Cytotoxic halogenated macrolides and modified peptides from the apratoxin-producing marine cyanobacterium Lyngbya bouillonii from Guam

Article abstract of DOI:10.1021/np1004032

Collections of the marine cyanobacterium Lyngbya bouillonii from shallow patch reefs in Apra Harbor, Guam, afforded three hitherto undescribed analogues of the glycosidic macrolide lyngbyaloside, namely, 2-epi-lyngbyaloside (1) and the regioisomeric 18E- and 18Z-lyngbyalosides C (2 and 3). Concurrently we discovered two new analogues of the cytoskeletal actin-disrupting lyngbyabellins, 27-deoxylyngbyabellin A (4) and lyngbyabellin J (5), a novel macrolide of the laingolide family, laingolide B (6), and a linear modified peptide, lyngbyapeptin D (7), along with known lyngbyabellins A and B, lyngbyapeptin A, and lyngbyaloside. The structures of 1-7 were elucidated by a combination of NMR spectroscopic and mass spectrometric analysis. Compounds 1-6 were either brominated (1-3) or chlorinated (4-6), consistent with halogenation being a hallmark of many marine natural products. All extracts derived from these L. bouillonii collections were highly cytotoxic due to the presence of apratoxin A or apratoxin C. Compounds 1-5 showed weak to moderate cytotoxicity to HT29 colorectal adenocarcinoma and HeLa cervical carcinoma cells.

Full text of DOI:10.1021/np1004032

1544
J. Nat. Prod. 2010, 73, 1544–1552
Cytotoxic Halogenated Macrolides and Modified Peptides from the Apratoxin-Producing
Marine Cyanobacterium Lyngbya bouillonii from Guam
Susan Matthew,†,‡ Lilibeth A. Salvador,†,‡ Peter J. Schupp,§ Valerie J. Paul,⊥ and Hendrik Luesch*,†
Department of Medicinal Chemistry, UniVersity of Florida, 1600 SW Archer Road, GainesVille, Florida 32610, UniVersity of Guam Marine
Laboratory, UOG Station, Mangilao, Guam 96923, and Smithsonian Marine Station, 701 Seaway DriVe, Fort Pierce, Florida 34949
ReceiVed June 17, 2010
Collections of the marine cyanobacterium Lyngbya bouillonii from shallow patch reefs in Apra Harbor, Guam, afforded
three hitherto undescribed analogues of the glycosidic macrolide lyngbyaloside, namely, 2-epi-lyngbyaloside (1) and
the regioisomeric 18E- and 18Z-lyngbyalosides C (2 and 3). Concurrently we discovered two new analogues of the
cytoskeletal actin-disrupting lyngbyabellins, 27-deoxylyngbyabellin A (4) and lyngbyabellin J (5), a novel macrolide of
the laingolide family, laingolide B (6), and a linear modified peptide, lyngbyapeptin D (7), along with known
lyngbyabellins A and B, lyngbyapeptin A, and lyngbyaloside. The structures of 1-7 were elucidated by a combination
of NMR spectroscopic and mass spectrometric analysis. Compounds 1-6 were either brominated (1-3) or chlorinated
(4-6), consistent with halogenation being a hallmark of many marine natural products. All extracts derived from these
L. bouillonii collections were highly cytotoxic due to the presence of apratoxin A or apratoxin C. Compounds 1-5
showed weak to moderate cytotoxicity to HT29 colorectal adenocarcinoma and HeLa cervical carcinoma cells.
Marine cyanobacteria have been attracting increasing attention
for probe and drug discovery due to the high incidence of
structurally novel bioactive secondary metabolites that complement
those known from terrestrial sources. These natural products are
predominantly modified peptides and depsipeptides, polyketides,
and peptide-polyketide hybrids, many of which are cyclic and
oftentimes halogenated.1 One intriguing characteristic of marine
cyanobacteria is that a single organism commonly produces several
distinct classes of natural products so that up to 10% of the genome
may be dedicated to secondary metabolism. One example of such
a “superproducer” is Lyngbya bouillonii.2,3 Various collections
carried out over the past two decades have consistently yielded
apratoxin A,4 lyngbyabellin A,5 lyngbyapeptin A,6 and, with
variable reproducibility, also lyngbyastatin 2,7,8 lyngbyabellin B,6
apratoxin B,9 and apramides A-G.10 Recently we found that the
secondary metabolite content of L. bouillonii may be depth-
dependent, because at the same site (Finger’s Reef, Guam) but at
greater depth (14 m instead of 2 m) apratoxin E rather than apratoxin
A was the major apratoxin produced by a morphologically identical
cyanobacterium.8 The latter, however, lacked the usually coexisting
snapping shrimp Alpheus frontalis that is known to use L. bouillonii
as food and tubular shelter.11-13
Previous investigations of L. bouillonii from Finger’s Reef in
Apra Harbor were guided by cancer cell viability assays, leading
to the isolation of the major cytotoxins. We have now meticulously
isolated other extract components that previously had escaped
isolation efforts due to low abundance, lower activity, and/or
complexity of the metabolite profile. A dereplication strategy was
utilized with the aid of LC-MS for initial characterization of
unknowns and identification of previously encountered compounds.
A sensitive 1 mm triple-resonance high-temperature superconduct-
ing (HTS) cryogenic probe was used to elucidate structures by
NMR.14 To investigate the chemical diversity of L. bouillonii within
Apra Harbor and to potentially identify new natural apratoxins to
complement our structure-activity relationship and mechanistic
studies,8,15 we examined another site (Western Shoals) that was a
habitat for an apparently similar cyanobacterium with largely
identical chemistry, yet it additionally yielded apratoxin C, previ-
ously isolated from a Palauan variety of this organism.9 Here we
describe the structure elucidation and initial biological evaluation
of seven new compounds (1-7) stemming from these collections,
all of which are related to known metabolites from L. bouillonii
(Figure 1). Six out of the seven compounds are halogenated, a
hallmark of many marine-derived metabolites. All extracts contained
apratoxin A as the major cytotoxic constituent (Figure 1).
Results and Discussion
Investigations of two L. bouillonii collections from Finger’s Reef
and one collection from Western Shoals (both sites located in Apra
Harbor, Guam) led to the isolation of lyngbyaloside, originally
identified from a L. bouillonii strain from Papua New Guinea,16
and three novel bromine-containing lyngbyaloside analogues, 2-epi-
lyngbyaloside (1), 18E-lyngbyaloside C (2), and 18Z-lyngbyaloside
C (3) (Figure 1). On the contrary, there was no evidence for the
presence of the structurally related macrolides lyngbouilloside17
and lyngbyaloside B18 produced by L. bouillonii varieties from
Papua New Guinea and Palau, respectively. In addition to lyng-
byabellins A and B,5,6,19 we also discovered two new members of
this structural class of L. bouillonii metabolites, 27-deoxylyngbya-
bellin A (4) and lyngbyabellin J (5), both bearing two chlorine atoms
characteristic of the lyngbyabellins (Figure 1). Laingolide B (6) is
a chlorinated analogue of the Papua New Guinea L. bouillonii
isolates laingolide20 and laingolide A21 (Figure 1), neither of which
has been identified yet in Guamanian or Palauan cyanobacteria.
The linear modified peptide lyngbyapeptin D (7) was found to be
coproduced with lyngbyapeptin A,6,22 albeit in minute quantities
relative to the parent compound, and readily decomposed to
compound 7a (Figure 1). The isolation was achieved by silica gel
column chromatography of extracts followed by one or more rounds
of reversed-phase HPLC.
The HRESI/APCIMS spectrum of 1 displayed two [M + Na] +
peaks at m/z 683.2410 and 685.2382 in equal intensity, suggesting
the presence of one bromine atom. Initial 2D NMR analysis of 1
in CDCl3 at 600 MHz (COSY, TOCSY, HSQC, HMBC) in
combination with HRESI/APCIMS analysis revealed that 1 has the
same planar structure as the co-isolated tricyclic glycoside macrolide
lyngbyaloside (Figure 1). The 1H and 13C NMR chemical shift data
for 1 were mostly identical to those for lyngbyaloside (Table 1).16
Significant differences were observed for H-4a and H-4b. NOESY
* To whom correspondence should be addressed. Tel: (352) 273-7738.
