Cycloforskamide, a cytotoxic macrocyclic peptide from the sea slug Pleurobranchus forskalii

Article abstract of DOI:10.1021/np400404r

A macrocylic dodecapeptide, cycloforskamide, was isolated from the sea slug Pleurobranchus forskalii, collected off Ishigaki Island, Japan. Its planar structure was deduced by extensive NMR analyses and was further confirmed by MS/MS fragmentation analyses. Finally, the absolute configuration was determined by total hydrolysis and chiral-phase gas chromatographic analysis. This novel dodecapeptide contains three d-amino acids and three thiazoline heterocycles and exhibits cytotoxicity against murine leukemia P388 cells, with an IC ₅₀ of 5.8 μM.

Full text of DOI:10.1021/np400404r

Note
pubs.acs.org/jnp
Cycloforskamide, a Cytotoxic Macrocyclic Peptide from the Sea Slug
Pleurobranchus forskalii
Karen Co Tan,† Toshiyuki Wakimoto,*,† Kentaro Takada,‡ Takashi Ohtsuki,§ Nahoko Uchiyama,§
Yukihiro Goda,§ and Ikuro Abe*,†
†Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku Tokyo 113-0033, Japan
‡Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
§National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan
S
* Supporting Information
ABSTRACT: A macrocylic dodecapeptide, cycloforskamide,
was isolated from the sea slug Pleurobranchus forskalii, collected
off Ishigaki Island, Japan. Its planar structure was deduced by
extensive NMR analyses and was further confirmed by MS/MS
fragmentation analyses. Finally, the absolute configuration was
determined by total hydrolysis and chiral-phase gas chromato-
graphic analysis. This novel dodecapeptide contains three D-
amino acids and three thiazoline heterocycles and exhibits
cytotoxicity against murine leukemia P388 cells, with an IC50 of
5.8 μM.
he sea slug Pleurobranchus forskalii (class Gastropoda,
Torder Notaspidea, family Pleurobranchidae) is widely
distributed in temperate, shallow, subtidal areas around the
Indo-Pacific and Mediterranean Seas. It belongs to a family of
shell-less mollusks that are opportunistic carnivores, which
scavenge on a variety of other invertebrates, such as sponges
and ascidians.1 Although these physically defenseless mollusks
are believed to secrete bioactive chemicals for their protection
and are therefore a vast resource of secondary metabolites,2
there have only been a limited number of studies on the
chemical components of P. forskalii. For instance, a cytotoxic
cyclic peptide, keenamide A, was isolated from this gastropod
collected from Manado, Indonesia,2 while lissoclimide-type
diterpenes were identified from samples collected in Philippine
waters.3 Furthermore, an ergot alkaloid peptide, ergosinine,
which has been isolated thus far only from terrestrial higher
plants and fungi, has also been found in P. forskalii.4 Likewise, a
potent neurotoxin, tetrodotoxin, has been detected in the gray
side-gilled sea slug, Pleurobranchaea maculata,5 which belongs
to the same family as P. forskalii.
molecular formula was established as C54H86N12O11S3 by
HRFABMS, which gave a pseudomolecular ion peak at
The marine mollusk P. forskalii was collected by hand in the
ocean near Ishigaki Island, Okinawa. The ethanolic extract of
the frozen mollusk (400 g) was partitioned between H2O and
CHCl3, and the organic layer was subjected to silica gel
chromatography, followed by reversed-phase HPLC on ODS,
to afford cycloforskamide (3.0 × 10−3%, by wet weight), as a
white powder.
