.27 brq (5.1)
1.50 d (5.1)
5.04 d (5.4)
2.27 m
1.00 d (7.0)
5.09 d (4.6)
2.04 m
1.55 m, 1.30 m
0.92 t (7.3)
0.98 d (6.8)
17.7
0.99 d (7.1)
a
1H NMR spectra were recorded at 500 MHz, and 13C spectra at 75 MHz. b Not observed.
with the presence of an isoleucic acid residue. 13C NMR shifts
(Table 2) and HMBC correlations (Figure 2) confirmed this
assignment, and a strong correlation from H-2′′ to C-1 of the
proline residue was used to connect the isoleucic acid to the
proline unit. The second new spin system consisted of another
downfield methine connected to a deshielded methyl resonance.
While no HMBC correlations were observed from these protons,
the distinctive chemical shifts (δH 5.27, brq; δC 71.1 and δH
1.50, d; δC 16.7) were indicative of a lactic acid residue, which
was later confirmed by HPLC analysis as described below. The
terminal carboxylic acid group was not observed in the 13C NMR
spectrum, likely due to broadening caused by partial deproto-
nation of this functionality.
The 1H and 13C NMR spectra of tumonoic acid H (5)
(C26H45NO7, m/z 484.3254, [M + H]+) were very similar to those
of 4, with the same proline and 2-methyldecanoyl units again
present; however, the abundance of secondary methyls (δH 1.00,
d; 1.00, d; 0.99, d; 0.98, d) indicated the presence of two
2-hydroxyisovaleric acid residues in place of the isoleucic and lactic
acids of 4. 2D NMR analysis (Figure 2) was consistent with such
a structure, and later chiral HPLC analysis confirmed the presence
of the hydroxyisovaleric acid residues.

Natural Products Chemistry of Blennothrix cantharidosmum
Journal of Natural Products, 2008, Vol. 71, No. 9 1533
Figure 3. Phylogenetic relationships of filamentous marine cyanobacteria from 16S rRNA nucleotide sequences, including new sequences
from Blennothrix cantharidosmum and Schizothrix sp., determined in this study (performed using partial 1082-nucleotide sequence).
Likewise, tumonoic acid I (6) (C27H47NO7, m/z 498.3413, [M
+ H]+) was very similar to both 4 and 5. A second set of
resonances characteristic of isoleucic acid (Table 2) indicated
the replacement of the lactic acid residue of 4 with an additional
isoleucic acid residue. Compound 6 showed a much smaller
methylene envelope than that of the other tumonoic acids,
indicating the presence of a shorter fatty acid chain. Additionally,
a distinctive secondary methyl resonance (δH 0.88, d; δC 20.1)
indicated the presence of a second methyl branch along the fatty
acid chain; this could be positioned at C-4 using 2D NMR

1534 Journal of Natural Products, 2008, Vol. 71, No. 9
Clark et al.
correlations to give the structure shown (Figure 2). On the basis
of the wide separation of the 1H NMR resonances (δH 1.64, 1.19)
of H-3 of the dimethyloctanoic acid moiety, and comparison
with literature examples, we propose that the C-2 and C-4 methyl
groups are in a syn-orientation.14
bacterial communication. Thus, investigations were carried out into
the possible effects of these compounds on bacterial quorum sensing
and biofilm formation. The tumonoic acids showed no activity in
an assay utilizing a mutated E. coli strain that produces green
fluorescence protein in response to 3-oxohexanoyl homoserine
lactone addition.16 However, in an assay using a wild-type strain
of Vibrio harVeyi that regulates bioluminescence via quorum
sensing, several of these tumonoic acids (2-5) displayed modest
inhibition of bioluminescence without affecting bacterial growth,
with tumonoic acid F (3) being the most active (IC50 of 62 µM).
Morphological and Phylogenetic Analysis. Given the dif-
ficulties in the taxonomic classification of cyanobacteria and relative
paucity of 16S rRNA sequences for Blennothrix species in the
literature, a detailed morphological and phylogenetic analysis of
Blennothrix strain PNG05-4 (PNG-5/21/05-4) was carried out. The
strain was morphologically identified as B. (Hydrocoleum) canthari-
dosmum Koma´rek & Anagnostidis. The thallus formed a soft,
gelatinous, thick, 2 cm high, brown-gray mat covering the reef
substrate. The cyanobacterium was filamentous, and filaments were
solitary or several were enclosed together in common sheaths.