Fax: (352) 273-7741. E-mail: luesch@cop.ufl.edu.
† University of Florida.
‡ Contributed equally.
§ University of Guam Marine Laboratory.
⊥ Smithsonian Marine Station.
10.1021/np1004032  2010 American Chemical Society and American Society of Pharmacognosy
Published on Web 08/12/2010

Apratoxin-Producing Cyanobacterium Lyngbya bouillonii
Journal of Natural Products, 2010, Vol. 73, No. 9 1545
Figure 1. Natural products isolated from shallow-water L. bouillonii found in Apra Harbor (Guam) and closely related analogues found in
Palau (lyngbyaloside B) or Papua New Guinea (laingolide and laingolide A).
correlations between H3-20/H-4b and H-2/H-4a suggested that 1
has the opposite configuration at C-2 compared with lyngbyaloside,
with CH3-20 occupying an axial position (Figure 2). The van der
Waals interaction between H-4b and CH3-20 causes deshielding
of the axial methylene proton H-4b relative to the corresponding
proton in lyngbyaloside (∆δ +0.5 ppm). This configuration allows
a shielding 1,3-interaction between H-2 and H-4a, causing an upfield
shift of H-4a relative to the H-4a resonance in lyngbyaloside (∆δ
-0.24 ppm) (Figure 2). Thus, the chemical shifts of the diaste-
reotopic methylene protons H-4a and H-4b are very similar (∆δ 0.33
ppm). Because the configuration of the other stereocenters in lyngby-
aloside (which on the basis of NMR data were the same as for
compound 1) had not been assigned yet,16 we attempted to use
proton-proton coupling constants and NOESY data to propose a
stereostructure for both molecules. The chemical shifts for H-6a and
H-6b differed substantially (∆δ 1.18 ppm), suggesting that CH3-21 is
equatorial and H-8 axial, by analogy with the analysis above for the
configuration of C-2: a shielding 1,3-diaxial interaction exists between
H-6b and H-8, leading to a relative upfield shift of the H-6b resonance,
while the van der Waals interaction of H-6a with CH3-21 causes the
H-6b signal to appear more downfield (Figure 2). The splitting pattern
occupies a pseudoequatorial position (Figure 2). A cis geometry of
the double bond at C-11 and C-12 was evident from the 3JH,H coupling
constant of 10.6 Hz. NOESY correlations between H-12/H3-22, H-13/
H-15, and H-14b/H-15 implied a pseudoequatorial orientation for CH3-
22 and axial position for H-15 (Figure 2). Therefore, we propose a
2R*,3S*,5R*,7R*,8S*,9S*,13R*,15R* configuration for compound 1
and consequently a 2S*,3S*,5R*,7R*,8S*,9S*,13R*,15R* configuration
for lyngbyaloside (Figure 1). For other lyngbyalosides, the glycoside
portion could be related to the aglycone on the basis of NOE or
ROESY correlations,16,18 some of which were also observed here for
1. The structure depicted for 1 is the more likely enantiomer since
L-rhamnose was also found in related metabolites, aurisides A and
B.23 Furthermore, the specific rotations for all lyngbyaloside-like
molecules have at least the same sign.
The HRESI/APCIMS spectrum of 2 showed a [M + Na]+ peak
at m/z 671.2399 and an isotope peak of equal intensity at m/z
673.2381, suggesting the presence of one bromine atom. The
molecular formula of 2 was deduced as C30H49BrO10, with 6 degrees
of unsaturation. Detailed 1H and 2D NMR analysis in CDCl3 (Table
2) revealed that 2 is closely related to lyngbyaloside B, with the
same molecular formula on the basis of HRESI/APCIMS. HSQC,
HMBC, COSY, and TOCSY spectra suggested the presence of an
ester moiety (δC 172.3), a hemiketal (δC 96.6), a hexapyranoside
3
and JH,H coupling constants (dt 10.9, 2.9 Hz) of H-9 together with
the absence of a NOESY correlation with H-7 suggested that H-9

1546 Journal of Natural Products, 2010, Vol. 73, No. 9
Matthew et al.
Table 1. NMR Data for 2-epi-Lyngbyaloside (1) in CDCl3 (600 MHz)
a
δC
δH (J in Hz)
HMBC
1, 3, 20
NOESY
3-OH, H-4a
1
2
3
174.2, C
49.0, CH
98.2, C
2.63, q (7.0)
3-OH
4a
4b
5.15, s
1.96, m
1.63, m
H-2, H-7
H-2,H-5
H3-20
39.8, CH2
5, 6
5
5
6a
6b
69.4, CH
36.0, CH2
4.21, m
2.20, m
1.00, m
1′
H-4a, H-6a, H-7, H-1′
H-5, H3-21, H-1′
7
8
9
10a
10b
11
12
13
14a
14b
15
16
17
18
19
20
21
70.6, CH
44.1, CH
72.3, CH
27.8, CH2
3.73, td (10.6, 1.6)
1.86, m
3.93, dt (10.9, 2.9)
2.20, m
3-OH, H-5
H-9
H-8, H-10a, H3-21
H-9
H-11
H-10b
H3-22
H-15
H-16
H-15
1.90, m
123.4, CH
139.3, CH
28.3, CH
43.3, CH2
5.45, dt (10.6, 7.9)
5.37, t (10.6)
2.48, m
9
10, 22
1.66, ddd (-14.1, 6.3, 3.3)
1.45, brdd (-14.1, 11.0)
5.54, brdd (11.0, 6.3)
5.73, dd (15.3, 6.3)
6.15, dd (15.3, 10.9)
6.69, dd (13.5, 10.9)
6.39, d (13.5)
1.25, d (7.1)
0.90, d (7.0)
1.05, d (6.6)
5.02, d (1.0)
3.52, dd (3.1, 1.0)
3.50, s
3.46, dd (9.4, 3.1)
3.46, s
3.12, brt (9.4)
3.55, s
3.59, dq (9.4, 6.2)
1.29, d (6.2)
13
73.1, CH
132.3, CH
128.7, CH
136.4, CH
110.0, CH
11.2, CH3
10.3, CH3
19.3, CH3
94.7, CH
77.6, CH
59.2, CH3
81.0, CH
57.8, CH3
82.1, CH
60.9, CH3
68.0, CH
17.6, CH3
H-13, H-14b
H-14a
H-18
15, 18
19
17
2, 3
H-17
H-4b
H-6a, H-9
H-12
22
1′
2′
12
3′, 5′
H-5, H-6a, 2′-O-Me
2′-O-Me
2′-O-Me
3′
2′
H-1′
3′-O-Me′
3′-O-Me
4′
3′
3′, 5′, 6′, 4′-O-Me
4′
4′-O-Me
5′
6′
4′, 5′
a Deduced from HSQC and HMBC experiments.
Figure 2. Proposed stereostructure and selected key NOESY correlations for 2-epi-lyngbyaloside (1). Configurational difference from
lyngbyaloside is indicated in red. Other groups and protons with critical stereoelectronical effects discussed in the text are shown in blue.
∆δ values for H-4a/b and H-6a/b relative to the corresponding resonances in lyngbyaloside are shown in blue, and ∆δ values between
diastereotopic protons in 1 are depicted in green.
3
group (δC/δH 94.6/4.97, 77.7/3.46, 80.9/3.42, 82.1/3.08, 67.9/3.53),
and a terminally brominated conjugated hexadiene side chain (δC/
δH 137.7/66.3, 127.7/5.99, 137.7/6.63, 106.3/6.17 for C/H-16 to
19). In contrast to lyngbyaloside B,18 2 lacks a methyl group at
C-6 of the aglycone but is O-methylated at C-2′ of the glycoside;
planar structures of 3 and 2 are identical. NOESY data and JH,H
coupling constants, however, indicated that the conjugated diene
side chain of 3 has a different configuration from that of 2. The
3JH,H coupling constants for H-18/H-19 (7.0 Hz), H-17/H-18 (10.2
Hz), and H-16/H17 (15.4 Hz) indicated an E,Z configuration of
the diene side chain (Figure 1). This was further confirmed by
NOESY correlations between H-18/H-19 and H-16/H-18. Thus,
compound 3 was a regioisomer of compound 2.