1
1175.5774 [M + H]+. The appearance of H NMR signals
between 6.5 and 8.5 ppm upon changing the NMR solvent
from CD3OD to CDCl3 indicated the presence of exchangeable
protons, presumably amide protons, suggesting the peptide
Cycloforskamide (1) showed m/z peaks at 1175 [M + H]+
and 1197 [M + Na]+ in the FABMS spectrum, and its
Received: May 21, 2013
Published: July 12, 2013
© 2013 American Chemical Society and
American Society of Pharmacognosy
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Journal of Natural Products
Note
Table 1. NMR Spectroscopic Data for 1 in CDCl3
position
δC, type
δH (J in Hz)
HMBC
NOESY
position
δC, type
δH (J in Hz)
HMBC
NOESY
NHallo‑Ile
L-Thr 1
L-Val 3
28
1
170.7, C
57.3, CH
69.6, CH
20.2, CH3
172.8, C
2
5.36, d (4.0)
4.28, m
1, 3
54
29
59.9, CH
27.0, CH
18.8, CH3
18.7, CH3
4.67, m
28
3
30
2.04, m
29, 31
29
4
1.30, d (5.6)
6.76 (br)
2, 3
5
31
0.83, m
NH
OH
L-Val 1
5
6
32
0.85, m
29
NH
L-Tzn 2
33
7.28, d (10.4)
33
173.5, C
172.2, C
79.6, CH
39.8, CH2
b
6
59.9, CH
33.3, CH
19.5, CH3
15.6, CH3
4.40, t (8.6)
2.08, m
5, 7, 8
34
5.11, dd (10.4, 2.6)
3.53, m
30, 33, 35, 36
7
35
33, 34, 36
a
8
0.92
7
D-Pro 1
36
a
9
0.92
7
181.7, C
62.2, CH
32.2, CH2
NH
L-Val 2
10
7.35, d (9.6)
10
NHVal2
37
4.93, d (5.6)
2.36, m
2.08, m
2.22, m
1.63, m
3.79, m
3.69, m
36, 38, 39
36, 39, 40
36
38
172.6, C
11
59.9, CH
30.8, CH
18.6, CH3
12.2, CH3
4.67, m
2.33, m
10, 12, 13
11
39
40
26.1, CH2
49.7, CH2
37, 38, 40
38
12
a
b
13
0.92
37, 38, 39
42
40
a
14
0.87
NH
L-Thr 2
15
8.35 (br)
NHVal1, 16
L-Ile
41
171.5, C
56.6, CH
40.5, CH
26.2, CH2
171.7, C
57.9, CH
70.1, CH
20.1, CH3
42
4.57, t (8.8)
1.63, m
1.40
41, 43, 44, 46
42
16
4.80, d (9.6)
4.04, d (2.1)
1.01, d (6.4)
7.98 (br)
15, 17, 18
16, 17
43
17
NHVal2
44
43, 45
43, 45
43, 44, 46
43, 44
47
18
1.03
a
NH
OH
L-Tzn 1
19
45
13.1, CH3
17.3, CH3
0.84
a
46
0.90
NH
L-Tzn 3
47
7.22, d (9.6)
48
171.4, C
79.2, CH
39.2, CH2
20
5.01, t (7.2)
3.71, m
19, 21, 22
19, 20, 22
171.5, C
79.2, CH
39.6, CH2
21
48
4.98, t (7.2)
3.62, m
47, 49, 50
47, 48, 50
NHIle
D-allo-Ile
22
49
178.7, C
57.8, CH
39.6, CH
28.5, CH2
D-Pro 2
50
23
4.76, d (4.6)
1.96, m
22, 24
176.2, C
b
24
51
60.4, CH
31.4, CH2
25.6, CH2
5.07, d (5.7)
2.09, m
48
25
1.38, m
24
52
50, 53
54
1.50, m
23, 24, 26, 27
24, 25
53
1.77, m
26
13.7, CH3
15.7, CH3
0.95, t (8.8)
1.96, m
a
27
0.89
24, 25
54
48.4, CH2
3.69, m
52, 53
2
NH
7.60 (br)
29
4.15, t (8.93)
a
b
Overlapped. Correlations observed only at 800 MHz.
nature of the isolated compound. The two doublet methyl
signals at δH 1.01 and 1.30 (Table 1), which showed HMBC
correlations with deshielded carbons at δC 70.1 and 69.6,
respectively, suggested the presence of two Thr residues. The
presence of branched-chain amino acids, two Ile and three Val
residues, was apparent due to the heavily overlapped upfield
region and was confirmed by COSY and HMBC experiments.