Furthermore, the trichomes were cylindrical and slightly constricted
at the crosswalls. Cells were disk-shaped, 21 µm wide and 3.1 µm
long. The end cells were cylindrical, with thickened outer cell walls.
The almost complete 16S rRNA gene sequence (1414 nucle-
otides) was PCR-amplified and sequenced (GenBank Acc. No.
EU253968). BLAST analysis of the 16S rRNA gene sequence
showed highest sequence identity (99.1%) with the B. (Hydroco-
leum) cantharidosmum strain GV58 from New Caledonia (GenBank
Acc. No. DQ883636).5 Due to the lack of full 16S rRNA sequences
for related species available in databases, including the Blennothrix
sp. from New Caledonia, a 1082-nucleotide stretch of the 16S rRNA
genes was used in the final multiple sequence alignment and
phylogenetic tree construction. The three main kinds of reconstruc-
tion methods [(1) the distance method by neighbor-joining (NJ),
(2) maximum parsimony (MP), and (3) likelihood method by
maximum-likelihood (ML)] were compared for their predicted
relationships. The overall topography of the different phylogenetic
trees was consistent with the ML method giving the most robust
bootstrap values; hence, this tree is displayed in Figure 3. The
clading was also stable using either the 1414- or the 599-nucleotide
stretches (see Supporting Information); however, use of the 1414
base pair stretch excluded several species of interest in the study.
In the final phylogenetic analysis, 43 16S rRNA cyanobacterial gene
sequences were included, including four from Microcoleus species.
Consistent with previous findings,5 species of Blennothrix and
Microcoleus are genetically very different and form separate clades
in the phylogenetic tree.
Stereochemical Investigations. After solving the planar
structures of 1-6, the next issues to be addressed were their absolute
configurations. This was determined for the proline residues of 1-7
by acid hydrolysis followed by derivatization with Marfey’s reagent
(FDVA) and subsequent LCMS analysis. This analysis revealed
that the proline unit in all seven compounds was L. The configura-
tions of the R-hydroxy acids of 4-6 were determined by chiral
HPLC. Analysis of the acid hydrolysates of 4-6 and comparison
with known standards revealed the following compositions: 4,
D-allo-isoleucic acid, L-lactic acid; 5, D-2-hydroxyisovaleric acid,
L-2-hydroxyisovaleric acid; 6, D-allo-isoleucic acid, L-isoleucic acid.
In order to determine the positions of the isomeric hydroxy acids
in 5 and 6, the natural products were derivatized with R-methyl-
benzylamine and the products subjected to base hydrolysis, yielding
only one free hydroxy acid. Analysis of these hydrolysates revealed
only D-2-hydroxyisovaleric acid for 5 and D-allo-isoleucic acid for
6, enabling the configurations of the hydroxy acids to be assigned
as shown.
The C-2 configuration of the fatty acid moiety of tumonoic acid
D (1) was determined by synthesis. Racemic 2-methyldecanoic acid
was first derivatized with L-proline methyl ester. The methyl ester
present in each component of the mixture was hydrolyzed under
basic conditions to yield two epimeric acids, 1 and 8. These were
separated using reversed-phase (RP) HPLC and characterized using
standard spectroscopic techniques. 1H and 13C NMR, IR, and [R]D
data were very similar for both epimers, and it was not possible to
conclusively distinguish the two solely on this basis. However, co-
injections of the synthetic epimers with natural 1 and analysis by
LCMS conclusively showed that the earlier eluting peak was the
natural epimer. Finally, each of the two synthetic tumonoic acids
was hydrolyzed under acidic conditions to give free 2-methylde-
canoic acid, which was purified by HPLC and its [R]D value
measured and compared with literature values.15 This sequence
revealed that 1 contained (2S)-2-methyldecanoic acid, whereas the
unnatural epimer 8 incorporated (2R)-2-methyldecanoic acid.