3
thus both compounds are constitutional isomers (Figure 1). JH,H
coupling constants and key NOESY correlations between 3-OH/
H-7, H-5/H-1′, H-11/H-14a, and H-11/H-14b suggested that the
configuration of the macrolide portion of 2 is likely the same as in
lyngbyaloside B. The 3JH,H coupling constants for H-18/H-19 (13.9
Hz), H-17/H-18 (10.6 Hz), and H-16/H-17 (14.6 Hz) and NOESY
correlations between H-15/H-16, H-16/H-18, and H-17/H-19 sug-
gested an E,E configuration of the conjugated diene in 2.
An identical molecular formula of C30H49BrO10 and isotope
pattern at m/z 671.2410 and 673.2379 [M + Na]+ were obtained
for 3. Interpretation of NMR spectra (Table 2) revealed that the
Compound 4 had a molecular formula of C29H40Cl2N4O6S2 based
on HRESI/APCIMS with [M + H]+ isotopic peaks at m/z 675/
677/679 in the ratio of 5:4:1, indicating the presence of two chlorine
atoms in the molecule. 1D and 2D NMR data suggested a close
relationship of 4 with lyngbyabellins (Table 3, Figure 1).5,6,19,24-26
COSY analysis combined with characteristic HMBC correlations of
the methyl protons at δ 2.05 (H3-8) to the gem-dichloro carbon at δ 90.0
(C-7) and the methylene carbon at δ 49.3 (C-6) established the aliphatic

Apratoxin-Producing Cyanobacterium Lyngbya bouillonii
Journal of Natural Products, 2010, Vol. 73, No. 9 1547
Table 2. NMR Data for Lyngbyalosides C (2 and 3) in CDCl3, (600 MHz)
18E-lyngbyaloside C (2)
18Z-lyngbyaloside C (3)
a
a
δC
δH (J in Hz)
HMBC
NOESY
δC
δH (J in Hz)
1
172.3, C
172.3, C
2a
2b
3
46.9, CH2
2.49, d (-12.1)
2.38, d (-12.1)
3
H-4b
3-OH, H-4a
47.0, CH2
2.53, d (-12.1)
2.41, d (-12.1)
96.6, C
96.6, C
3-OH
4a
4b
4.57, brs
2.09, m
1.28, m
2, 4
4, 6
2
H-2b, H-7
H-2b, H-5
H-2a
4.56, s
2.15, m
1.33, m
41.5, CH2
41.4, CH2
5
6a
69.2, CH
37.7, CH2
4.10, m
1.89, m
1′
4, 7
H-4a, H-6a, H-7, H-1′
H-5
69.1, CH
37.9, CH2
4.10, m
1.92, m
6b
1.15, m
1.17, m
7
8a
69.8, CH
31.4, CH2
3.79, t (10.1)
1.70, m
9
11
3-OH, H-5
H-11
69.9, CH
31.5, CH2
3.79, t (10.1)
1.70, m
8b
1.45, m
1.46, m
9a
32.4, CH2
1.45, m
10, 12
H3-20
32.3, CH2
1.46, m
9b
1.32, m
1.38, m
10
11
12a
12b
13
37.0, CH
65.5, CH
44.1, CH2
1.48, m
4.26, m
2.77, d (-15.6)
1.44, dd (-15.6, 5.3)
11, 12
13
13, 14
10, 11
H-11
37.0, CH
65.7, CH
44.2, CH2
1.55, m
4.30, m
2.80, d (-15.4)
1.47, dd (-15.4, 6.0)
H-8a, H-10a, H-12a, H-14a, H3-20
H-11, H3-20, H3-21
86.2, C
86.3, C
14a
14b
15a
15b
16
17
18
19
20
38.6, CH2
1.97, m
1.63, m
2.17, m
2.18, m
5.76, dt (14.6, 5.9)
5.99, dd (14.6, 10.6)
6.63, dd (13.9, 10.6)
6.17, d (13.9)
0.80, d (7.0)
1.50, s
4.97, brs
3.46, dd (3.0, 1.0)
3.49, s
3.42, dd (9.0, 3.0)
3.48, s
3.08, dd (10.0, 9.0)
3.52, s
3.53, dq (10.0, 6.2)
1.25, d (6.2)
13, 16
16, 17
H-11
38.6, CH2
2.03, m
1.67, m
2.28, m
2.26, m
26.7, CH2
H-16, H-17
27.0, CH2
135.9, CH
127.7, CH
137.7, CH
106.3, CH
13.5, CH3
23.3, CH3
94.6, CH
77.7, CH
58.9, CH3
80.9, CH
57.6, CH3
82.1, CH
60.8, CH3
67.9, CH
17.8, CH3
14, 15, 18
15
16, 19
17, 18
11
10, 11
2′, 3′, 5′
3′, 2′-O-Me
H-15, H-18
H-15, H-19
H-16
138.8, CH
126.2, CH
132.6, CH
105.6, CH
13.3, CH3
23.4, CH3
94.5, CH
77.8, CH
58.9, CH3
81.0, CH
57.5, CH3
82.2, CH
60.7, CH3
68.0, CH
17.6, CH3
5.96, dt (15.4, 7.0)
6.44, dd (15.4, 10.2)
6.60, dd (10.2, 7.0)
6.04, d (7.0)
0.81, d (6.7)
1.50, s
4.97, d (1.0)
3.46, dd (2.8, 1.0)
3.50, s
3.44, dd (9.4, 2.8)
3.49, s
3.10, brt (9.4)
3.54, s
H-17
H-9a/9b, H-11, H-12a
H-12a
H-5, H-5′
H-3′, 3′-O-Me
21
1′
2′
2′-O-Me
3′
H-2′, H-5′
H-2′
3′-O-Me
4′
3′, 4′-O-Me
4′-O-Me, H-5′, H3-6′
H-4′
4′-O-Me
5′
4′
4′, 5′
H-1′, H-3′, H-4′, H3-6′
H-4′, H-5′
3.56, dq (9.4, 6.0)
1.26, d (6.0)
6′
a Deduced from HSQC and HMBC experiments.
chain as 7,7-dichloro-3-acyloxy-2,2-dimethyloctanoate.5,6,19 Further 2D
NMR analysis revealed that the molecule also has two 2-alkylthi-
azole-4-carboxylic acid units (C-11 to C-14 and C-22 to C-25), a
glycine moiety, and an isoleucine-derived unit as in lyngbyabellin
A, but an R-hydroxyisovaleric instead of the R,ꢀ-dihydroxyisova-
leric acid derived moiety in lyngbyabellin A (Table 3, Figure 1).
The molecular formula of 4 (C29H40Cl2N4O6S2) indicated the loss
of one oxygen atom compared to lyngbyabellin A, consistent with
the lack of the hydroxy group at C-27, as evident from our NMR
analysis. Otherwise NMR data were virtually identical to those for
lyngbyabellin A, indicating that the relative configuration of both
compounds was the same. Therefore compound 4 had to be the
deoxy derivative of lyngbyabellin A and was named 27-deoxylyng-
byabellin A (4). The specific rotation of 4 matched reported values
for natural5 and synthetic27,28 lyngbyabellin A, suggesting that it
had the exact absolute configuration as lyngbyabellin A.
The LRESIMS spectrum of 5 showed a characteristic isotope
pattern for a dichlorinated compound at m/z 886/888/890 (5:4:1).