Among the heavily overlapping signals between δH 3.5 and 4.0,
at least two protons had HMQC correlations with carbons at δC
49.7 and 48.4, reminiscent of aminomethylene groups,
suggesting the presence of at least two Pro residues. As the
NMR spectra recorded at 800 MHz revealed well-resolved
signals in the corresponding region, these assignments were
further corroborated. Some signals in the same region at δH
3.53, 3.71, and 3.62, displaying HMQC correlations with δC
39.8, 39.2, and 39.6, respectively, were assumed to be
methylene protons attached to an electronegative atom,
probably sulfur. These protons showed COSY correlations
with three presumed α-protons at δH 5.11, 5.01, and 4.98,
respectively. This sulfur-containing heterocycle was later
inferred to be thiazoline (Tzn), by corroboration with the
HMBC data. Of the three Tzn rings, two derive from the
carbonyl of Pro residues, as indicated by the HMBC correlation
between the proline’s β-protons and the thiazoline’s imine
carbon. Incidentally, the Ile-Pro1 and Thr1-Pro2 amide bonds
were both inferred to adopt a trans configuration, as indicated
by the NOESY correlation between the Pro’s δ-protons and the
preceding amino acid’s α-proton.6,7 The final Tzn ring derives
from an Ile carbonyl, as indicated by the HMBC correlation
between the Ile α-H at δH 4.76 and the Tzn imine carbon at δC
178.7. The rest of the amino acid sequence was determined by
either HMBC or NOESY correlations (Figure 1), except for the
Thr2−Tzn1 connection.
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Note
To determine the absolute configuration of 1, it was first
subjected to total acid hydrolysis, to obtain its component
amino acids. The Tzn moiety is converted to Cys in this
process.10 N-Trifluoroacetyl/methyl ester derivatives of the
hydrolysate were subsequently subjected to a chiral-phase GC
analysis, and the retention times were compared with those of
standard amino acids derivatized in the same manner. Through
this experiment, all three Val, both Thr, one of the two Ile, and
all three Cys residues were found to be in the L-form. On the
basis of the latter result, Tzn should also exist in the L-form. In
contrast, the Pro and allo-Ile residues were both found to be in
the D-form. On the basis of these results, the two Ile residues,
one preceding Tzn and another preceding Pro, had opposite
configurations at the α-position. However, the α-carbon
preceding a Tzn ring is reportedly extremely labile to
epimerization, due to the adjacent imine bond.11−13 Thus, it
can be inferred that D-allo-Ile precedes Tzn, while L-Ile precedes
Pro. In accordance with the sequence of the planar structure, it
also follows that the Pro residues, which both precede a Tzn
ring, exhibit the D-configuration. In order to rule out the
possibility that the D-amino acids were racemized hydrolytic
products, 1 was treated with ozone to cleave the imine bond of
Tzn prior to hydrolysis, thus preventing racemization.10,14 The
results established that the intact peptide contains D-allo-Ile and
exclusively D-Pro. Wipf et al. reported that upon treatment of
the unnatural [L-Val]-lissoclinamide 7 with pyridine, the α-H of
Val, which neighbors Tzn, spontaneously epimerizes, yielding
the naturally observed epimer, lissoclinamide 7.12 Thus, the
thermodynamically stable conformation of the macrocyclic
peptide would reinforce the epimerization to the D-config-
uration in preference to the L-isomer, by the stereochemically
labile site adjacent to the thioimide functionality.15 Therefore,
the complete structure of cycloforskamide was established as 1.
Cycloforskamide showed moderate cytotoxicity against P388
murine leukemia cells, with an IC50 of 5.8 μM. From an
ecological point of view, because patellamides are also cytotoxic
and are metal chelators, they may serve as feeding deterrents
and/or play a detoxification role by chelating toxic metals, such
as copper and zinc.16 In addition, the biosynthetic genes for
cyanobactin production have been ascribed to a symbiotic
cyanobacterium, Prochloron didemni.8,9 Therefore, we envision
that 1 could be a symbiont-derived peptide conferring
beneficial effects to the host organism. Experiments are
currently being performed to clarify whether P. forskalii hosts
the cyanobacteria that produce 1 or acquires them from its diet.
