Lyngbyabellins K-N from two Palmyra atoll collections of the marine cyanobacterium Moorea bouillonii

Article abstract of DOI:10.1002/ejoc.201200691

Five lipopeptides of the lyngbyabellin structure class, four cyclic (1-3 and 5) and one linear (4), were isolated from the extracts of two collections of filamentous marine cyanobacteria obtained from the Palmyra Atoll in the Central Pacific Ocean. Their

Full text of DOI:10.1002/ejoc.201200691

Job/Unit: O20691
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Date: 13-08-12 16:33:40
Pages: 11
FULL PAPER
DOI: 10.1002/ejoc.201200691
Lyngbyabellins K–N from Two Palmyra Atoll Collections of the Marine
Cyanobacterium Moorea bouillonii
Hyukjae Choi,[a][‡] Emily Mevers,[b][‡] Tara Byrum,[a] Frederick A. Valeriote,[c] and
William H. Gerwick*[a,d]
Dedicated to the memory of Ernesto Fattorusso
Keywords: Natural products / Peptides / Structure elucidation / Biological activity
Five lipopeptides of the lyngbyabellin structure class, four
cyclic (1–3 and 5) and one linear (4), were isolated from the
extracts of two collections of filamentous marine cyanobacte-
ria obtained from the Palmyra Atoll in the Central Pacific
Ocean. Their planar structures and absolute configurations
were elucidated through a combination of spectroscopic and
chromatographic analyses as well as chemical synthesis of
fragments. In addition to structural features typical of the
lyngbyabellins, such as two thiazole rings and a chlorinated
2-methyloctanoate residue, these new compounds possess
several unique aspects. Of note, metabolites 2 and 3 possess
rare monochlorination on the 3-acyloxy-2-methyloctanoate
residue, whereas lyngbyabellin N (5) has an unusual N,N-
dimethylvaline terminus. Lyngbyabellin N also possesses a
leucine statine residue and shows strong cytotoxic activity
against the HCT116 colon cancer cell line (IC50
40.9Ϯ3.3 n ).
=
M
amide,[7] the jamaicamides,[8] and the malyngamides.[9] In
general, these structurally diverse metabolites exhibit a
range of interesting biological activities, such as antican-
cer,[10] antifeedant,[11] molluscicidal,[12] anti-inflamma-
tory,[13] and neuromodulatory.[14] The lyngbyabellins are an-
other family of metabolites produced by Moorea sp. that
are non-ribosomal peptide synthetase/polyketide synthase
(NRPS/PKS) derived peptides and have a recognizable
architecture composed of thiazole rings, hydroxy acid resi-
dues, and an acyl group with distinctive chlorination at the
penultimate carbon atom.[15] Several of the lyngbyabellins
are reported to exhibit moderate to potent cytotoxicity to
various cancer cell lines and to exert this activity through
interference with the actin system.[15]
Introduction
Over the last 20 years, marine cyanobacteria have
emerged as exceptionally prolific producers of biologically
active secondary metabolites, rivaling the metabolic rich-
ness of the actinobacteria.[1] Because they lack other more
visible defense mechanisms, such as a hardened exterior or
a cryptic habitat, and have an overall macroscopic structure,
it is thought that cyanobacteria derive value from the bio-
synthesis of these structurally intriguing secondary metabo-
lites for their chemical defense.[2] The genus Moorea (for-
mally Lyngbya spp) is one of the chemically most prolific
and has yielded important metabolites such as the apratox-
ins,[3] antillatoxin A,[4] lyngbyatoxin A,[5] curacin A,[6] barb-
In the present work, a number of filamentous marine
cyanobacteria were collected from the Palmyra Atoll (ap-
proximately 1000 miles SSW of Hawaii) in 2008 and 2009,
and their extracts were evaluated in several biological as-
says. A few of the reduced complexity fractions from two
extracts, both subsequently identified as Moorea bouillonii,
were found to be highly cytotoxic to H-460 human lung
cancer cells in vitro [52% survival at 30 μg/mL (fraction F
from the 2009 collection), and 20% survival at 3 μg/mL
(fraction H from the 2008 collection)], and these were cho-
sen for further investigation. Bioassay-guided fractionation
of these extracts yielded five new peptides, lyngbyabellin K–
N (1–5), and these were structurally fully defined by spec-
trochemical methods. Additionally, the known metabolites
[a] Center for Marine Biotechnology and Biomedicine, Scripps
Institution of Oceanography, University of California San Diego,
9500 Gilman Dr. MC 0212, La Jolla, CA 92093, USA
Fax: +1-858-534-0529
Homepage: http://gerwick.ucsd.edu/
[b] Department of Chemistry and Biochemistry, University of
California San Diego,
9500 Gilman Dr., La Jolla, CA 92093, USA
[c] Division of Hematology & Oncology, Department of Internal
Medicine, Henry Ford Hospital,
Detroit, MI 48202, USA
[d] Skaggs School of Pharmacy and Pharmaceutical Sciences,
University of California San Diego,
La Jolla, CA 92093, USA
[‡] H. C. and E. M. contributed equally to this work
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201200691.
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H. Choi, E. Mevers, T. Byrum, F. A. Valeriote, W. H. Gerwick
FULL PAPER
apratoxins F and G were also isolated from these fractions
Two downfield-shifted singlets of protons at δ = 8.13 and
and accounted for the majority of the remarkable cytotoxi- 8.17 ppm with attached carbon atoms (signals at δ = 128.9
city of the crude fractions.
and 128.5 ppm) were attributed to two 2,4-disubstituted thi-
azole rings, accounting for two more degrees of unsatura-
tion. The final degree of unsaturation was thus deduced to
constitute an overall macrocyclic structure for 1.
Results and Discussion
The two thiazole ring structures were confirmed by
HMBC correlations from 12-H (δ = 8.13 ppm) to C-11 (δ
= 145.8 ppm) and C-13 (δ = 169.8 ppm), and 18-H (δ =
8.17 ppm) to C-17 (δ = 146.4 ppm) and C-19 (δ =
169.1 ppm) (Table 1). The HMBC correlations from 12-H
and 18-H of the two thiazole rings to carbonyl carbon
atoms C-10 (δ = 160.2 ppm) and C-16 (δ = 161.0 ppm) indi-
cated that carboxylic acid derivatives were directly attached
to the 4-position of each of thiazole ring. In addition,
COSY correlations between 14-OH (δ = 5.73 ppm), 14-H (δ
= 5.39 ppm), and 15-Ha (δ = 4.74 ppm) and 15-Hb (δ =
4.65 ppm), as well as HMBC correlations from 15-Ha and
15-Hb to C-13 and C-16, established that a 1,2-dihydroxy-
ethyl moiety formed a linkage between the two thiazole-4-
carboxylate groups (Figure 1).
Collection and Isolation of Lyngbyabellins K–N (1–5)
A collection of tube-like mats of the filamentous marine
cyanobacterium M. bouillonii (PAL 8/3/09-1) was obtained
by SCUBA diving in the north lagoon at Strawn Island,
Palmyra Atoll, USA, in August 2009. The ethanol-pre-
served collection was subsequently repeatedly extracted
with CH2Cl2/CH3OH (2:1) and then fractionated by silica-
gel vacuum column chromatography (VLC) to produce nine
fractions (A–I). Fraction F possessed moderate anticancer
activity against H-460 human lung cancer cells (52% sur-
vival at 30 μg/mL). Thus, this fraction was further purified
by reverse-phase (RP) HPLC to afford lyngbyabellins K (1,
10 mg, 0.26%) and M (4, 2 mg, 0.05%) as well as mixtures
of two epimers of lyngbyabellin L (2 and 3). This last mix-
ture was subjected to chiral HPLC to ultimately yield pure
lyngbyabellin L (2, 0.7 mg, 0.018%) and 7-epi-lyngbyabel-
lin L (3, 0.6 mg, 0.015%).