HRESI/APCIMS ([M + Na]+ m/z 886.2198) implied a molecular
formula of C37H51Cl2N3O12S2. NMR analysis (Table 4) suggested
the presence of a gem-dichloro moiety (δC 90.0 for C-7), and
HMBC correlations ascertained that this moiety is part of a 7,7-
dichloro-3-acyloxy-2-methyloctanoate residue, found in certain
lyngbyabellin-type compounds.24-26,29 HMBC correlations of a
highly shielded methyl group (CH3-23: δC 7.88, δH 0.92) allowed
for the assignment of the R,ꢀ-dihydroxy-ꢀ-methylpentanoic acid
(Dhmpa, C-19 to C-24) unit in 5. The presence of two disubstituted
thiazole rings was determined from HMBC correlations of two
methine singlets at δH 8.24 (H-18) and 8.12 (H-12) to nonprotonated
heteroaromatic carbons C-17/19 (δC 145.9/165.2) and C-11/13 (δC
146.3/165.8), respectively. The cyclic core structure of 5 (Figure
1) was constructed on the basis of HMBC correlations between
H-20/C-1, H-15/C-16, and H-14/C-13 (Table 4). The planar cyclic
core of 5 is identical to that of lyngbyabellin C;24 however, acylation
of the hydroxy group at C-14 was evident from the low-field signal
for H-14 (δH 6.45) and HMBC correlation of H-14 to C-25 (Table
4). HMBC and COSY analysis revealed a bis-acylated γ-amino-
ꢀ-hydroxy acid putatively derived from condensation of valine and
acetate (Table 4). Although no HMBC correlations were observed
between C-32 and H-28 or 28-NH, the chemical shift of C-32 (δC
173.4) was indicative of an amide functionality. HMBC and COSY
correlations suggested that C-32 was derived from a butyric acid
moiety. Similarly, the hydroxy group was found to be acetylated.
The deduced planar structure of 5 (Figure 1) is closely related to
lyngbyabellins C-I24-26 and hence given the trivial name lyng-
byabellin J. The absolute configuration of 5 was established using
enantioselective analysis after ozonolysis in combination with acid
or base hydrolysis. Peaks corresponding to (2R,3S)-Dhmpa and
D-glyceric acid were observed in the base hydrolysate. The
configuration of the γ-amino-ꢀ-hydroxy acid unit at C-27 and C-28
was established by comparing the coupling constants of the
R-methylene signal (H-26a/b), in accordance with the recently
proposed NMR-based technique.30 H-26a showed a small coupling
(3JH,H ) 3.9 Hz) to H-27, while H-26b gave a large coupling (3JH,H
) 7.0 Hz), suggesting a syn arrangement at C-27 and C-28. The
absolute configuration at C-28 was established by acid hydrolysis

1548 Journal of Natural Products, 2010, Vol. 73, No. 9
Matthew et al.
Table 3. NMR Data for 27-Deoxylyngbyabellin A (4) in CDCl3
(600 MHz)
Table 4. NMR Data for Lyngbyabellin J (5) in CDCl3 (600
MHz)
a
a
C/H no.
δC
δH (J in Hz)
COSY
HMBC
C/H no.
δC
δH (J in Hz)
COSY
HMBC
1, 3
1
2
3
173.1, C
46.5, C
78.2, CH 5.30, dd
(10.3, 1.3)
29.5, CH2 1.71, m
1.40, m
22.3, CH2 1.60, m
49.3, CH2 2.22, m
2.01, m
1
2
3
4a
4b
5
6a
6b
7
173.9, C
43.0, CH
74.4, CH
31.3, CH2 1.81, m
1.75, m
21.0, CH2 1.69, m
49.1, CH2 2.20, m
2.10, m
3.00, dq (8.4, 6.8) H3-9
5.25, m
H-4a, H-4b
5
5
4, 6
4a
4b
5
6a
6b
7
H-3, H-4b, H2-5
H-3, H-4a, H2-5
H-4, H-6a, H-6b
H2-5, H-6b
H-6a, H-6b
H-5, H-6b
H-5, H-6a
3, 4, 6
5, 6, 7
H2-5, H-6a
90.7, C
37.3, CH3 2.09, s
14.4, CH3 1.22, d (6.8)
90.0, C
8
9
6, 7
1, 3
8
9
37.1, CH3 2.05, s
24.1, CH3 1.28, s
20.2, CH3 1.34, s
160.5, C
146.9, C
127.5, CH 8.06, s
168.3, C
7
H-2
b
1, 2, 3
1, 2, 3
10
11
12
13
14
15a
10
11
12
13
14
15
146.3, C
128.5, CH
165.8, C
70.4, CH
64.4, CH
8.12, s
11, 13
12, 14
6.45, t (6.3)
4.95, dd
(-11.3, 5.2)
4.50, dd
H-15a, H-15b
H-14, H-15b
13, 25
14, 16
55.1, CH 5.23, dd
15-NH, H-16
16, 17, 19
(7.7, 7.5)
15b
H-14, H-15a
14, 16
15-NH
16
7.26, d (10.4) H-15
(-11.3, 6.8)
40.1, CH 1.96, m
H-15, H-17a, H-17b 15, 17, 19
H3-19
16
17
18
19
20
21
22
23
24
25
26a
160.3, C
145.9, C
130.0, CH
165.2, CH
74.4, CH
74.0, C
17a
17b
18
19
20
25.4, CH2 1.50, m
1.13, m
H-16, H-17b, H3-18 16, 18
H-16, H-17a, H3-18
8.24, s
5.70, s
1.64, m
17, 19
1
11.2, CH3 0.92, t (7.3)
14.9, CH3 0.77, d (6.8)
168.3, C
H-17
H-16
17
16
31.0, CH
H-23
H-22
21a
42.9, CH2 4.61, dd
(-16.9, 8.8)
21-NH
20, 22
23, 25
7.9, CH3 0.92, t (6.2)
21
21
21.5, CH
169.4, C
1.07, s
21b
3.75, dd
(-16.9, 4.8)
7.97, br
36.6, CH2 2.79, dd
H-26b, H-27
H-26a, H-27
25, 27
25, 27
25, 26
21-NH
22
23
24
25
H-21
(-16.1, 3.9)
2.70, dd
(-16.1, 7.0)
5.22, m
160.6, C
148.7, C
26b
27
124.7, CH 8.20, s
168.5, C
69.6, CH
55.1, CH
28.0, CH
H-26a, H-26b,
H-28
26
76.7, CH 5.85, d (9.9)
H-27
1, 25, 27,
28, 29
26, 28, 29
28
28-NH
29
30
31
4.15, m
5.65, d (10.1)
1.85, m
H-27, 28-NH
H-28
27
32.9, CH 2.34, m
H-26, H3-28,
H3-29
H3-30, H3-31
H-29
H-29
28
29
29
20.0, CH3 0.93, d (7.5)
16.5, CH3 0.87, d (7.4)
173.4, C
28
29
18.7, CH3 0.95, d (6.6)
18.6, CH3 1.10, d (6.6)
H-27
H-27
26, 27
26, 27
32
33
34
35
36
38.9, CH2 2.12, t (7.4)
19.2, CH2 1.60, m
13.8, CH3 0.87, t (7.9)
170.7, C
H-34
H2-33, H2-35
H2-34
32
34
36
a Deduced from HSQC and HMBC experiments.
(leading to partial dehydration of the liberated hydroxy acid),
subsequent ozonolysis, oxidative workup, and analysis of the
liberated valine unit. Enantioselective HPLC-MS established the
presence of L-Val in the hydrolysate. Comparison of the coupling
constant between H-2 and H-3 (8.4 Hz) with data for lyngbyabellin
E (9.5 Hz) and hectochlorin (7.4 Hz) suggested that 5 should have
the same relative configuration at C-2 and C-3 and likely the same
absolute configuration, as all lyngbyabellins isolated to date have
a 3S configuration. These data support the absolute configuration
of lyngbyabellin J (5) as 2S,3S,14R,20R,21S,27R,28S.