Figure 1. HMBC and NOESY correlations of 1.
Despite the absence of 2D NMR correlations, tandem mass
spectrometric data provided evidence that these two amino
acids were indeed connected. In particular, the daughter ion at
m/z 975 suggested the loss of 200 amu, which can be assigned
to a Val-Thr fragment (Figure 2). Meanwhile, another daughter
EXPERIMENTAL SECTION
Figure 2. MS/MS fragmentation data for 1.
■
General Experimental Procedures. Optical rotations were
measured on a Jasco DIP-1000 digital polarimeter. The UV spectrum
was obtained by a Gene Spec III UV/vis spectrometer. H and 13C
1
NMR, COSY, HMQC, HMBC, and NOESY (mixing time, 250 μs)
spectra in CDCl3 were recorded at 293 K on either a JEOL ECX-500
(1H 500 MHz, 13C 125 MHz) NMR spectrometer with a 5 mm broad
band tunable probe or an ECA-800 (1H 800 MHz, 13C 200 MHz)
NMR spectrometer equipped with a 5 mm C13/H1 dual cold probe
with a z-axis gradient, utilizing residual solvent signals for referencing.
FABMS and FABMS/MS experiments were performed on a JEOL
JMS-700T spectrometer, using m-nitrobenzyl alcohol as the matrix.
The geometry of the spectrometer was BEBE, with an accelerating
voltage of 10 kV. The selected ion was collided with helium gas in the
collision chamber, which was floated. The gas was introduced to cause
the dissociation at a pressure that reduced the intensity of precursor
ions to 30%.
ion appeared at m/z 862, reflecting the loss of 313 amu, which
could be assigned to a Val-Thr-Tzn fragment. Hence, the planar
structure of 1 was determined to be an N-to-C macrocyclic
Tzn-containing dodecapeptide. The structure of 1 is similar to
those of the patellamides and lissoclinamides, and 1 thus
appears to belong to a class of ribosomally synthesized, post-
translationally modified peptides (RiPPs) called cyanobactins.
The characteristic structural features of cyanobactins include N-
to-C macrocyclization and heterocyclization to form thiazoline,
methyloxazoline, and oxazoline, or their oxidized counter-
parts.8,9 The 36-membered ring size of 1 is comparable to that
of wewakazole from Lyngbya majuscula.10
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Journal of Natural Products
Note
Animal Material. The Pleurobranchus forskalii sea slug was
collected at a depth of 2 m in the ocean near Ishigaki Island, Japan,
in May 2012. The animal was kept frozen until use. A voucher
specimen (Pf-I-0512) was deposited at the Laboratory of Natural
Products Chemistry, Graduate School of Pharmaceutical Sciences, The
University of Tokyo.
ASSOCIATED CONTENT
* Supporting Information
Detailed experimental procedures and spectroscopic data. This
material is available free of charge via the Internet at http://
pubs.acs.org.
■
S
Isolation. A portion of the frozen animal (400 g) was crushed and
extracted with EtOH (2.5 L). The ethanolic extract was evaporated in
vacuo and partitioned between H2O (500 mL) and CHCl3 (500 mL ×
5). The CHCl3-soluble material (660 mg) was subjected to flash
chromatography on a silica gel column, eluted with a stepwise gradient
of MeOH (0−20%) in CHCl3. The fractions eluted with around 1%
MeOH in CHCl3 were then loaded on a Cosmosil MS-II column (⦶
250 × 10 mm). A linear gradient was employed using aqueous MeOH,
starting from 70%, increasing its concentration to 100% for a period of
10 min, and further washing with the organic solvent for 15 min. The
flow rate was set at 3.2 mL/min, while UV detection was performed at
210 nm. The major peak, eluting between 14.5 and 15.3 min, yielded
1.2 mg of compound 1. In an effort to increase the yield, an additional
200 g portion of the mollusk was extracted and purified in the same
manner as above. Furthermore, the side fractions from the silica gel
chromatography and HPLC separation were analyzed for the presence
of 1 and were purified by the HPLC method described above. They
were subsequently combined with the original sample, after giving
NMR and mass spectrometric data indistinguishable from those of 1.