An additional sample of M. bouillonii (PAL 8/16/08-3)
was collected by SCUBA diving in 2008 from reefs 9–15 m
deep surrounding Palmyra Atoll. This ethanol-preserved
material was repeatedly extracted (CH2Cl2/CH3OH, 2:1)
and fractionated by using normal-phase VLC to yield nine
fractions. Three polar fractions (100% EtOAc, 25% EtOAc/
CH3OH, and 75% EtOAc/CH3OH) were strongly cytotoxic
to H-460 cancer cells (20% survival at 3 μg/mL) and were
thus fractionated with reverse-phase solid-phase extraction
(RP-SPE) followed by preparative thin-layer chromatog-
raphy (prepTLC) to yield 4.3 mg of highly purified lyngbya-
bellin N (5) as a pale yellow oil. These same fractions also
contained several known apratoxins, and these compounds
were responsible for the majority of the cytotoxicity.[3]
Further inspection of the 1H NMR spectrum of 1 re-
vealed a series of upfield and highly coupled resonances
reflective of an aliphatic chain. Additionally, a downfield
methyl singlet at δ = 2.12 (8-H3) showed HMBC corre-
lations to the signal of a quaternary carbon atom at δ =
90.4 ppm (C-7) as well as to a signal of a methylene carbon
atom at δ = 49.5 ppm (C-6). The chemical shift of C-7 (δ =
90.4 ppm) was indicative of a gem-dichloro substituent, as
observed in dolabellin,[16] hectochlorin,[17] and the lyngbya-
bellins,[15] and thus, accounted for the two chlorine atoms
in the molecular formula of 1. This moiety was extended to
include an additional six carbon atoms (C-1 to C-5, and C-
9, see Table 1) by integrated reasoning of COSY, HSQC,
and HMBC data and identified this moiety as 7,7-dichloro-
3-acyloxy-2-methyloctanoate
(DCAMO).
Additional
HMBC correlations from 3-H of the DCAMO residue to a
carbonyl carbon, C-10, of the first thiazole-4-carboxylate
unit, allowed connection between these atoms through an
ester bond.
Sequential COSY correlations between adjacent methine
protons 20-H (δ = 5.53 ppm) and 21-H (δ = 2.24 ppm) to
both doublet methyl groups 22-H3 and 23-H3, along with
Structures of Lyngbyabellins K–N (1–3, 5) and 7-epi-
Lyngbyabellin L (4)
Lyngbyabellin K (1), a pale yellow oil, showed an ion HMBC correlations from these two methyl groups back to
cluster at m/z = 578.99/580.96/582.96 in a ratio of 100:85:25 C-20 and C-21, defined the side chain of a 2-hydroxyisoval-
by low-resolution (LR) ESIMS, indicating the presence of eric acid (HIVA) residue. The HMBC correlations from 20-
two chlorine atoms in the molecule. The molecular formula H to C-1 (δ = 174.3 ppm) and C-19 (δ = 169.1 ppm) sup-
of 1 was determined to be C23H28Cl2N2O7S2 by interpret- ported the position of this HIVA-derived residue between
ation of high-resolution (HR) ESITOFMS data (m/z [M + C-19 and C-1, as shown in Figure 1, and completed the
Na]+ = 601.0609). The IR spectrum of 1 displayed absorp- assignment of all atoms from the molecular formula of
tion bands at 3421, 1737, and 1616 cm–1, indicating the lyngbyabellin K (1). Additionally, from the above dis-
presence of hydroxy, ester, and amide functionalities, cussion, the sequence of residues in the macrocyclic ring
respectively. The 13C NMR spectra of 1 in CDCl3 showed was defined as DCAMO–HIVA–thiazole-1-carboxylate–
five downfield-shifted signals of quaternary carbon atoms glyceric acid–thiazole-2-carboxylate–DCAMO.
(δ = 174.3, 169.8, 169.1, 161.0, and 160.2 ppm) that made
To define the absolute configuration at each stereocenter,
double bonds with O or N and two carbon–carbon double lyngbyabellin K (1) was crystallized from CH3CN. A nee-
bonds (δ = 146.4, 145.8, 128.9, and 128.5 ppm), accounting dle-shaped crystal (monoclinic, 0.21ϫ0.11ϫ0.05 mm) was
for seven of the ten degrees of unsaturation.
subjected to single-crystal X-ray diffraction analysis. Re-
2
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Lyngbyabellins K–N from the Marine Cyanobacterium Moorea bouillonii
Table 1. NMR spectral data for lyngbyabellin K–M (1–4) in CDCl3 at 500 (1H) and 125 MHz (13C).
Position
1
δC
2
δC
3
δC
4
δC
δH multiplicity (J)
COSY
HMBC
δH
δH
δH
[ppm] [ppm] [Hz]
[ppm] [ppm] [ppm] [ppm] [ppm] [ppm]
1
2
3
174.3
43.2
75.1
174.6
42.7
75.6
174.6
42.6
75.4
174.4
44.4
75.4
3.18 dt (9.4, 7.3)
5.36 br. s
3, 9
2, 4a, 4b
1, 3, 9
1, 10
3.31
5.33
3.31
5.34
2.84
3.80
5.51
1.93
3-OH
4a
4b
5a
5b
6a
6b
7
30.7
20.5
49.5
1.92 m
1.73 m
1.75 m
3, 4b, 5
3, 4a, 5
4, 6a, 6b
29.9
20.5
40.1
1.91
1.72
1.61
1.55
1.77
1.66
4.02
1.49
1.30
30.3
21.0
40.3
1.95
1.67
1.63
1.52
1.77
1.66
3.98
1.49
1.30
34.4
21.5
50.0
3, 5, 6
3, 4, 6, 7
1.86
2.24
2.30 m
2.15 m
5, 6b
5, 6a
4, 5, 7, 8
4, 5, 7, 8
90.4
37.6
15.3
58.2
25.5
15.3
58.5
25.4
15.1
90.8
37.7
15.0
8
9
2.12 s
1.30 d (7.3)
6, 7
1, 2, 3
2.16
1.26
2
10
11
12
13
14
14-OH
15a
15b
16
17
18
19
20
21
22
23
24
25
160.2
145.8
128.5
169.8
70.1
160.0
145.7
128.3
169.7
70.1
160.4
145.9
128.2
169.2
70.0
161.8
147.6
128.2
171.2
70.30
8.13 s
10, 11, 13
8.15
8.14
8.16
5.39 br. s
5.73 br. s
4.74 dd (11.3, 2.3)
4.65 dd (11.3, 3.0)
14-OH, 15a, 15b
5.35
6.25
4.76
4.58
5.33
6.24
4.76
4.57
5.41
6.68
4.95
4.30
14
14
14
69.1
13, 16
13, 14, 16
69.9
69.4
70.26
161.0
146.4
128.9
169.1
77.5
33.6
19.3
160.8
146.1
128.9
169.9
77.6
34.3
19.2
160.7
145.5
128.9
169.7
77.4
34.8
19.0
161.7
145.9
129.2
170.5
77.5
34.4
19.5
16.8
61.8
14.7
8.17 s
16, 17, 19
8.16
8.16
8.30
5.53 d (6.6)
2.37 m
0.99 d (6.5)
1.07 d (6.5)
21
1, 19, 21, 22, 23
19, 20, 22, 23
20, 21, 23
5.51
2.30
1.04
1.06
5.51
2.32
1.03
1.07
5.99
2.33
1.13
0.98
4.42
1.41
20, 22, 23
21
21
17.9
20, 21, 22
17.4
17.3
sulting from the presence of heavy atoms (chlorine and sul-
Because the planar structures of 2 and 3 are closely re-
fur) in compound 1, the absolute configuration could be lated to that of 1, except for the alterations at C-7, similar
deduced as (2S,3S,14R,20S) [final R(F2) = –0.02(2), see the 13C NMR shifts, optical rotations, and circular dichroism
Supporting Information].