Compound 6 was deduced to have a molecular formula of
C20H32ClNO3 on the basis of NMR and HRESI/APCIMS (m/z
370.2147 for [M + H]+). The presence of one chlorine atom was
consistent with the [M + H]+ isotope ion cluster at m/z 370/372 in
a 3:1 ratio. The final structure assignment of 6 was achieved through
1D and 2D NMR experiments including COSY, HSQC, HMBC,
and NOESY (Table 5). This compound possessed a 15-membered
macrocyclic lactone with enamide and tert-butyl functionalities
observed for the two laingolides reported from the same species
collected at Laing Island in Papua New Guinea (Figure 1).20,21
Compound 6, hence termed laingolide B, was more similar to
laingolide A21 because of the lack of a methyl group at C-4;
however, the methyl group at C-7 in laingolide A was replaced by
an exocyclic vinyl chloride, a group commonly found in cyano-
bacterial compounds such as in many ubiquitously occurring
malyngamides.31-35 The 13C NMR chemical shifts were charac-
37
21.0, CH3 2.04, s
a Deduced from HSQC and HMBC experiments. b Not observed.
teristic for an exomethylene group (δC7 138.0, δC17 112.8), yet the
signal for H-17 integrated only for one proton due to the chlorine
substitution. HMBC correlations from protons at H2-8 (δ 2.32/2.36)
to C-6, C-7, and C-17 as well as from the olefinic proton singlet at
H-17 (δ 5.80) to C-6 (δ 30.0), C-7 (δ 138.0), and C-8 (δ 33.3)
allowed unambiguous placement of this functionality (Table 5). The
E geometry of the vinyl chloride in 6 was deduced on the basis of
NOESY cross-peaks from H-17 to H-8b and H-9. Laingolide B
(6) underwent degradation over time similar to its reported
analogues, thus hampering the determination of the absolute
configuration at its two asymmetric centers.
The LRESIMS spectrum of lyngbyapeptin D (7) showed a
pseudomolecular ion peak for [M + Na]+ at m/z 706.5, consistent
with the molecular formula C36H53N5O6S postulated on the basis
1
of NMR data (Table 6). The H NMR spectrum of 7 showed two
O-Me singlets for methyl groups deshielded by anisotropy from
unsaturated systems (δH 3.49 and 3.74) and three N-Me singlets
for secondary amides (δH 2.95, 2.85, and 2.66) and suggested the
presence of five C-Me groups, one of which was deshielded by an
adjacent π system (δΗ 2.19) and attached to a nonprotonated carbon
(singlet). 2D NMR analysis inclusive of COSY, HSQC, and HMBC
(Table 6) suggested that compound 7 was comprised of three

Apratoxin-Producing Cyanobacterium Lyngbya bouillonii
Journal of Natural Products, 2010, Vol. 73, No. 9 1549
Table 5. NMR Data for Laingolide B (6) in CDCl3 (600 MHz)
b
C/H no.a
δC
δH (J in Hz)
COSY
HMBC
1
2
175.8, C
35.0, CH
35.7, CH2
3.02, ddq (10.8, 10.6, 6.5)
1.63, m
1.44, m
1.40, m
1.18, m
H-3a/3b, H3-16
1, 3, 4, 16
1, 2, 4, 5, 16
3a
3b
4a
4b
5a/5b
6a
6b
7
H-2, H-3b, H-4a/4b
H-2, H-3a, H-4a/4b
H-3a/3b, H-4b, H-5
H-3a/3b, H-4a
26.0, CH2
3, 5, 6
26.0, CH2
30.0, CH2
1.40, 2H, m
2.46, m
1.90, m
H-4a/4b, H-6a/6b
H-5, H-6b, H-17
H-5, H-6a, H-17
4, 5, 7, 8, 17
138.0, C
8a
8b
9
33.3, CH2
2.32, m
2.26, m
4.96, dd (9.6, 1.8)
H-8b, H-9, H-17
H-8a, H-9, H-17
H-8a/8b
6, 7, 9, 17, 18
7, 9, 10, 11, 19
11, 13, 14
77.6, CH
171.7, C
37.2, CH2
11
12a
12b
13
14
16
17
18
19
20
3.09, ddd (-11.4, 5.6, 1.5)
2.94, dd (-11.4, 10.8)
5.10, ddd (13.8, 10.8, 5.6)
6.97, d (13.8)
1.16, d (6.5)
5.80, s
H-12b, H-13, H-14
H-12a, H-13, H-14
H-12a/12b, H-14
H-12a/12b, H-13
H-2
103.6, CH
133.1, CH
17.7, CH3
112.8, CH
34.3, C
11, 12, 14
1, 12, 13, 20
1, 2, 3
H-6a/6b, H-8a/8b
6, 7, 8
(25.8 × 3), CH3
(0.93, s) × 3
9, 18
1, 13
30.3, CH3
3.10, s
a Nomenclature for laingolide B adapted from laingolide and laingolide A.20,21 b Deduced from HSQC and HMBC experiments.
Table 6. NMR Data for Lyngbyapeptin D (7) in CDCl3 (600 MHz)
a
unit
C/H no.
δC
δH (J in Hz)
COSY
HMBC
Mba
1
166.8, C
2
3
4
89.8, CH
5.12, s
H3-4
H-2
b
19.0, CH3
51.0, CH3
169.0, C
2.19, s
3.49, s
O-Me
1
N,O-diMe-Tyr
2
54.4, CH
34.6, CH2
5.75, dd (9.5, 6.2)
3.25, dd (-13.3, 9.5)
2.71, dd (-13.3, 6.2)
H-3a, H-3b
H-2, H-3b
H-2, H-3a
1, 3
2, 4, 5/9
3a
3b
4
129.8, C
5/9
6/8
7
130.7, CH
114.0, CH
158.6, C
7.17, d (8.2)
6.76, d (8.2)
H-6/8
H-5/9
6/8, 7
5/9, 7
O-Me
N-Me
1
54.5, CH3
31.1, CH3
171.1, C
3.74, s
2.95, s
7
2, 1 (Mba)
N-Me-Leu
2
50.7, CH
37.9, CH2
5.48, m
1.65, m
1.50, m
1.35, m
0.93, d (6.5)
0.93, d (6.5)
2.85, s
H-3a, H-3b
H-2, H-3b, H-4
H-2, H-3a
H-3a/3b, H3-5, H3-6
H-4
1, 3
2, 4
3a
3b
4
5
6
24.5, CH
23.3, CH3
22.1, CH3
30.0, CH3
169.6, C
5, 6
3, 4, 6
3, 4, 5
2, 1 (N,O-diMe-Tyr)
H-4
N-Me
1
N-Me-Val
2
3
4
5
59.9, CH
27.6, CH
18.7, CH3
19.1, CH3
30.3, CH3
171.4, C
4.88, d (11.5)
2.08, m
0.57, d (6.5)
0.88, d (6.5)
2.66, s
H-3
1, 3, 1 (N-Me Leu)
2
2, 3, 5
2, 3, 4
H-2, H3-4, H3-5
H-3
H-3
N-Me
2
2, 1 (N-Me-Leu)
Pro-thz
4
5
6
7a
7b
8a
8b
9a
9b
141.1, CH
119.0, CH
58.3, CH
31.8, CH2
7.69, d (2.5)
7.24, d (2.5)
5.45, m
2.31, m
2.24, m
2.13, m
1.99, m
3.88, m
3.77, m
H-5
H-4
H-7a, H-7b
H-6, H-7b, H-8a, H-8b
H-6, H-7a
H-7a, H-8b, H-9a, H-9b
H-7a, H-8a, H-9a, H-9b
H-8a, H-8b, H-9b
H-8a, H-8b, H-9a
2
24.3, CH2
47.5, CH2
a Deduced from HSQC and HMBC experiments. b Not observed.
methylated proteinogenic amino acid residues, namely, N,O-diMe-
tyrosine, N-Me-leucine, and N-Me-valine, and a proline-derived
pyrrolidine substituted with a thiazole ring (Pro-thz). In addition 7
showed evidence for the presence of a 3-methoxy-2-butenoic acid
(Mba) moiety. HMBC analysis together with NOESY experiments
enabled the sequencing of all units as depicted for 7 (Figure 1),
and the NOESY cross-peak between the O-Me and olefinic (H-2)
signal of the Mba unit indicated an E configuration of the double
bond. Thus compound 7 is a close analogue of lyngbyapeptin A
where an N-Me-Ile was replaced with N-Me-Val (Figure 1).6,22

1550 Journal of Natural Products, 2010, Vol. 73, No. 9
Matthew et al.
obtained by Gerwick and co-workers, who previously probed the
effect of hydroxylation at that position in other lyngbyabellins and
found no major differences.26 The configuration of the hydroxy
acid-derived unit esterified to the 7,7-dichloro-3-acyloxy-2-methyl-
octanoic acid residue (here Dhmpa) did not have a profound effect
on the activity, as previously observed.25 Furthermore, the cyto-
toxicity of cyclic and acyclic lyngbyabellins appears to be
similar.25,26 As mentioned above, laingolide B (6) and lyngbyapep-
tin D (7) decomposed during our characterization studies, preventing
us from testing any bioactivity of these compounds.