Although we did not conduct similar reaction sequences on the
other new tumonoic acids, we propose on biogenetic grounds that
4 and 5 share the same absolute configuration at C-2. As the known
tumonoic acid A (7) possesses the opposite 2R configuration, we
have not assigned the absolute configurations of the fatty acid
portions for 2, 3, or 6. Intriguingly, our isolate of tumonoic acid A
(7) was of 2R configuration, as in the original report {[R]D -79.4
(c 0.3, CHCl3); lit. [R]D -79 (c 1.1, CHCl3)12}, and this is opposite
the configuration at C-2 in compound 1.
Tumonoic acids A-C were originally isolated in 1999 along with
two methyl esters and were not reported to possess any significant
biological activities.12 The new tumonoic acids differ from these
previously isolated examples in the greater diversity of fatty acids
incorporated. All three tumonoic acids in the original isolation
possess the same 2,4-dimethyl-3-hydroxydodec-4-enoic acid moiety,
while those in the current study include a range of unusual fatty
acids.
Biological Activities. The tumonoic acids A (7) and D-I (1-6)
were assayed for anticancer, antimalarial, anti-Chagas, antileish-
mania, and antimicrobial (MRSA) activity. Tumonoic acid I (6)
displayed moderate activity in the antimalarial assay (IC50 ) 2 µM),
whereas none of the other naturally occurring analogues showed
any activity in this assay at a concentration of 10 µg/mL. It was
interesting to observe only 6 displaying such activity given the
strong structural similarities between 6 and the other isolated acids,
especially 4 and 5. It was also noted that several of these tumonoic
acids bore a close resemblance to homoserine lactones involved in
Schizothrix is a former genus that has also been assigned as a
subgenus of Microcoleus on the basis of the presence of multiple
trichomes within a sheath,7 and thus it bears a close resemblance
to Blennothrix. However, the 16S rRNA gene data for Schizothrix
species available in GenBank is even less extensive than that for
Blennothrix, with only a 599-nucleotide stretch being currently
available (Schizothrix calcicola UTEX, GenBank Acc. No.
AY271841). Hence, in the interests of further clarifying the
taxonomic relationships between these genera, a Schizothrix sp.
PNG5-22 (PNG-5/20/05-2) from our own collection was morpho-
logically and phylogenetically identified according to previously
described taxonomic methods, and its 16S rRNA gene sequence
was also included in this study. The thallus of this specimen was
soft, mucilaginous, hemispherical, 4 cm tall, and dark yellowish-
brown. The cyanobacterium was filamentous, with several cylindri-
cal trichomes within the same sheaths. The cells were isodiametric,
1.2 µm wide by 3.2 µm long.
A 1370-nucleotide stretch of the Schizothrix PNG5-22 16S rRNA
gene was PCR-amplified and sequenced (GenBank Acc. No.
1060443), and a 1082-nucleotide stretch included in the phyloge-

Natural Products Chemistry of Blennothrix cantharidosmum
Journal of Natural Products, 2008, Vol. 71, No. 9 1535
µL of 10× PfuUltra II 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 II fusion HS DNA polymerase (cat. 600760),
and 20.5 µL of dH2O. The PCR reaction was performed in an Eppendorf
Mastercycler gradient as follows: initial denaturation for 2 min at 95
°C, 30 cycles of amplification using 20 s at 95 °C, 20 s at 50 °C (16S
rDNA)/56 °C (ITS), and 15 s at 72 °C, and final elongation for 3 min
at 72 °C. PCR products were subcloned using the Zero Blunt TOPO
PCR cloning kit (cat. K2800-20SC) from Invitrogen into pCR-Blunt
II TOPO vector, transformed into TOPO cells, and cultured on LB-
Kanamycin plates. Plasmid DNA was isolated using the QIAprep spin
miniprep kit (cat. 27106) from Qiagen and sequenced in both directions
using pCR-Blunt II TOPO vector specific primers M13F and M13R
and the internal middle primers 359F and 758R as previously
described.22
Phylogenetic Analysis. The 16S rRNA gene sequences were visually
inspected, and a total DNA stretch of 1414/1082 nucleotides was aligned
together with 43 cyanobacterial species representing 14 major genera
of the order Oscillatoriales, obtained from GenBank (http://ww-
w.ncbi.nlm.nih.gov). The multiple sequence alignments were performed
using ClustalX, with standard gap opening and extension penalties
without gaps, and the aligned 16S rRNA gene sequences were used to
generate phylogenetic trees in MEGA 4.0. The phylogenetic relations
of the cyanobacterial 16S rRNA genes were compared by (1) the
distance method by neighbor-joining (NJ), (2) maximum parsimony
(MP), and (3) the likelihood method by maximum-likelihood (ML).