(CD) absorption measurements for all three compounds
Lyngbyabellin L (2) and 7-epi-lyngbyabellin L (3) showed indicate that they are of the same enantiomeric series at all
essentially identical sodiated parent ion clusters by HR ESI- comparable centers (see the Supporting Information).
TOF MS at m/z = 567.1000 and 569.0980 in a ratio of Therefore, the absolute configurations of C-2, C-3, C-14,
78:32, indicating that both possessed the same molecular and C-20 of 2 and 3 were assigned to be the same as those
formula of C23H28ClN2O7S2, containing a single chlorine for lyngbyabellin K (1), namely, (2S,3S,14R,20S).
1
atom each. The H and 13C NMR spectra of 2 possessed
Several different approaches were employed to determine
several resonances similar to those of 1 (Table 1); thus con- the absolute configuration of C-7 in lyngbyabellin L (2) and
firming the presence of two extended 2,4-disubstituted thi- its C-7 epimer (3). First, a detailed analysis of the 1H NMR
azole units, an HIVA unit, and a 1,2-dihydroxyethyl moiety spectroscopic data for compound 2, using a DQF-COSY
1
(Figure 1). However, in the H and 13C NMR spectra of 2, experiment, revealed all of the vicinal 1H–1H coupling con-
a new doublet methyl signal appeared at δ = 1.49 ppm (8- stants
in
the
7-chloro-3-acyloxy-2-methyloctanoate
H3) and a downfield-shifted methine signal was observed at (CAMO) residue (Figure 2a). The large vicinal coupling
δ = 4.02 (7-H) and 58.2 ppm (C-7). These new features were constants (Ͼ 7 Hz) of 2-H–3-H and 3-H–4-Hb in 2 indi-
accompanied by several missing resonances relative to 1, cated that these protons were anti to one another. Con-
namely, the distinctive methyl group singlet at δ = 2.12 ppm versely, the relatively small vicinal coupling constant of 3-
(8-H3) and the signal of the quaternary gem-dichloro car- H–4-Ha (J = 3 Hz) showed that they were in a gauche rela-
bon atom at δ = 90.4 ppm (C-7). Interpretation of these tionship. NOESY correlations between 2-H/9-H3–4-Ha
data in combination with the altered molecular formula indicated 4-Ha and 4-Hb were pro-S and pro-R, respec-
suggested that 2 was a monochloro species at C-7. The spec- tively. The large vicinal coupling constants between 4-Ha
troscopic and 2D NMR linkage data as well as the HR and 5-Hb, as well as the NOESY correlation between 5-Ha
ESITOFMS results for compound 3 were almost identical and 3-H, allowed assignment of 5-Ha and 5-Hb as pro-S
to those of 2, and thus, compound 3 was deduced to be and pro-R, respectively. Similarly, a large vicinal coupling
stereoisomeric with compound 2 at C-7.
constant observed between 5-Ha and 6-Hb and NOESY
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try of C-12–C-13 as the erythro rotamer B-3. Heteronuclear
coupling constants between C-3/4-Ha (2JC-3,4-Ha = 0 Hz)
and C-3/4-Hb (2JC-3,4-Hb = –5.9 Hz) also indicated that 4-
Ha and the oxygen substituent at C-3 were in an anti rela-
tionship, whereas 4-Hb and the C-3 oxygen atom were
gauche. The small heteronuclear coupling constants be-
3
tween 4-Ha/C-6, 5-Ha/C-3, 5-Hb/C-3 (3J4-Ha,C-6 = 1.5, JC-
3
= 1.0, JC-3,5-Hb = 0.8 Hz) revealed that the paired
3,5-Ha
protons and carbon atoms were gauche to one another (Fig-
ure 2b). Additional small heteronuclear coupling constants
between 5-Ha/C-7 and 5-Hb/C-7 (3J5-Ha,C-7
= 2.1,
3J5-Hb,C-7 = 1.7 Hz) confirmed that C-7 was gauche to both
5-Ha and 5-Hb. Further small heteronuclear coupling con-
stants between 7-H/C-5, 6-Ha/C-8, and 6-Hb/C-8 indicated
that these paired atoms were in gauche relationships. Thus,
based on this J-based configuration analysis and the abso-
lute configuration of C-2 and C-3, the absolute configura-
tion at C-7 in 2 was deduced to be (R); this was consistent
with previous analyses of homonuclear coupling constants
and NOESY correlations. These assignments were sup-
ported by a reversal in chemical shifts for the pro-R and
pro-S protons attached to C-5 in 2 (δ = 1.55 and 1.61 ppm)
relative to those in compound 3 (δ = 1.63 and 1.52 ppm),
which we interpret to reflect deshielding effects to these
nonequivalent protons from the chlorine atom at C-7 and
oxygen atom at C-3[19] (Figure 2c).
The LR ESIMS results for lyngbyabellin M (4) showed a
parent ion cluster at m/z = 624.95/626.95/628.93 in the ratio
of 100:80:20, indicating the presence of two chlorine atoms,
similar to metabolite 1. The molecular formula of 4 was
determined to be C25H34N2O8Cl2S2 by HR ESITOFMS:
m/z [M + Na]+ = 647.1029 (calcd. for C25H34Cl2N2O8S2Na
647.1026). The 1H and 13C NMR spectra of 4 showed many
features similar to those of 1. However, the 3-H signal in
lyngbyabellin K (1) was at δ = 5.36 ppm, whereas the signal
of this proton in 4 was observed at δ = 3.80 ppm. The latter
proton in 4 showed a unique COSY correlation with an
additional exchangeable proton whose signal was found at
Figure 1. Structures of lyngbyabellin K–N (1–5) and lyngbyabel-
lin H (6).
correlation between 4-Hb and 6-Hb indicated 6-Ha and 6- δ = 5.51 ppm. Furthermore, in lyngbyabellin M a new
Hb to be pro-R and pro-S. Finally, a large coupling constant methylene quartet (24-H2) and methyl triplet (25-H3) were
between 6-Ha and 7-H, small vicinal coupling constant be- observed at δ = 4.42 and 1.41 ppm, respectively, and these
tween 6-Hb and 7-H, and NOESY correlations between 7- were connected to one another by COSY. An HMBC corre-
H with 5-Ha and 5-Hb and 8-H3 with 6-Ha and 6-Hb, re- lation from 24-H2 to C-10 confirmed that the ester linkage
vealed that the absolute configuration at C-7 of 2 was (R). between C-3 and C-10 of 1 had been cleaved and the carb-
In a similar fashion, a detailed coupling constant analysis oxylic acid at C-10 in 4 was esterified with an ethyl moiety.
for the DCAMO residue of 3 revealed that the absolute Owing to the similarities in 13C NMR shifts at most carbon
configuration at C-7 was (S) (Figure 2a).