The final combined yield of 1 was 3.0 mg.
AUTHOR INFORMATION
Corresponding Author
*E-mail: wakimoto@mol.f.u-tokyo.ac.jp, abei@mol.f.u-tokyo.ac.
jp. Phone: +81-3-5841-4740. Fax: +81-3-5841-4744.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful to Prof. K. Tsuchiya, Tokyo University of
Marine Science and Technology, for the species identification.
K.C.T. is grateful to the Ajinomoto Scholarship Foundation.
This work was partly supported by a Grant-in-Aid from the
Ministry of Education, Culture, Sports, Science and Technol-
ogy (MEXT), Japan.
REFERENCES
■
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Cycloforskamide (1): white solid; [α]22 +13 (c 0.07, MeOH);
D
1
UV (MeOH) λmax (log ε) 227 nm (2.60); H and 13C NMR (Table
1); HRFABMS m/z 1175.5774 [M + H]+ (calcd for C54H87N12O11S3,
1175.5779, Δ 0.6 mmu).
Amino Acid Analysis by Chiral-Phase GC. Cycloforksamide
(100 μg) was hydrolyzed with 6 M HCl (500 μL) at 110 °C for 24 h.
The reaction mixture was treated with 5−10% HCl/MeOH (500 μL)
at 100 °C for 30 min and was then treated with trifluoroacetic
anhydride (TFAA)/CH2Cl2 (1:1, 500 μL) at 100 °C for 5 min. The
chiral-phase GC analysis of the N-trifluoroacetyl (TFA)/methyl ester
derivatives was performed using a CP-Chirasil-D-Val column (Alltech,
0.25 mm × 25 m; N2 as the carrier gas; program rate 50−200 °C at 4
°C/min), and peaks were observed at tR = 6.08, 7.03, 8.26, 8.53, 9.28,
and 15.32 min. To confirm the absolute configuration of the
thiazoline-based amino acids, a methanolic solution of the peptide
(100 μg in 3 mL) was cooled to −78 °C, and then a stream of ozone
was bubbled through this solution until the reaction mixture turned
pale blue. Oxidative workup was performed by adding 7 drops of 30%
hydrogen peroxide, and the reaction mixture was allowed to stand at
room temperature for 1 h. After removing the solvent under nitrogen,
acid hydrolysis, Me/TFAA derivatization, and chiral-phase GC analysis
were conducted as above. Standard amino acids were also converted to
the TFA/Me derivatives by the same procedure. Retention times
(min) were as follows: L-Val (6.01), D-Val (6.58), L-Thr (6.98), D-Thr
(7.77), L-allo-Thr (10.55), D-allo-Thr (11.54), L-Ile (8.24), D-Ile
(8.90), L-allo-Ile (7.60), D-allo-Ile (8.43), L-Pro (8.69), D-Pro (9.28), L-
Cys (15.88), D-Cys (16.11). Thus, the presence of L-Val, L-Thr, L-Ile,
D-allo-Ile, D-Pro, and L-Cys (L-Tzn) was confirmed.
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Cytotoxicity Assay against P388. P388 murine leukemia cells
were cultured in RPMI 1640 (Wako Chemicals) medium,
supplemented with 10 μg/mL of penicillin/streptomycin (Invitrogen)
and 10% fetal bovine serum (MP Biomedicals), at 37 °C under a 5%
CO2 atmosphere. To each well of the 96-well microplate, containing
100 μL of 1 × 105 cells/mL tumor cell suspension, was added 100 μL
of test solution (samples were dissolved in MeOH), and the plates
were incubated for 3 days. After the addition of 50 μL of 3-(4,5-
dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT)
solution (1 mg/mL) to each well, the plates were incubated for 4 h
under the same conditions. The mixtures were centrifuged, and the
supernatants were removed by suction. The precipitates thus obtained
were dissolved in DMSO, and the absorbance at 570 nm was measured
with a microplate reader. The IC50 for the positive control,
doxorubicin, was 210 nM.
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