The evolutionarily distant cyanobacterium Nostoc punctiforme PCC73102
(GenBank Acc. No. AF027655) from the order Nostocales was used
as an outgroup.
netic analysis described above. Interestingly, this Schizothrix sp.
PNG5-22 appeared genetically very different from members of both
Microcoleus and Blennothrix (Figure 3). Thus, it appears that the
taxonomic groupings originally created using phycological criteria
and nomenclature are supported by this genetic-based analysis, and
this raises concerns about the recent regrouping of genera under
the bacteriological system.7
Conclusions. The tumonoic acids were originally isolated from
a Lyngbya majuscula/Schizothrix calcicola assemblage, as well as
a collection of L. majuscula, all collected in Guam.12 Given the
uncertainties involved in cyanobacterial taxonomy and the overall
morphological resemblance between Blennothrix, Schizothrix, and
Lyngbya, it is possible that the original tumonoic acid isolation was
in fact from a Lyngbya/Blennothrix assemblage.17 However, in the
absence of 16S rRNA data from the previous work it is impossible
to make a definitive assignment. The current study illustrates the
inadequacy of morphological identification in cyanobacterial tax-
onomy and demonstrates the complementary power of molecular
taxonomy in the classification of these organisms. Precise taxonomic
investigations are important in guiding chemical investigations of
some of the less well studied cyanobacteria, and these can lead to
the isolation of new metabolites such as those described herein.
Experimental Section
General Experimental Procedures. Optical rotations were measured
on a JASCO P1010 polarimeter. IR and UV spectra were recorded on
a Nicolet IR-100 FT-IR and Beckman-Coulter DU800 spectrophotom-
eter, respectively. NMR spectra were recorded on Varian Unity 300
and 500 MHz spectrometers with the solvent CDCl3 (δH at 7.26, δC at
77.0) used as an internal standard. High-resolution mass spectra were
acquired using an Agilent ESI-TOF mass spectrometer. LCMS analyses
were carried out on a Finnigan LCQ advantage mass spectrometer,
attached to a Finnigan Surveyor HPLC system. HPLC was performed
using Waters 515 pumps and a Waters 996 photodiode array detector,
under computer control using Empower software. Chemicals were
acquired from VWR, except for 2-methyldecanoic acid, which was
purchased from Narchem, and 1-ethyl-(3-dimethylaminopropyl)carbo-
diimide hydrochloride (EDAC ·HCl), purchased from Chem-Impex
International.
Collection. The Blennothrix and Schizothrix specimens were col-
lected by scuba in Papua New Guinea in May 2005. The Blennothrix
sp. PNG05-4 (PNG-5/21/05-4) was collected to the west of Duke of
York Island, near Kibil, at a depth of 30 ft (GPS coordinates S
04°07.270′, E 152°26.666′). The Schizothrix sp. PNG5-22 (PNG-5/20/
05-2) was collected on a reef to the west of the Credner Islands, near
Kerawara, at a depth of 40 ft (GPS coordinates S 04°12.922′, E
152°22.926′). The samples were stored in EtOH-seawater (1:1) at -20
°C until extraction, with a small (∼100 mg) tissue sample preserved
in 5 mL of RNA-later (Ambion) for genetic analysis. Voucher
specimens are available from WHG.
Morphological Characterization. Preliminary morphological and
taxonomic characterizations of the Blennothrix and Schizothrix speci-
mens were performed using a field microscope (RFM-2 microscope
from Richardson Technologies Inc.) and the taxonomic field guide by
Littler and Littler,18 while more accurate measurements and charac-
terizations were made with an Olympus BH-2 light microscope. The
following morphological parameters were evaluated: thallus morphol-
ogy, cell length, cell width (and cell length/width ratios), constriction
of cell walls, presence of specialized cells such as heterocysts, akinetes,
or calyptra, size and shape of trichomes, and characteristics of end cells.