positions and similar optical rotations, we propose that
Finally, the prochirality of each proton attached to C-4, lyngbyabellin M (4) is of the same enantiomeric series as
C-5, and C-6, as well as the absolute configuration at C-7 lyngbyabellin K (1).
in 2, was confirmed by J-based configurational analysis, as
The relative configuration at C-2 and C-3 of the
shown in Figure 2c.[18] A large homonuclear coupling con- DCAMO residue in lyngbyabellin M (4) was assigned as
stant, as measured by a DQF-COSY experiment, between erythro by comparing 3J2-H,3-H of 4 (6.2 Hz) to the coupling
2-H and 3-H (3J2-H,3-H = 11.0 Hz) indicated an anti rela- constants of model diastereomers of methyl 3-hydroxy-2-
tionship between these protons. A HETLOC experiment methyloctanoate (3J2-H,3-H for the erythro isomer 6.3 Hz;
was used to measure a large heteronuclear coupling con- 3J2-H,3-H for the threo isomer = 3.6 Hz).[16] The absolute
stant between 2-H and C-3 (2J2-H,C-3 = –6.4 Hz), and in configuration at these two centers in compound 4 were re-
conjunction with NOESY correlations between 9-H3 and 3- vealed by NMR spectroscopic analyses of the di-Mosher
H/4-Ha led to the assignment of the relative stereochemis- esters produced by esterification of the C-3 and C-14 hy-
4
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Lyngbyabellins K–N from the Marine Cyanobacterium Moorea bouillonii
Figure 2. Approaches to the determination of the relative configuration of the C-7 stereocenter in the DCAMO residue of lyngbyabellin L
(2) and 7-epi-lyngbyabellin L (3): (a) homonuclear coupling constant and NOE-based determination of relative configuration, (b) J-based
configuration analysis on lyngbyabellin L (2), and (c) deshielding effects to inequivalent protons from the chlorine atom at C-7 and oxygen
atom at C-3 of lyngbyabellin L (2) and 7-epi-lyngbyabellin L (3). [a] The coupling constant is not observed due to overlap. [b] The coupling
constant is not detected due to a weak signal.
droxy groups. Calculation of ΔδS-R values for protons near
The HR ESITOFMS results for lyngbyabellin N (5)
C-3 and C-14 allowed assignment of the absolute configura- showed a parent ion cluster nearly identical to those of the
tion as (3S) and (14R), respectively (Figure 3). Finally, the other lyngbyabellin analogues with an [M + H]+ ion at m/z
absolute configuration of the HIVA residue in 4 was deter- = 905.2997/907.2977/909.2950 (calcd. for C40H59Cl2N4O11S2
mined to be l by GC–MS analysis of the methyl ester deriv- 905.2993) in a ratio of 100:80:20, indicating the presence of
ative following acid hydrolysis and comparison with stan- two chlorine atoms and 13 degrees of unsaturation; 3 more
1
dards, and thus, the complete absolute configuration of 4 than in compound 1. The H and 13C NMR spectra of 5
was determined to be (2S,3S,14R,20S).
again showed features similar to those of 1; however, there
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Date: 13-08-12 16:33:40
Pages: 11
H. Choi, E. Mevers, T. Byrum, F. A. Valeriote, W. H. Gerwick
FULL PAPER
(3R,4S)-Sta-d-FDAA (93.1 min)]. From the retention time
of the natural product statine derivative (93.29 min), it was
clear that this residue was of the (3R,4S) configuration.
The absolute configuration of the DiMeVal residue in
compound 5 was determined by comparing the chiral GC–
MS retention time of the methylated residue liberated by
acid hydrolysis with authentic standards. The two stan-
dards, l-N,N-DiMeVal and d-N,N-DiMeVal, were synthe-
sized from l- and d-Val, respectively, according to literature
procedures.[22] The two standards each possessed distinctly
Figure 3. ΔδS–R values of the di-Mosher esters of lyngbyabellin M
(4).
was a significant increase in the number of new signals, sug- different retention times in GC–MS [l-DiMeVal (63.7 min)
gesting the addition of several more residues. In the 1H and d-DiMeVal (64.2 min)]. The methylated residue from
NMR spectrum, the broad singlet at δ = 5.73 ppm was no the acid hydrolysate gave a single peak at 63.8 min; thus
longer present; thus suggesting that 5 was similar to other indicating the l configuration.
known lyngbyabellins, which contain attached residues es-
The lyngbyabellin family of compounds are known to
terified to C-14.[15] Furthermore, there were three ad- exhibit moderate to potent cytotoxicity against a number
ditional downfield methyl groups (δ = 2.77, 2.72, and of different cancer cell types through the promotion of actin
1.91 ppm) and two amide protons (δ = 9.38 and 8.81 ppm). polymerization.[15] Thus, after completion of the structural
From the 13C NMR spectra, there were an additional three analysis, compounds 1–5 were evaluated in an H-460 hu-
ester/amide carbonyl groups (δ = 165.8, 168.9, and man lung carcinoma cell cytotoxicity assay. Compound 5
169.4 ppm), accounting for the remaining degrees of unsat- showed strong yet variable cytotoxicity (IC50 = 0.0048–
uration. COSY and HMBC correlations established the 1.8 μm, perhaps due to solubility problems), whereas com-
presence of an N,N-dimethylvaline (DiMeVal) residue and pounds 1–4 were inactive. However, in the HCT116 colon
an O-acetylated leucine statine. HMBC correlations from cancer cell line, reproducible IC50 values were obtained for
14-H (δ = 6.27 ppm) to C-24 (δ = 168.9 ppm) and 27-NH (δ lyngbyabellin N (40.9Ϯ3.3 nm),[23] confirming the potent
= 8.81 ppm) to C-32 (δ = 165.8 ppm) completed the planar cytotoxic effect of this new member of the lyngbyabellin
structure of 5, as depicted in Figure 1.
class of compounds and suggesting that the side chain of
The planar structure of lyngbyabellin N (5) is closely re- lyngbyabellin N is an essential structural feature for this po-
lated to that of lyngbyabellin H (6), except for the replace- tent activity. However, this trend is not entirely consistent
ment of the polyketide portion with a DiMeVal residue. A within this structure class, because other lyngbyabellin ana-
comparison of optical rotations and carbon chemical shifts logues without the side chain exhibit sub-micromolar ac-
(macrocyclic lactone portion) strongly supported the as- tivity against HT29 and HeLa cells.[15]
sumption that the absolute configurations of the macro-
It is interesting to note the increasing structural complex-
cyclic lactone in 5 and 6 were identical, and thus, we as- ity in the lyngbyabellin family of metabolites, with that of
signed the stereoconfiguration of lyngbyabellin N as lyngbyabelling N (5) being the most intricate to date. While
(2S,3S,14R,20S). Further support of this configuration was it has the recognizable core of the lyngbyabellins, the side
obtained by a comparison of the CD absorption curve of 5 chain and DiMeVal terminus resemble those of the dolasta-
with those of 1, 2, and 3. All of these CD curves were nearly tin 10 and coibacin A families of metabolites and, in this
identical, confirming that 5 has the (2S,3S,14R,20S) config- regard, it has a hybrid structure between these cyanobacte-
uration.