In summary, L. bouillonii from Apra Harbor, Guam, continues
to be a source of novel secondary metabolites, particularly
halogenated compounds. In addition to the previously reported
chemical diversity of structures from this cyanobacterium, the
various compounds described here document the tremendous
biosynthetic potential of cyanobacteria to produce small “focused”
libraries of structural analogues, which may be exploited for drug
discovery.
Figure 3. ESIMS/MS of the lyngbyapeptin D degradation product
7a.
Table 7. Cytotoxic Activity (IC50, µM) of Lyngbyalosides and
Lyngbyabellins from Apra Harbor (Guam) against Two Cancer
Cell Lines
Experimental Section
General Experimental Procedures. Optical rotations were measured
on a Perkin-Elmer 341 polarimeter. UV spectra were recorded on a
SpectraMax M5 (Molecular Devices). H and 2D NMR spectra were
1
structural class
lyngbyalosides
compound
HT29
HeLa
recorded in CDCl3 on a Bruker Avance II 600 MHz spectrometer
equipped with a 1 mm triple-resonance high-temperature supercon-
ducting (HTS) cryogenic probe14 using residual solvent signals (δH 7.26;
δC 77.0 ppm CDCl3) as internal standards. HSQC and HMBC
experiments were optimized for JCH ) 145 and JCH ) 7 Hz,
respectively. HRMS data were obtained using an Agilent LC-TOF mass
spectrometer equipped with an APCI/ESI multimode ion source
detector. Enantioselective LC-MS data were obtained on a 3200 Q
TRAP (Applied Biosystems) connected to a Shimadzu LC system.
ESIMS/MS data were obtained on a 3200 Q TRAP by direct infusion
using a syringe driver.
lyngbyaloside
37
35
2-epi-lyngbyaloside (1)
18E-lyngbyaloside C (2)
18Z-lyngbyaloside C (3)
lyngbyabellin A
27-deoxylyngbyabellin A (4)
lyngbyabellin B
38
13
>100
0.047
0.012
1.1
33
9.3
53
0.022
0.0073
0.71
0.041
1
n
lyngbyabellins
lyngbyabellin J (5)
0.054
Compound 7 suffered decomposition to 7a by O-demethylation and
subsequent tautomerization of the enol to the corresponding ketone
(Figure 1). This was verified by HRESI/APCIMS analysis of the
decomposition product (7a), which showed a pseudomolecular ion
peak at m/z 692.3450 for [M + Na]+, in agreement with the
molecular formula C35H51N5O6S. The structure and amino acid
sequence was further confirmed by ESIMS/MS of 7a (Figure 3).
The absolute configuration of 7a (and thus indirectly of 7) was
established using enantioselective HPLC-MS analysis following
ozonolysis, oxidative workup, and acid hydrolysis. Peaks corre-
sponding to N-Me-L-Val, N-Me-L-Asp (from degradation of N,O-
diMe-L-Tyr), N-Me-L-Leu, and L-Pro were detected, indicating the
all-S configuration as in lyngbyapeptins A-C.6,22,24
Biological Material. The cyanobacterium Lyngbya bouillonii from
Apra Harbor, Guam, was collected by snorkeling or scuba in shallow
waters at Finger’s Reef and Western Shoals. At both sites the
cyanobacterium was encountered with the shrimp Alpheus frontalis.
Finger’s Reef collections were carried out in May 2005 (VP) and in
May 2007 (1-4 m depth, SL-1). The VP collection is a re-collection
of VP417 (16S rRNA gene sequence has been deposited under GenBank
accession nos. AY049750 and AY049751),9 and a voucher specimen
is located at the Smithsonian Marine Station. A voucher sample of
SL-1 has been given the accession number GH0011398 in the GUAM
herbarium. A morphologically identical cyanobacterium containing the
shrimp was collected from Western Shoals in January 2007 (1-3 m
depth, WSL-1). A specimen of the WSL-1 collection is deposited in
the GUAM herbarium (accession number GH0011397).
Dereplication Strategy. Cyanobacterial samples were fractionated
as described below. Fractions from Si gel chromatography were
analyzed by HPLC-PDA-MS and 1H NMR and compared with authentic
standards of all compounds previously isolated from L. bouillonii from
Apra Harbor, Guam.4-10,25 Novel chlorinated and brominated com-
pounds were rapidly identified on the basis of their characteristic isotope
clusters of pseudomolecular ions, and the close relationship to known
lyngbyalosides and lyngbyabellins was established by 1H NMR analysis.
Extraction and Isolation. L. bouillonii collected in 2005 from
Finger’s Reef (VP) was extracted with CH2Cl2 and MeOH (2:1) and
the extract (10 g) chromatographed on silica gel with CH2Cl2 containing
increasing concentrations of i-PrOH to afford 16 fractions. Fraction 5
(2% i-PrOH in CH2Cl2; 100 mg) and fractions 6 and 7 (5% i-PrOH in
CH2Cl2; 71 and 70 mg) were individually subjected to semipreparative
HPLC (Phenomenex Phenyl-hexyl, 250 × 10 mm, 5 µ, 2.0 mL/min;
PDA detection) using a MeOH-H2O linear gradient (90-100% MeOH
for 30 min and 100% MeOH for 10 min). Fractions were pooled on
the basis of retention times, 1H NMR analysis, and low-resolution MS
measurements to afford mixtures of lyngbyabellins A and B, lyng-
byapeptin A, apratoxin A, and mixtures of lyngbyalosides (tR 14-16
min; ∼4 mg). Lyngbyalosides were purified by reversed-phase semi-
preparative HPLC (Phenomenex Synergi Hydro-RP, 250 × 10 mm, 5
µm, 2.0 mL/min; PDA detection) using the same program as described
above to afford 2-epi-lyngbyaloside (1) (tR 14.3 min, 0.5 mg), 18E-
Lyngbyalosides17,18 and lyngbyabellins5,6,19,26 have been re-
ported to exhibit weak to moderate cytotoxicity toward cancer cells,
respectively. Consequently we tested these compounds for anti-
proliferative activity against two cancer cell lines (Table 7). As
expected, lyngbyalosides were only weakly active. Lyngbyaloside
and its 2-epimer 1 had almost identical IC50 values of 33-38 µM
against HT29 colorectal adenocarcinoma and HeLa cervical car-
cinoma cells, indicating that the configuration at C-2 is not critical
for cytotoxicity. Compound 2 was approximately 3-fold more active
and the most potent of the lyngbyalosides tested, while the C-18
regioisomer 3 was the least active and approximately 5-fold less
potent against HeLa cells compared with 2 (Table 7). Minor toxicity
was noted against HT29 cells at the highest concentration tested
(100 µM). However, it was insufficient to provide an IC50 value,
suggesting that the orientation of the bromine atom is a considerable
determinant for cytotoxicity.
The lyngbyabellins are known to disrupt the actin cytoskeleton5,26
and exhibited mostly nanomolar cytotoxicity in our assays. Lyng-
byabellin B was the least active of this structural class, with IC50
values near 1 µM (Table 7). Lyngbyabellin A, its deoxy analogue
4, and lyngbyabellin J (5) possessed similar activity, with 4 being
slightly more potent (Table 7). The comparable activity for
lyngbyabellin A and deoxy analogue 4 is consistent with data

Apratoxin-Producing Cyanobacterium Lyngbya bouillonii
Journal of Natural Products, 2010, Vol. 73, No. 9 1551
lyngbyaloside C (2) (tR 15.5 min, 2.5 mg), 18Z-lyngbyaloside C (3)
(tR 14.7 min, 0.2 mg), and lyngbyaloside (tR 16.0 min, 0.2 mg).