Morphological identification was made in accordance with the phyco-
logical systems by Gomont,19 Geitler,20 and Koma´rek and Anagnos-
tidis.21
DNA Extraction, 16S rDNA Gene PCR-Amplification, and
Cloning. Genomic DNA was extracted from 40 mg of cleaned
cyanobacterial tissues using the Wizard Genomic DNA purification kit
(cat. A1120) according to the manufacturer’s specifications (Promega,
Madison, WI). The isolated genomic DNA was further purified using
Genomic-tips 20/G from Qiagen (cat. 10223). The 16S rRNA genes
were PCR-amplified from isolated DNA using the cyanobacteria-
specific primers, 106F and 1509R, as previously described.22 The
reaction volumes were 25 µL containing 0.5 µL of DNA (50 ng), 2.5
Extraction and Isolation. Approximately 64.5 g (dry weight) of
the alga was extracted repeatedly with CH2Cl2-MeOH (2:1) to yield
1.15 g of crude extract. The extract was fractionated by VLC using a
stepwise gradient with solvents of increasing polarity, from 100%
hexanes to 100% MeOH. The polar fractions eluting with 3:1
EtOAc-MeOH and 100% MeOH possessed activity against the H-460
cell line. These were further fractionated by RP solid-phase extraction
on Strata C18-E 500 mg cartridges using a stepwise gradient from 10%
MeOH-H2O to 100% MeOH. Further purification was carried out via
RP HPLC [Phenomenex Synergy-Hydro (4 µm particles, 21.2 × 250
mm), gradient elution from 45% CH3CN-H2O to 75% CH3CN-H2O
over 30 min], yielding the tumonoic acids D (1, 19.6 min, 1.8 mg), E
(2, 14.2 min, 1.7 mg), F (3, 24.5 min, 3.4 mg), G (4, 28.6 min, 8.0
mg), H (5, 31.1 min, 1.1 mg), I (6, 32.7 min, 5.5 mg), and A (7, 18.3
min, 7.6 mg) and a mixture containing lyngbyastatins 1 and 3 (33.8
mg, 15-17 min).
Tumonoic acid D (1): colorless oil; [R]D -24.3 (c 0.81, MeOH);
IR (neat) νmax 2957, 2927, 2856, 1725, 1614, 1468, 1447, 1195 cm-1
;
1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 75 MHz) data,
see Table 1; HRESI(+)MS m/z [M + H]+ 284.2211 (calcd for
C16H30NO3, 284.2220).
Tumonoic acid E (2): colorless oil; [R]D -24.8 (c 0.94, MeOH);
IR (neat) νmax 3381, 2960, 2927, 2874, 1718, 1619, 1448, 1201 cm-1
;
1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 75 MHz) data,
see Table 1; HRESI(+)MS m/z [M + H]+ 326.2321 (calcd for
C18H32NO4, 326.2326).
Tumonoic acid F (3): colorless oil; [R]D -173.3 (c 1.62, MeOH);
IR (neat) νmax 2959, 2927, 2856, 1746, 1626, 1461, 1461, 1373, 1238,
1
1195 cm-1; H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 75
MHz) data, see Table 1; HRESI(+)MS m/z [M + H]+ 384.2736 (calcd
for C21H38NO5, 384.2744).
Tumonoic acid G (4): colorless oil; [R]D -33.6 (c 0.69, MeOH);
IR (neat) νmax 2961, 2927, 2858, 1744, 1640, 1617, 1459, 1263, 1193,
1
1098 cm-1; H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 75
MHz) data, see Table 2; HRESI(+)MS m/z [M + H]+ 470.3106 (calcd
for C25H44NO7, 470.3112).
Tumonoic acid H (5): colorless oil; [R]D -42.6 (c 0.69, MeOH);
IR (neat) νmax 2963, 2928, 2855, 1741, 1642, 1610, 1463, 1427, 1208,
1192, 1023 cm-1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3,
75 MHz) data, see Table 2; HRESI(+)MS m/z [M + H]+ 484.3254
(calcd for C26H46NO7, 484.3269).
Tumonoic acid I (6): colorless oil; [R]D -40.6 (c 3.0, MeOH); IR
(neat) νmax 2964, 2929, 2878, 1742, 1647, 1625, 1460, 1198, 1132 cm-1
;
1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 75 MHz) data,
see Table 2; HRESI(+)MS m/z [M + H]+ 498.3417 (calcd for
C27H48NO7, 498.3413).

1536 Journal of Natural Products, 2008, Vol. 71, No. 9
Clark et al.