rial natural product classes. Additionally, from a biosyn-
The absolute configuration of the leucine statine in 5 was thetic logic perspective, these more complex lyngbyabellins
determined by LC–MS analysis of the acid hydrolysate ap- are perplexing, because they possess two logical points for
propriately derivatized with Marfey’s reagent (d-FDAA). the initiation of molecule construction: the polyketide chain
The four standards, (3S,4S)-statine (Sta), (3R,4S)-Sta, represents one such point and DiMeVal the second. Thus,
(3S,4R)-Sta, and (3R,4R)-Sta, were all synthesized from these complex lyngbyabellins may indeed represent the hy-
their corresponding amino acids (l- and d-leucine, respec- bridization and cojoining of two natural product structure
tively), and according to literature procedures, converted classes. An additional point of interest is the occurrence
into their respective N-benzyl-protected aldehydes.[20] Each of monochlorinated carbon atoms in the polyketide section
standard was then treated with tert-butyl 2-bromoacetate in (compounds 2 and 3). Previously, the Walsh group[24]and
the presence of nBuLi to yield a mixture of diastereomeric ourselves[25] independently showed that the monochloro
protected leucine statines. However, these diastereomers species was not encountered in the process of chlorination
were inseparable by HPLC and were thus converted into of the methyl group of barbamide (only the di- and tri-
tert-butyloxycarbonyl (Boc) protected analogues, which chloro species are formed). Hence, the occurrence of a
were readily purified by RP HPLC.[21] Hydrolysis, followed monochloromethylene moiety in these two metabolites sug-
by derivatization with Marfey’s reagent, yielded the four gests the functioning of a radical halogenase with differing
standards, which each possessed a distinct retention time in catalytic properties from any of the currently characterized
LC–MS [(3R,4R)-Sta-d-FDAA (78.2 min), (3S,4R)-Sta-d- or partially characterized halogenases. It is still unclear
FDAA (80.9 min), (3S,4S)-Sta-d-FDAA (92.2 min), and whether lyngbyabellin M (4) is an extraction artefact or a
6
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Job/Unit: O20691
/KAP1
Date: 13-08-12 16:33:40
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Lyngbyabellins K–N from the Marine Cyanobacterium Moorea bouillonii
goon at Strawn Island, Palmyra Atoll, USA, in August 2008 (5°
naturally occurring compound. Whereas lyngbyabellin M
(4) has an ethyl ester at C-10, previously reported linear
lyngbyabellins have a methyl ester at C-16. The position of
the ethyl ester in 4 is consistent with the proposed biosyn-
thetic pathway of the lyngbyabellins, as predicted from our
knowledge of hectochlorin biosynthesis.[26] It has been
shown that methyl esters of hybrid polyketides/non-ribo-
somal peptides are produced by an unusual S-adenosyl-l-
methionine (SAM) dependent methyltransferase in myxo-
53Ј 55.76ЈЈ N, 162° 05Ј 0.092ЈЈ W). The lyngbyabellin N producing
cyanobacterium (collection code: PAL 8/16/08-3) was collected by
SCUBA diving on reefs 9–15 m deep around the Palmyra Atoll,
USA. The environmental samples were stored in EtOH/H2O (1:1)
at –20 °C, while genetic materials were preserved in RNA stabiliza-
tion solution at –20 °C (RNAlater, Ambion Inc.). Morphological
characterization was performed by using an Olympus IX51 epifluo-
rescent microscope (100ϫ) equipped with an Olympus U-CMAD3
camera. Taxonomic identification of cyanobacterial specimens was
bacteria.[27] Methyl and ethyl esters have been found as nat- performed in accordance with current phycological systems.[29,30]
urally occurring compounds in cyanobacterial extracts,[28]
and thus, may be formed by pathway termination reactions
similar to those in myxobacteria.
Isolation of Lyngbyabellins K, L, and M and 7-epi-Lyngbyabellin L
(1–4): The cyanobacterial tissue (PAL 8/3/09-1, morphologically
identified as M. bouillonii) was repetitively extracted with 2:1
CH2Cl2/CH3OH to afford 3.9 g of crude extract. A portion of the
extract (3.2 g) was fractionated by silica gel VLC with a stepwise
gradient solvent system of increasing polarity starting from 10%
EtOAc in hexanes to 100% CH3OH to produce nine fractions
(A–I). The fraction eluting with 80% EtOAc in hexane (fraction F)
was subsequently separated by RP HPLC (Phenomenex Fusion RP
4 μ, 250ϫ10 mm, 65% CH3CN/H2O at 3 mL/min) to give pure
lyngbyabellin K (1, 10 mg, 0.26%), lyngbyabellin M (4, 2 mg,
0.05%), and a mixture of lyngbyabellin L and 7-epi-lyngbyabel-
lin L. The mixtures were subjected to chiral HPLC (Chiral-AGP,
150ϫ4.0 mm, 5 μm, UV: 210 nm), and lyngbyabellin L (2, 0.7 mg,
0.018%) and 7-epi-lyngbyabellin L (3, 0.6 mg, 0.015%) were eluted
with 13% CH3CN in H2O.
Conclusions
A chemical investigation of anticancer-active extracts of
M. bouillonii collected in the Palmyra Atoll led to the isola-
tion of the five new lyngbyabellins 1–5. Their planar struc-
tures and absolute configurations were determined by a
combination of various spectroscopy, chromatography and
synthetic chemistry techniques. While compounds 1–4 were
inactive in the H-460 cytotoxicity assay, compound 5 exhib-
ited strong yet variable cytotoxicity (IC50 = 0.0048–1.8 μm).
We do not understand the basis for this variable level of
activity; however, it most likely relates to solubility issues in
the assay buffer. These new lyngbyabellin metabolites pos-
sess several unique structural features, a monochlorometh-
ylene group and two conceptual points of chain initiation,
which reflect unique biosynthetic reactions not yet charac-
terized or understood.
Isolation of Lyngbyabellin N (5): 1 L of cyanobacterial tissue (pre-
viously identified as M. bouillonii)[3] was repetitively extracted with
2:1 CH2Cl2/CH3OH to afford 4.2 g of crude extract. The extract
was fractionated by silica gel VLC with a stepwise gradient solvent
system of increasing polarity starting from 10% EtOAc in hexanes
to 100% CH3OH to produce nine fractions (A–I). The fraction
eluting with 75% EtOAc in CH3OH (fraction H) was subsequently
separated by using 5 g RP SPE with a stepwise gradient solvent
system decreasing in polarity starting from 55% CH3CN in H2O
to 100% CH2Cl2 to produce five fractions (1–5). The fraction elut-
ing with 70% CH3CN in H2O (fraction 3) was further separated
by prepTLC, with an isocratic solvent system of 100% EtOAc, to
yield pure lyngbyabellin N (5, 5.4 mg, 0.12%).
Experimental Section
General Experimental Procedures: Optical rotations were measured
with a JASCO P-2000 polarimeter; CD spectra were recorded in
EtOH by using a JASCO J-810 spectropolarimeter. UV and IR
spectra were recorded with a Beckman Coulter DU800 spectropho-
tometer and a Nicolet ThermoElectron Nicolet IR100 FTIR spec-
trometer as KBr plates, respectively. NMR spectra were recorded
Lyngbyabellin K (1): Pale yellow oil. [α]2D5 = –28.0 (c = 0.5, MeOH).