The L. bouillonii sample collected from Finger’s Reef in 2007 (SL-
1) was freeze-dried and extracted with EtOAc-MeOH (1:1) to yield
16.1 g of crude material. The extract was subjected to flash chroma-
tography on silica gel, eluting with CH2Cl2 followed by increasing
concentrations of i-PrOH in CH2Cl2 and finally with MeOH. The
fractions that eluted with 4% i-PrOH (305 mg), 6% i-PrOH (58 mg),
and 8% i-PrOH (24.8 mg) were subjected to semipreparative reversed-
phase HPLC (Phenomenex Ultracarb, ODS 250 × 10 mm, 5 µm, 3.0
mL/min; PDA detection; isocratic 80% MeCN(aq) for 30 min;
80-100% MeCN for 30-40 min; and 100% MeCN for 40-60 min)
to afford several impure fractions (tR 3-8 min, 13 mg; tR 9-12 min,
7.4 mg; tR 13-17 min, 3 mg; tR 17 min, 6.4 mg; tR 20-21 min, 4.6
mg) along with a lyngbyapeptin A-enriched fraction (tR 12.0 min; 20.6
mg), mixtures of lyngbyalosides (tR 16-17.0 min; 6.4 mg), laingolide
B (6) (tR 21.0 min; 3.5 mg), and apratoxin A (tR 22.0 min; 1.0 mg).
The collection of L. bouillonii from Western Shoals (WSL-1) was
freeze-dried and then extracted with EtOAc-MeOH (1:1) to yield 5.5 g
of organic extract, which was subjected to flash chromatography on
silica gel as described above. The fractions eluted with 4% i-PrOH
(209 mg), 6% i-PrOH (50 mg), and 8% i-PrOH (15.7 mg) were further
separated by semipreparative reversed-phase HPLC as described above
for SL-1 to yield several impure fractions (tR 3-11 min; 18.6 mg) along
with semipure lyngbyapeptin A (tR 12.5 min; 13.8 mg), lyngbyalosides
mixtures (tR 16-17.0 min; 6.2 mg), laingolide B (6) (tR 21.0 min; 4.0
mg), and apratoxin A (tR 22.0 min; 1.1 mg). Another fraction that eluted
with 4% i-PrOH from silica gel (50 mg) also yielded minor amounts
of apratoxin C (tR 19.0 min; 0.5 mg) in addition to the above-mentioned
compounds.
13C NMR, COSY, and HMBC data, see Table 4; HRESI/APCIMS m/z
[M + Na]+ 886.2198 (calcd for C37H5135Cl2N3O12S2Na, 886.2183), m/z
886/888/890 (100:80:23 [M + Na]+ ion cluster).
Laingolide B (6): colorless, amorphous solid; [R]20D +170 (c 0.07,
MeOH); UV (MeOH) λmax (log ε) 240 (4.13) nm; 1H NMR, 13C NMR,
COSY, and HMBC data, see Table 5; HRESI/APCIMS m/z [M + H]+
370.2147 (calcd for C20H3335ClNO3, 370.2147), m/z 370/372 (100:33
[M + H]+ ion cluster).
Lyngbyapeptin D (7): colorless, amorphous solid; [R]20 -60 (c
D
0.01, MeOH); UV (MeOH) λmax (log ε) 210 (4.59), 250 (4.42) nm; 1H
NMR, 13C NMR, COSY, and HMBC data, see Table 6; LRESIMS
m/z [M + Na]+ 706.5 (calcd for C36H53N5O6SNa, 706.3614).
Compound 7a: 1H NMR (600 MHz, CDCl3) δ 7.73 (d, J ) 3.0 Hz,
1H), 7.27 (d, J ) 2.8 Hz, 1H), 7.16 (d, J ) 8.4 Hz, 1H), 6.76 (d, J )
8.9 Hz, 1H), 5.75 (dd, J ) 9.2, 5.9 Hz, 1H), 5.48 (m, 2H), 4.89 (d, J
) 11.9 Hz, 1H), 3.75-3.94 (m, 2H), 3.74 (s, 1H), 3.53 (d, J ) 7.3 Hz,
1H), 3.23 (dd, J ) 13.2, 9.7 Hz, 1H), 3.07 (m, 1H), 2.95 (s, 3H), 2.86
(s, 3H), 2.80 (s, 1H), 2.76 (d, J ) 5.5 Hz, 1H), 2.68 (m, 1H), 2.66 (s,
3H), 2.21-2.29 (m, 3H), 2.19 (s, 3H), 2.05-2.18 (m, 2H), 1.90-2.03
(m, 3H), 1.25 (s, 1H), 0.86-0.98 (m, 10H), 0.55 (d, J ) 6.47 Hz, 3H);
HRESI/APCIMS m/z [M + Na]+ 692.3450 (calcd for C35H51N5O6SNa,
692.3458).
Absolute Configuration of 5. A sample of 5 (200 µg) was dissolved
in CH2Cl2 and ozonized at -78 °C for 30 min. The ozonized sample
was divided into two portions, and the solvent was evaporated. One
portion of the ozonized sample was subjected to base hydrolysis using
1 N KOH(aq) and MeOH (1:1) at 80 °C for 3 h. This was analyzed by
enantioselective HPLC (CHIRALPAK MA (+) (4.6 × 50 mm), 0.5
mL/min, detection at 254 nm). The retention times for standard
D-glyceric acid and L-glyceric acid were 8.6 and 7.4 min, respectively,
using 0.5 mM CuSO4. The base hydrolysate was determined to contain
D-glyceric acid (8.6 min). Another portion of the base hydrolysate was
analyzed using enantioselective HPLC-MS for 2,3-dihydroxy-3-meth-
ylpentanoic acid (Dhmpa) [column, Chirobiotic TAG (4.6 × 250 mm),
Supelco; solvent, MeOH-10 mM NH4OAc (60:40, pH 5.58); flow rate,
0.5 mL/min; detection by ESIMS in negative ion mode (MRM scan)].
(2R,3S)-Dhmpa eluted at 6.3 min. The retention times (tR, min; MRM
ion pair) of the authentic standards were as follows: (2S,3R)-Dhmpa
(4.2; 147f57), (2S,3S)-Dhmpa (4.9), (2R,3R)-Dhmpa (5.6), (2R,3S)-
Dhmpa (6.3). MS parameters were as follows: Dhmpa: DP -25.0, EP
-4.0, CE -25.0, CXP -4.0, CEP -8.0; CUR 40, CAD medium, IS
-4500, TEM 750, GS1 65, GS2 65. The absolute configuration of the
Dhmpa unit was further confirmed by enantioselective HPLC (CHIRAL-
PAK MA (+) (4.6 × 50 mm), 0.5 mL/min, detection at 254 nm) using
a mobile phase of 2 mM CuSO4-CH3CN (95:5). The retention times
(min) of Dhmpa standards were (2R,3R)-Dhmpa (19.0), (2R,3S)-Dhmpa
(29.0), (2S,3R)-Dhmpa (46.0), and (2S,3S)-Dhmpa (47.0). The base
hydrolysate was found to contain (2R,3S)-Dhmpa (29.0).
Another portion of the ozonized sample was hydrolyzed using 6 N
HCl at 110 °C for 20 h. The hydrolysate was dried under N2 and the
residue dissolved in MeOH and ozonized at -78 °C for 30 min. After
evaporation of the solvent under N2, the residue was dissolved in
H2O2-HCOOH (1:2) and left to stir overnight at room temperature
before refluxing at 100 °C for 1 h. The solvent was removed, and the
sample was analyzed by enantioselective HPLC-MS [column, Chiro-
biotic TAG (4.6 × 250 mm), Supelco; solvent, MeOH-10 mM
NH4OAc (40:60, pH 5.30); flow rate, 0.5 mL/min; detection by ESIMS
in positive ion mode (MRM scan)]. L-Val eluted at 8.20 min. The
retention times (tR, min; MRM ion pair) of the authentic amino acids
were as follows: L-Val (8.20; 118f72), D-Val (15.1). MS parameters
were as follows: Val: DP 5.7, EP 9.0, CE 40.0, CXP 8.0, CEP 10.0;
CUR 40, CAD high, IS 4500, TEM 750, GS1 65, GS2 65.