It should be noted that for many of the tumonoic acids, a second,
minor set of NMR signals was observed (<10% height of major peaks
in all cases). These were attributed to minor isomers caused by
cis-trans proline isomerization.
A small subsample of each of the synthetic tumonoic acids was
hydrolyzed at 110 °C in dioxane-6 M HCl (1:1) for 18 h. The
hydrolysate was extracted with EtOAc, the organic layer reduced under
vacuum, and the residue purified by HPLC using a Phenomenex
Synergi-Hydro (4 µm, 21 × 250 mm) column, eluting with a gradient
from 50% CH3CN-H2O to 100% CH3CN at a flow rate of 4 mL/min.
The optical rotations of the purified methyldecanoic acids were
measured and compared with literature values.15 Compound 1 yielded
(2S)-2-methyldecanoic acid ([R]D +12.3 (CHCl3, c 0.26); lit. +5.1),
while the unnatural epimer 8 gave (2R)-2-methyldecanoic acid
([R]D -9.8 (CHCl3, c 0.32); lit. -5.1).
Absolute Configuration of the Proline Residues of Tumonoic
Acids D-I. The tumonoic acids (∼0.1 mg each) were hydrolyzed in
6 N HCl at 110 °C for 18 h and then extracted twice with EtOAc. The
aqueous layers were dried under a stream of N2 before derivatization
with 1-fluoro-2,4-dinitrophenyl-5-L-valerylamide (FDVA) in acetone
(50 µL) and 0.1 M NaHCO3 (100 µL) in sealed vials at 90 °C for 5
min. The resulting solutions were analyzed using RP HPLC using an
HP LiChrospher 100 RP-18 (5 µm, 4 × 125 mm) column, eluting with
50% CH3CN-H2O at a flow rate of 0.8 mL/min. The L-proline
derivative eluted at 7.7 min, with the D-proline derivative eluting at
8.9 min.
Chiral HPLC Analysis. Isoleucic acids were synthesized according
to literature methods.23 The organic layers from the acid hydrolysates
of 4, 5, and 6 were evaporated to dryness and then redissolved in 2
mM CuSO4. Analysis was carried out using three separate conditions.
Condition 1: 100% 2 mM CuSO4, 0.8 mL/min, Phenomenex Chirex
3126 (4.6 × 250 mm) column; elution times (tR, min) for standards:
L-lactic acid (22.1), D-lactic acid (29.2) min. Condition 2: 12.5% CH3CN
in 2 mM CuSO4, 0.8 mL/min, Phenomenex Chirex 3126 (4.6 × 250
mm) column; elution times (tR, min) for standards: L-2-hydroxyisova-
leric acid (7.8), D-2-hydroxyisovaleric acid (12.1) min. Condition 3:
12.5% CH3CN in 2 mM CuSO4, 0.8 mL/min, Phenomenex Chirex 3126
(4.6 × 50 mm) column; elution times (tR, min) for standards: L-allo-
isoleucic acid (18.3), L-isoleucic acid (21.4), D-allo-isoleucic acid (28.5),
D-isoleucic acid (34.1).
epi-Tumonoic acid D (8): colorless oil; [R]D -120 (c 0.67, CDCl3);
IR (neat) νmax 2958, 2930, 2857, 1736, 1642, 1601, 1468, 1450, 1333,
1
1191 cm-1; H NMR (CDCl3, 500 MHz) δH 9.26 (br s, COOH), 4.60
(d, J ) 4.7 Hz, H-2′), 3.63 (br t, J ) 8.6 Hz, H-5′a), 3.53 (ddd, J )
9.2, 8.7, 6.1 Hz, H-5′b), 2.57 (dq, J ) 6.8, 6.6 Hz, H-2), 2.41 (m,
H-3′a), 2.03 (m, H-3′b), 2.01 (m, H-4′, 1.67 (m, H-3a), 1.41 (m, H-3b),
1.28-1.25 (m, H-4 - H-9), 1.15 (d, J ) 6.6 Hz, 2-Me), 0.87 (t, J )
6.8 Hz, H-10); 13C NMR (CDCl3, 75 MHz) δC 179.1 (C-1), 172.8 (C-
1′), 59.9 (C-2′), 47.7 (C-5′), 38.1 (C-2), 33.5 (C-3), 31.8 (C-8), 29.6,
29.4, 29.2 (C-5-C-7), 27.5 (C-4), 27.4 (C-3′), 24.8 (C-4′), 22.6 (C-9),
17.2 (2-Me), 14.0 (C-10); ESI(+)MS m/z 284 [M + H]+
.