UV (MeOH): λmax = 236 nm (logε = 3.76). CD (MeOH): λmax (Δε)
= 211 (+1.11), 227 (–1.45), 238 (–0.58), 245 (–0.69), 265 nm
(+0.28). IR (KBr): νmax = 3421, 3127, 2965, 2935, 2880, 1737, 1616,
˜
1481, 1379, 1320, 1211, 1163, 1096 cm–1. 1H, 13C and 2D NMR:
Table 1. HR ESIMS: m/z[M + Na]+ = 601.0609 (calcd. for
C23H28Cl2N2O7S2Na 601.0607, Δ +0.2 mmu).
with chloroform as the internal standard (δC = 77.2 ppm; δH
=
7.26 ppm) with a Varian 500 MHz spectrometer (500 and 125 MHz
for 1H and 13C NMR, respectively) or a Bruker 600 MHz spec-
trometer (600 and 150 MHz for 1H and 13C NMR, respectively)
equipped with a 1.7 mm MicroCryoProbe. For the HETLOC ex-
periments, 128 scans (4 K) and 512 experiments were obtained with
zero-filling at F1 to 4 K. A mixing time of 60–80 ms was used. HR
ESI mass spectra were obtained with an Agilent 6230 ESI-TOF
mass spectrometer. X-ray diffraction data was obtained by using
state-of-the-art Bruker single-crystal diffractometers with CCD de-
tectors. HPLC was carried out by using a Waters 515 pump system
with a Waters 996 PDA detector. Acid hydrolysis was performed by
using a Biotage (Initiator) microwave reactor equipped with high-
pressure vessels.
Lyngbyabellin L (2): Pale yellow oil. [α]2D5 = –33.0 (c = 0.5, MeOH).
UV (MeOH): λmax = 236 nm (logε = 3.69). CD (MeOH) λmax (Δε)
= 210 (+0.19), 225 (–1.98), 239 (–0.70), 245 (–0.74), 265 nm
(+0.17). IR (KBr): νmax = 3440, 3116, 2962, 2921, 1738, 1480, 1367,
˜
1320, 1211, 1100 cm–1. 1H, 13C and 2D NMR: Table 1 and Sup-
porting Information. HR ESIMS: m/z[M + Na]+ = 567.1000
(calcd. for C23H29ClN2O7S2Na 567.0997, Δ +0.3 mmu).
7-epi-Lyngbyabellin L (3): Pale yellow oil. [α]2D5 = –34.8 (c = 0.3,
MeOH). UV (MeOH): λmax = 236 nm (logε = 3.59). CD (MeOH)
λmax (Δε) = 210 (+0.25), 225 (–1.42), 239 (–0.51), 245 (–0.52),
265 nm (+0.12). IR (KBr): ν
= 3731, 3623, 2962, 2920, 2846,
˜
max
Cyanobacterial Collections and Taxonomic Identification: The
lyngbyabellins K–M producing cyanobacterium (collection code:
PAL 8/3/09-1) was collected by SCUBA diving in the north of la-
1737, 1458, 1368, 1319, 1211, 1101 cm–1. 1H, 13C and 2D NMR:
Table 1 and Supporting Information. HR ESIMS: m/z[M + Na]+
= 567.1000 (calcd. for C23H29ClN2O7S2Na 567.0997, Δ +0.3 mmu).
Eur. J. Org. Chem. 0000, 0–0
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Date: 13-08-12 16:33:40
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H. Choi, E. Mevers, T. Byrum, F. A. Valeriote, W. H. Gerwick
FULL PAPER
Lyngbyabellin M (4): Pale yellow oil. [α]2D5 = –4.5 (c = 0.5, MeOH).
UV (MeOH): λmax = 234 nm (logε = 3.71). IR (KBr): νmax = 3377,
X-ray Crystallography: Compound 1 (8 mg), dissolved in CH3CN
(400 μL), was transferred into a tube and the tube was placed in a
˜
2963, 2926, 2853, 1730, 1644, 1465, 1334, 1224, 1175 cm–1. 1H, 13C, vial. The vial was sealed and monitored for crystal growth over a
and 2D NMR: Table 1 and Supporting Information. HR ESIMS:
m/z[M + Na]+ 647.1029 (calcd. for C25H34Cl2N2O8S2Na 647.1026,
Δ +0.3 mmu).
month, then colorless needle-shaped crystals were observed.
Crystal Data for Lyngbyabellin K (1): Monoclinic, space group
P21/c, unit cell dimensions a = 10.6419(8) Å, b = 5.8776(5) Å, c =
23.859(2) Å, α = 90°, β = 101.928(6)°, γ = 90°, V = 1460.1(2) Å3,
Lyngbyabellin N (5): Pale yellow oil. [α]2D7 = –24.0 (c = 1.05,
MeOH). UV (MeOH): λmax = 202 (logε = 4.36), 235 nm (logε =
3.99). CD (MeOH): λmax (Δε) = 212 (–0.64), 224 (–1.60), 237
Z
=
2, ρcalcd.
=
1.411 gcm–3, crystal dimensions
0.21ϫ0.11ϫ0.05 mm. A total of 7218 reflections were collected
covering the indices –12 Յ h Յ 12, –6 Յ k Յ 6, –27 Յ l Յ 10. 4362
reflections were symmetry-independent, with Rint = 0.0358. Final
R(F2) = –0.02(2). Relevant data collection parameters and results
of structural refinement for this structural determination are given
in the Supporting Information. CCDC-882823 [lyngbyabellin K
(1)] contains the supplementary crystallographic data for this pa-
per. These data can be obtained free of charge from The Cambridge
Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_
request/cif.
(–0.27), 248 (–1.36), 268 nm (+0.03). IR (KBr): νmax = 3436, 2961,
˜
2933, 1742, 1677, 1467, 1372, 1321, 1233, 1166, 1097, 1037 cm–1.
1H, 13C, and 2D NMR: Table 2. HR ESIMS: m/z[M + H]+
905.2997 (calcd. for C40H59Cl2N4O11S2 905.2993, Δ +0.4 mmu).
=
Table 2. NMR spectral data for lyngbyabellin N (5) in [D6]DMSO
at 500 (1H) and 125 MHz (13C).
Position
δC
δH multiplicity (J) COSY
HMBC
Ozonolysis, Acid Hydrolysis, and Chiral GC–MS: A portion
(100 μg) of 4 was dissolved in CH2Cl2 (500 μL) at Ϫ78 °C, and O3
was bubbled through the sample for 5 min. The pale blue solution
was dried under N2 (g). The products were resuspended in 6 n HCl
(200 μL) and allowed to react at 110 °C for 2 h. The acid hydroly-
sates were dried under N2 (g), treated with an excess of CH2N2 at
room temperature for 30 min, and dried under N2 (g). The products
were dissolved in CH2Cl2 and injected into chiral GC–MS (Chira-
sil-Val, Agilent Technologies J&W Scientific, 30 mϫ0.25 mm) un-
der the following conditions: the initial oven temperature was
32 °C, kept for 15 min, followed by a ramp from 32 to 60 °C at a
rate of 10 °C/min, followed by another ramp to 200 °C, at a rate
of 15 °C/min and kept at 200 °C for 5 min. The retention time of
products resulting from the acid hydrolysate of 4 matched the syn-
thetic (2S)-HIVA standard [9.7 min; (2R)-HIVA: 10.2 min]. Syn-
thetic standards of (2S)-HIVA and (2R)-HIVA were methylated
with an excess of CH2N2, dried under N2 (g), resuspended in
CH2Cl2, then analyzed with chiral GC–MS. The retention time of
products resulting from the acid hydrolysate of 4 matched the syn-
thetic (2S)-HIVA standard [9.7 min; (2R)-HIVA: 10.2 min].