The final purification of the semipure compounds from either WSL-1
or SL-1 collection was achieved by semipreparative reversed-phase
HPLC (Phenomenex Phenyl-hexyl, 250 × 10 mm, 5 µm, 2.0 mL/min;
PDA detection) using a MeOH-H2O linear gradient (90-100% MeOH
for 30 min and 100% MeOH for 10 min), yielding laingolide B (6) (tR
14.9 min; 3.5 mg), lyngbyapeptin D (7) (tR 15.5 min; 0.2 mg), 27-
deoxylyngbyabellin A (4) (tR 18.0 min; 0.3 mg), lyngbyaloside C
mixtures (tR 14.5 min; ∼1 mg), semipure lyngbyabellin J (tR 12.3 min;
1 mg), lyngbyabellin A (tR 13.3 min; 2 mg), lyngbyabellin B (tR 14.7
min; 1 mg), and lyngbyapeptin A (tR 16.5 min; 8 mg). In addition to
the above-mentioned compounds, repurification of the semipure frac-
tions obtained from the SL-1 collection (tR 9-12 min, 7.4 mg, and tR
17 min, 6.4 mg) also yielded lyngbyapeptin D (7) (tR 15.5 min; 0.2
mg) and 27-deoxylyngbyabellin A (4), (tR 18.0 min; 0.3 mg),
respectively. The lyngbyabellin J-containing mixture was further
purified using semipreparative reversed-phase HPLC (Phenomenex
Synergi Hydro-RP, 250 × 10 mm, 5 µm, 2.0 mL/min; PDA detection)
using a MeCN-H2O linear gradient (70-100% MeOH for 30 min and
100% MeOH for 10 min) to yield lyngbyabellin J (5) (tR 13.3 min; 0.4
mg).
2-epi-Lyngbyaloside (1): colorless, amorphous solid; [R]20 -24
D
(c 0.05, MeOH); UV (MeOH) λmax (log ε) 220 (3.40), 240 (3.57) nm;
1H NMR, 13C NMR, HMBC, and NOESY data, see Table 1; HRESI/
APCIMS m/z [M + Na]+ 683.2410 (calcd for C31H4979BrO10Na,
683.2401), 685.2382 (calcd for C31H4981BrO10Na, 685.2389) (100:100
[M + Na]+ ion cluster).
18E-Lyngbyaloside C (2): colorless, amorphous solid; [R]20D -13
(c 0.13, MeOH); UV (MeOH); λmax (log ε) 220 (3.15), 240 (3.23) nm.
1H NMR, 13C NMR, HMBC, and NOESY data, see Table 2; HRESI/
APCIMS m/z [M + Na]+ 671.2399 (calcd for C30H4979BrO10Na,
671.2401), 673.2381 (calcd for C30H4981BrO10Na, 673.2387) (100:100
[M + Na]+ ion cluster).
18Z-Lyngbyaloside C (3): colorless, amorphous solid; [R]20D -21
(c 0.01, MeOH); UV (MeOH) λmax (log ε) 220 (3.13), 240 (3.28) nm;
1H and 13C NMR data, see Table 2; HRESI/APCIMS m/z [M + Na]+
671.2410 (calcd for C30H4979BrO10Na, 671.2401), 673.2381 (calcd for
C30H4981BrO10Na, 673.2379) (100:100 [M + Na]+ ion cluster).
Acid Hydrolysis of 7a and Enantioselective Amino Acid Analysis
by LC-MS. A sample of 7a (50 µg) was dissolved in CH2Cl2 and
subjected to ozonolysis at room temperature for 30 min. The solvent
was evaporated, and the residue was treated with 0.6 mL of
H2O2-HCOOH (1:2) at 70 °C for 20 min. The resulting oxidation
product was concentrated to dryness and subsequently hydrolyzed with
0.6 mL of 6 N HCl at 110 °C for 20 h. The hydrolyzed product was
dried, reconstituted in 100 µL of H2O, and analyzed by enantioselective
HPLC-MS [column, Chirobiotic TAG (4.6 × 250 mm), Supelco;
solvent, MeOH-10 mM NH4OAc (40:60, pH 5.23); flow rate, 0.5 mL/
min; detection by ESIMS in positive ion mode (MRM scan)]. N-Me-
L-Val, N-Me-L-Leu, and L-Pro eluted at tR 14.1, 16.6, and 19.0 min,
27-Deoxylyngbyabellin A (4): colorless, amorphous solid; [R]20
D
-75 (c 0.02, MeOH); UV (MeOH) λmax (log ε) 210 (4.11), 230 (4.00),
280 (3.04) nm; 1H NMR, 13C NMR, COSY, and HMBC data, see Table
3; HRESI/APCIMS m/z [M
+
H]+ 675.1843 (calcd for
C29H4135Cl2N4O6S2, 675.1845), m/z 675/677/679 (100:80:23 [M + H]+
ion cluster).
Lyngbyabellin J (5): colorless, amorphous solid; [R]20D +25 (c 0.02,
MeOH); UV (MeOH) λmax (log ε) 202 (4.59), 236 (3.72) nm; 1H NMR,

1552 Journal of Natural Products, 2010, Vol. 73, No. 9
Matthew et al.
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respectively. The retention times (tR, min; MRM ion pair) of the
authentic amino acids were as follows: N-Me-L-Val (14.1; 132f86),
N-Me-D-Val (47.9), N-Me-L-Leu (16.6; 146f100), N-Me-D-Leu (117.2),
L-Pro (19.0; 116f70), D-Pro (61.5). Compound-dependent parameters
were as follows: N-Me-Val: DP 29.4, EP 4.2, CE 17.4, CXP 2.7; N-Me-
Leu: DP 32.4, EP 4.0, CE 17.1, CXP 2.49; Pro: DP 35.0, EP 7.7, CE
22.7, CXP 5.0. Source gas parameters used were as follows: CUR 30,
CAD high, IS 4500, TEM 450, GS1 40, GS2 40. N-Me-Asp derived
from N-Me-Tyr was detected using the negative ion mode with the
same LC conditions (tR 5.6 min in the hydrolysate). The retention times
(tR, min; MRM ion pair) of the authentic standards were as follows:
N-Me-L-Asp (5.6; 146f102), N-Me-D-Asp (13.2). The MS parameters
used were as follows: DP -24.4, EP -2.0, CE -17.3, CXP -13.4,
CUR 30, CAD high, IS -4500, TEM 450, GS1 40, GS2 40.
Cell Viability Assay. HT29 colorectal adenocarcinoma and HeLa
cervical carcinoma cells were cultured in Dulbecco’s modified Eagle
medium (DMEM, Invitrogen) supplemented with 10% fetal bovine
serum (FBS, Hyclone) under a humidified atmosphere with 5% CO2 at
37 °C. HeLa cells (3000/well) and HT29 cells (12 500/well) were
seeded in 96-well plates. Upon 24 h incubation, cells were treated with
varying concentrations of 1-5. The cells were incubated for an
additional 48 h, and cell viability was measured using MTT reagent
according to the manufacturer’s instructions (Promega). Data shown
are from duplicate experiments.
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Acknowledgment. This research was supported by the National
Institutes of Health (NIGMS grant P41GM086210 to H.L. and V.J.P.)
and the James & Esther King Biomedical Research Program (Grant
No. 06-NIR07 to H.L.). P.J.S. acknowledges NIH MBRS SCORE grant
S06-GM-44796 for support. The authors gratefully acknowledge NSF
for funding through the External User Program of the National High
Magnetic Field Laboratory (NHMFL), which supported some of the
NMR studies at the Advanced Magnetic Resonance Imaging and
Spectroscopy (AMRIS) facility in the McKnight Brain Institute of the
University of Florida (UF). The 600 MHz, 1 mm triple-resonance HTS
cryogenic probe was developed through collaboration between UF,
NHMFL, and Bruker Biospin. We thank P. Williams for providing
standards of 2,3-dihydroxy-3-methylpentanoic acid. This is contribution
#660 of the University of Guam Marine Laboratory and contribution
#829 from the Smithsonian Marine Station at Fort Pierce.
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Supporting Information Available: NMR spectra for compounds
1-7. This material is available free of charge via the Internet at http://
pubs.acs.org.
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