Biological Activity. All metabolites were tested for activity in
antimalarial,24 antileishmania,25 anti-Chagas,26 cytotoxicity (NCI-H460
lung tumor,27,28 L1210, colon 38, H-116, H-125M, CEM, and CFU-
GM cell lines29), antibacterial (MRSA),30 and quorum sensing assays.
Procedures for all assays except quorum sensing are given in the
appropriate references.
Quorum sensing assays were carried out using marine broth (MB)
containing 1 g of yeast extract and 5 g of peptone per liter of synthetic
seawater (Instant Ocean). For agar plates, 15 g of agar (USB) per liter
of H2O was used. 4-Bromo-5-(bromomethylene)-2-(5H)-furanone was
synthesized as reported previously.31 The 3-oxohexanoylhomoserine
lactone (OHHL) is commercially available (Sigma-Aldrich, lot #
102K3851).
Vibrio harVeyi BB120 produces bioluminescence in response to the
autoinducers HAI-1, AI-2, and CAI-1. V. harVeyi was cultured in MB
at 28 °C while shaking. An overnight culture of V. harVeyi BB120 in
MB was diluted to an OD600 of 0.1. An aliquot (100 µL) of the diluted
culture was added to 5 mL of MB. Test compounds dissolved in DMSO
were added to the wells of an opaque microtiter plate (Nunc A/S,
Denmark; 0.0076 to 250 µM final concentration; 0.5% DMSO
concentration; negative control ) DMSO alone). The synthesized
brominated furanone was utilized as a positive control for inhibition
of luminescence (0.0076 to 250 µM). The diluted V. harVeyi culture
was added to the wells and mixed thoroughly. The plates were incubated
for 4 h and read on a Packard Lumicount microtiter plate reader
(Packard, UK). Relative luminescence units were normalized by the
optical density (600 nm) values obtained by transferring 100 µL to a
clear-bottom microtiter plate.
Escherichia coli JB525 is E. coli MT102 harboring the gfp plasmid
pJBA132. This mutant produces an unstable green fluorescent protein
in response to a C6-C8 N-acyl-homoserine lactone autoinducer. E.
coli JB525 was cultured in LB4 broth at 30 °C. Inhibition of
fluorescence was determined using a method modified from Anderson
et al.16 An overnight culture of E. coli JB525 in LB4 broth was diluted
to an OD450 of 0.25 with fresh media. Test compounds dissolved in
DMSO, or DMSO alone, were added to the wells of an opaque
microtiter plate (Nunc A/S, Denmark; 32 nM OHHL; 0.0076 to 250
µM test compounds; 1% DMSO concentration). The diluted E. coli
culture was added to the wells and mixed thoroughly. The plates were
incubated with shaking at 30 °C for 90 min. Fluorescence was
determined using a Packard Fluorocount microtiter plate reader (λ )
480 nm excitation, λ ) 515 nm emission). Relative fluorescence values
were normalized by optical density values obtained by transferring 100
µL to a clear-bottom microtiter plate (λ ) 450 nm, SpectraMax
Multimode microplate reader, Molecular Devices).
The hydrolysates of 4, 5, and 6 were analyzed by the methods above,
revealing the presence of L-lactic acid and D-allo-isoleucic acid in 4;
L-2-hydroxyisovaleric acid and D-2-hydroxyisovaleric acid in 5; and
L-isoleucic acid and D-allo-isoleucic acid in 6.