[ppm] [ppm] [Hz]
1
2
3
4a
4b
5a
6a
6b
7
173.2
42.9
74.5
30.0
2.84 dd (9.4, 6.8) 3, 9
1, 3, 9
5.13 m
1.80 m
1.69 m
1.24 m
2.27 m
2.20 m
2, 4a, 4b
3
3
6
4a, 4b, 5
4a, 4b, 5
3. 6
3, 6
4
7, 8
7, 8
29.0
48.3
92.1
37.0
14.6
159.4
145.4
129.7 8.44 s
165.5
70.2
63.6
8
9
2.09 s
6, 7
1, 2, 4
1.16 d (7.1)
2
10
11
12
13
14
15a
15b
16
17
18
19
20
21
22
23
24
25a
25b
26
27
28a
28b
29
30
31
27-NH
32
33
34
35
36
37
38
33-NH
39
40
10, 11, 13
6.27 t (6.1)
15
15
14
14
4.81 dd (11.2, 5.2) 14
4.57 dd (11.7, 6.8) 14
160.2
145.0
130.2 8.46 s
167.2
76.2
31.9
18.4
17.9
168.9
34.0
17, 19
Ozonolysis and Acid Hydrolysis of Lyngbyabellin N (5): A portion
(1 mg) of 5 was dissolved in CH2Cl2 (1 mL) at –78 °C, and O3 was
bubbled through the sample for 10 min. The pale blue solution was
dried under N2 (g). The products were resuspended in 6 n HCl
(500 μL) and allowed to react at 160 °C in a microwave reactor for
5 min.
5.55 d (8.1)
21
1, 19, 21, 22, 23
20, 23
20, 21, 23
20, 21, 22
2.24 m
20, 22, 23
0.80 d (6.5)
1.00 d (6.5)
21
21
2.91 m
26
24
24, 26
Modified Marfey’s Analysis to Determine the Configuration of the
Statine Units: An aliquot (ca. 500 μg) of the acid hydrolysate was
dried under N2 (g) and dissolved in 1 m sodium hydrogen carbonate
(1 mL), and 1% d-FDAA (12 μL) in acetone was added. The solu-
tion was maintained at 40 °C for 90 min, at which time the reaction
was quenched by the addition of CH3CN, and 10 μL of the solution
was analyzed by LC–ESIMS. The Marfey’s derivatives of the hy-
drolysate and standards were analyzed by RP HPLC by using a
Phenomenex Luna 5 μ C18 column (4.6ϫ250 mm). The HPLC
conditions began with 10% CH3CN/H2O acidified with 0.1% for-
mic acid (FA) followed by a gradient profile to 50% CH3CN/H2O
acidified with 0.1% FA over 85 min at a flow of 0.4 mL/min, moni-
toring from 200 to 600 nm. The retention times of the authentic
acid d-FDAA derivatives were 78.2, 80.9, 92.2, and 93.1 min for
(3R,4R)-, (3S,4R)-, (3S,4S)-, and (3R,4S)-Sta-d-FDAA, respec-
tively; the hydrolysate product gave a peak with a retention time of
93.3 min, indicating an absolute configuration of (3R,4S). Corre-
spondingly, (3S,4S)-, (3S,4R)-, (3R,4R)-, and (3R,4S)-statine were
2.70 m
26
71.6
47.9
37.5
5.12 m
25a, 25b, 27
4.35 t (4.4)
1.41 m
26, 28a, 28b, NH-1
29
29, 31
29, 31
1.23 m
29
24.4
24.0
20.8
1.55 m
28, 30, 31
0.90 d (6.4)
0.81 d (6.2)
8.81 d (8.7)
29
29
27
28, 29, 31
28, 29, 31
165.8
71.8
26.3
19.5
16.5
41.5
41.0
3.63 t (6.7)
2.31 m
34
32, 34, 35, 36
32, 33, 35, 36
33, 34, 36
33, 34, 35
33, 38
33, 35, 36
1.07 d (6.8)
0.94 d (6.6)
2.72 d (4.0)
2.77 d (4.2)
9.38 br. s
34
34
33, 37
169.4
20.8
1.91 s
39
8
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Pages: 11
Lyngbyabellins K–N from the Marine Cyanobacterium Moorea bouillonii
served by ESIMS, and the 1H NMR chemical shift was assigned
synthesized from l- (1 g) and d-leucine (1 g), respectively, according
to a procedure reported by Reetz et al. to yield the benzyl-protected
aldehyde.[20] An aliquot (3.38 mmol) of the benzyl-protected alde-
hydes dissolved in tetrahydrofuran (15 mL) was then added to a
solution of tert-butyl acetate (3.72 mmol) and freshly prepared lith-
ium diisoproplyamine (5.06 mmol). The reaction mixture was
warmed to –40 °C for 1 h and then quenched with NaHCO3
(85 mL), filtered, and the aqueous layer was separated and washed
with Et2O (2ϫ 20 mL). The organic layer was then washed with
brine and dried with NaSO4 and filtered to yield diastereomeric
tert-butyl 4-(dibenzylamino)-3-hydroxy-6-methylheptanoate. The
mixture of diastereomers were inseparable; thus, the method out-
lined by Andrés et al. was used, where the benzyl-protected amine
was converted into the Boc-protected derivative allowing for purifi-
cation of a small amount of each of the diastereomers.[21] Each of
the diastereomers were identified by a comparison of 1H NMR
spectra of each of the pure standards to the known compounds
reported in the literature.[21] An aliquot (2 mg) of each protected
statine was treated with 6 n HCl (1 mL) and heated to 160 °C in a
microwave reactor for 5 min. Each of the hydrolysate products was
dried with N2 (g) and then treated with Marfey’s reagent as men-
tioned above to yield the four Marfey-derived statine standards.
by COSY.
3,14-Di-(S)-MTPA Ester of Lyngbyabellin M (6): Pale yellow
amorphous solid. 1H NMR (CDCl3, 600 MHz): δ = 8.16 (s, 1 H,
12-H), 7.96 (s, 1 H, 13-H), 6.78 (dd, J = 7.6, 2.9 Hz, 1 H, 14-H),
5.98 (d, J = 5.4 Hz, 1 H, 20-H), 5.48 (td, J = 7.6, 3.4 Hz, 1 H, 3-
H), 5.07 (dd, J = 12.3, 2.9 Hz, 1 H, 15-Ha), 4.79 (dd, J = 12.3,
7.6 Hz, 1 H, 15-Hb), 4.43 (q, J = 7.1 Hz, 2 H, 24-H2), 3.05 (dq, J
= 7.6, 7.2 Hz, 1 H, 2-H), 2.41 (m, 1 H, 21-H), 2.12 (m, 1 H, 6-Ha),
2.07 (m, 1 H, 6-Hb), 2.06 (s, 3 H, 8-H3), 1.73 (m, 1 H, 5-Ha), 1.68
(m, 1 H, 5-Hb), 1.59 (m, 2 H, 4-H2), 1.41 (t, J = 7.1 Hz, 3 H, 25-
H3), 1.28 (d, J = 7.2 Hz, 3 H, 9-H3), 0.96 (d, J = 6.8 Hz, 3 H, 22-
H3), 0.95 (d, J = 6.8 Hz, 3 H, 23-H3) ppm. LR ESIMS: m/z =
1057.02 [M + H]+, 1079.12 [M + Na]+.
3,14-Di-(R)-MTPA Ester of Lyngbyabellin M (7): Pale yellow
amorphous solid. 1H NMR (CDCl3, 600 MHz): δ = 8.22 (s, 1 H,
12-H), 7.88 (s, 1 H, 18-H), 6.72 (dd, J = 7.1, 3.1 Hz, 1 H, 14-H),
5.95 (d, J = 5.4 Hz, 1 H, 20-H), 5.47 (m, 1 H, 3-H), 4.98 (dd, J =
12.3, 3.1 Hz, 1 H, 15-Ha), 4.78 (dd, J = 12.3, 7.1 Hz, 1 H, 15-Hb),
4.44 (q, J = 7.1 Hz, 2 H, 24-H2), 3.05 (m, 1 H, 2-H), 2.42 (m, 1 H,
21-H), 2.19 (m, 1 H, 6-Ha), 2.14 (m, 1 H, 6-Hb), 2.11 (s, 3 H, 8-
H3), 1.78 (m, 1 H, 5-Ha), 1.68 (m, 1 H, 5-Hb), 1.76 (m, 2 H, 4-
H2), 1.42 (t, J = 7.1 Hz, 3 H, 25-H3), 1.16 (d, J = 7.2 Hz, 1 H, 9-
H), 0.95 (d, J = 6.8 Hz, 6 H, 22-H3, 23-H3) ppm. LR ESIMS: m/z
= 1056.99 [M + H]+, 1079.07.58 [M + Na]+.
Preparation and GC–MS Analysis of HIVA: An aliquot (ca. 500 μg)
of the hydrolysate was treated with an excess of CH2N2 at room
temperature for 30 min and dried under N2 (g). Correspondingly,
l- and d-HIVA were synthesized as described above. The products
were dissolved in CH2Cl2 and injected into a chiral GC–MS instru-
ment with a Chiralsil-Val column (Agilent Technologies J&W Sci-
entific, 30 mϫ0.25 mm) under the following conditions: the initial
oven temperature was 32 °C, kept for 15 min, followed by a ramp
from 32 to 60 °C at a rate of 10 °C/min, followed by another ramp
to 200 °C, at a rate of 15 °C/min and kept at 200 °C for 5 min. The
retention time of products resulting from the acid hydrolysate of 4
matched those of the synthetic (2S)-HIVA standard [9.7 min; (2R)-
HIVA, 10.2 min].
Cytotoxicity Assay: H-460 cells were added to 96-well plates at
3.33ϫ104 cells/mL of Roswell Park Memorial Institute (RPMI)
1640 medium with 10% fetal bovine serum (FBS) and 1% penicil-
lin/streptomycin. The cells, in a volume of 180 μL per well, were
incubated overnight (37 °C, 5% CO2) to allow recovery before
treatment with test compounds. Compounds were dissolved in
DMSO to a stock concentration of 10 mg/mL. Working solutions
of the compounds were made in RPMI 1640 medium without FBS,
with a volume of 20 μL added to each well to give a final com-
pound concentration of either 30 or 3 μg/mL. An equal volume of
RPMI 1640 medium without FBS was added to wells designated
as negative controls for each plate. Plates were incubated for ap-
proximately 48 h before being stained with 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT). By using a Thermo-
Electron Multiskan Ascent plate reader, plates were read at 570 and
630 nm. Concentration response graphs were generated by using
GraphPad Prism (GraphPad Software Inc., San Diego, CA).
Preparation and GC–MS Analysis of DiMeVal: The methylated hy-
drolysate product of 5 was analyzed by chiral GC–MS using a Cy-
closil
B
column (Agilent Technologies J&W Scientific,
30 mϫ0.25 mm) under the following conditions: the initial oven
temperature was 34 °C and kept for 68 min, followed by a ramp
from 34 to 100 °C at a rate of 30 °C/min and kept at 100 °C for
5 min. Synthetic standards of (2S)- and (2R)-DiMeVal were first
methylated by dissolving each starting material (10 mg) in H2O
(433 μL), followed by the addition of formaldehyde (27 μL) and
10% Pd/C (10.4 mg). The mixture was then treated with H2 (g) for
16 h. After 16 h, the reaction mixtures were brought to a boil and
then concentrated in vacuo. Each of the synthetic standards were
then treated with CH2N2 for 5 min and then dried with N2 (g),
resuspended in CH2Cl2, then analyzed by chiral GC–MS. The re-
tention time of products resulting from the acid hydrolysate of 5
matched that of the authentic (2S)-DiMeVal standard [63.7 min;
(2R)-DiMeVal, 64.2 min].
Supporting Information (see footnote on the first page of this arti-
1
cle): H NMR, 13C NMR, COSY, gHSQC, and HMBC spectra in
1
CDCl3 for lyngbyabellin K and M (1 and 4). H NMR spectra in
CDCl3 for the Mosher derivatives of lyngbyabellin M (4). 1H
NMR, 13C NMR, gHSQC, COSY, HMBC, DQF-COSY, NOESY,
and HETLOC spectra in CDCl3 for lyngbyabellin L (2) and 7-epi-
lyngbyabellin L (3). 1H NMR, 13C NMR, gHSQC, COSY, HMBC,
and TOCSY spectra in [D6]DMSO for lyngbyabellin N (5). 1H
NMR and 13C NMR in CDCl3 for lyngbyabellin N (5). ORTEP
representation of lyngbyabellin K (1) crystal, and crystal data. CD
spectra for lyngbyabellin K, L, N (1, 2, and 5) and 7-epi-lyngbyab-
ellin L (3).
Preparation of the α-Methoxy-α-(trifluoromethyl)phenylacetic Acid
(MTPA) Ester of Lyngbyabellin M: Duplicate samples of com-
pound 4 (0.5 mg) were dried and dissolved in anhydrous pyridine
(1 mL), and a catalytic amount of 4-(dimethylamino)pyridine was
added. Separately and into each vial, 15 μL of (R)-MTPA chloride
and 15 μL of (S)-MTPA chloride were added. The reaction vials
were stored at 40 °C with stirring for 48 h. The acylation products
were purified by using RP HPLC (Phenomenex Jupiter 5 μ C18,
4.6ϫ250 mm, 85% CH3OH/H2O at 1 mL/min). The m/z values of
the two diastereomeric MTPA derivatives of compound 4 were ob-
Acknowledgments
We thank A. C. Jones, A. R. Pereira, and R. C. Coates for assist-
ance with the collection of the lyngbyabellin-producing cyanobacte-
rial samples from the Palmyra Atoll, C. E. Moore for assistance
with crystallographic data analysis of lyngbyabellin K, as well as
Eur. J. Org. Chem. 0000, 0–0
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
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Job/Unit: O20691
/KAP1
Date: 13-08-12 16:33:40
Pages: 11
H. Choi, E. Mevers, T. Byrum, F. A. Valeriote, W. H. Gerwick
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Growth Regulation & Oncogenesis Training Grant NIH/NCI
(T32A009523-24) for a fellowship to E. M.
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Published Online: ᭿
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Eur. J. Org. Chem. 0000, 0–0

Job/Unit: O20691
/KAP1
Date: 13-08-12 16:33:40
Pages: 11
Lyngbyabellins K–N from the Marine Cyanobacterium Moorea bouillonii
Cytotoxic Lipopeptides
Two independent collections of the marine
cyanobacterium Moorea bouillonii led to
the isolation of five lipopeptides of the
lyngbyabellin structure class. Their struc-
tures were elucidated by various spec-
troscopy, synthesis, and chromatography
techniques. Lyngbyabellin N showed strong
cytotoxic activity against the HCT116 co-
lon cancer cell line (IC50 = 40.9Ϯ3.3 nm).
H. Choi, E. Mevers, T. Byrum,
F. A. Valeriote, W. H. Gerwick* ..... 1–11
Lyngbyabellins K–N from Two Palmyra
Atoll Collections of the Marine Cyanobac-
terium Moorea bouillonii
Keywords: Natural products / Peptides /
Structure elucidation / Biological activity
Eur. J. Org. Chem. 0000, 0–0
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
11