To define the positions of the hydroxy acids in 5 and 6, both were
separately derivatized using L-(-)-R-methylbenzylamine using the
following method: 0.2 mg of the natural product was dissolved in 100
µL of CH2Cl2, the solution was cooled to 0 °C, and solutions of L-(-)-
R-methylbenzylamine (12 µL), triethylamine (12 µL), EDAC ·HCl (18
µL), and DMAP (12 µL) (all solutions 5 mg/mL) were added
sequentially. The mixture was allowed to stir overnight, with LCMS
analysis indicating complete conversion to the desired products. The
crude products were evaporated to dryness, resuspended in CH2Cl2,
and washed sequentially with 0.1 M HCl, H2O, 1 M NaHCO3, H2O,
and brine. The organic layers were evaporated to dryness and the
residues resuspended in MeOH-1 M NaOH (1:1) and left to stir
overnight at room temperature. The MeOH was removed under N2,
and the aqueous residue acidified with 2 M HCl and then extracted
twice with EtOAc. The EtOAc was removed under N2, and the samples
were suspended in 2 mM CuSO4 and analyzed by the relevant chiral
HPLC methods as described above, revealing the presence of free D-2-
hydroxyisovaleric acid in 5 and D-allo-isoleucic acid in 6.
Synthesis of Tumonoic Acid D Epimers. Commercially available
racemic 2-methyldecanoic acid (50 mg) was dissolved in 2 mL of
CH2Cl2, the mixture was cooled to 0 °C, and L-proline methyl ester
(30 mg), triethylamine (30 mg), EDAC·HCl (51 mg), and DMAP (33
mg) were added sequentially. The mixture was allowed warm to room
temperature over 30 min and then stirred for 18 h, with LCMS analysis
indicating complete conversion to the desired products. The crude
products were evaporated to dryness and resuspended in EtOAc, the
slurry was filtered, and then the organic solubles were washed as above
to yield the crude product (52 mg). The mixture was then hydrolyzed
in EtOH-1 M NaOH (1:1) overnight at room temperature. The aqueous
layer was acidified with 1 M HCl and extracted twice with EtOAc,
and the organic layer was evaporated to dryness to yield the epimeric
acids 1 and 8 (40 mg).
The two epimeric products were separated by RP HPLC using a
Phenomenex Jupiter (10 µm, 21 × 250 mm) column, eluting with 45%
CH3CN-H2O at a flow rate of 4 mL/min to give tumonoic acid D (1,
14.2 mg) and the unnatural epimer 8 (13.8 mg). Co-injections with
natural 1 were used to confirm which was the natural isomer, using
the following HPLC conditions: HP LiChrospher 100 RP-18 (5 µm, 4
× 125 mm) column, eluting with 40% CH3CN-H2O at a flow rate of
0.8 mL/min. Elution times (tR, min): 1 (27.5), 8 (31.7).
Acknowledgment. We gratefully acknowledge the government of
Papua New Guinea for permission to make these collections, and T. L.
Simmons, R. Grindberg, and C. Sorrels for collection of the organisms.
We also thank S. Kelly, J. Wingerd, D. Kyle, and L. D. Urena for
conducting biological assays, and A. Pereira for recording the optical
rotation of tumonoic acid A. Financial support for this work came from

Natural Products Chemistry of Blennothrix cantharidosmum
Journal of Natural Products, 2008, Vol. 71, No. 9 1537
(15) Wallace, P. A.; Minnikin, D. E.; McCrudden, K.; Pizzarello, A. Chem.
Phys. Lipids 1994, 71, 145–162.
(16) Anderson, J. B.; Heydorn, A.; Hentzer, M.; Eberl, L.; Geisenberger,
O.; Christensen, B. B.; Molin, S.; Givskov, M. App. EnViron.
Microbiol. 2001, 67, 575–585.
the National Cancer Institute (CA100851) and the International
Cooperative Biodiversity Groups Program (grant number 1U01 TW
006634-01).
Supporting Information Available: Additional phylogenetic trees
using the full 1400-nucleotide sequences and truncated 599-nucleotide
sequence. NMR spectra showing all 1H NMR, 13C NMR, gCOSY,
gHSQC, and gHMBC spectra for tumonoic acids D-I (1-6). This
material is available free of charge via the Internet at http://pubs.acs.org.
(17) While cellular dimensions of the Blennothrix cantharidosmum and
Schizothrix sp. collections made in the current study were quite
dissimilar, dimensions are known to vary greatly for Schizothrix.19-21
.
(18) Littler, D. S.; Littler, M. M. South Pacific Reef Plants; Offshore
Graphics, Inc.: Washington, D.C., 2000.
(19) Gomont, M. Ann. Sci. Nat. Bot. Paris 1892, 15, 263–368.
(20) Geitler, L. In Rabenhorst’s Kryptogamen-Flora Von Deutchland,
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