Amphidinolide X, a novel 16-membered macrodiolide from dinoflagellate Amphidinium sp

Article abstract of DOI:10.1021/jo0343634

A novel cytotoxic 16-membered macrodiolide, amphidinolide X (1), has been isolated from a marine dinoflagellate Amphidinium sp. (strain Y-42). The gross structure of 1 was elucidated on the basis of spectroscopic data including one-bond and long-range ¹³C-¹³C correlations. The relative and absolute stereochemistries were determined by combined analyses of NOESY data and ¹H-¹H and ¹H-¹³C coupling constants of 1 and NMR data of the degradation products. Amphidinolide X (1) is the first macrodiolide consisting of polyketide-derived diacid and diol units from natural sources. The biosynthetic origins of 1 were investigated by means of feeding experiments with ¹³C-labeled acetates.

Full text of DOI:10.1021/jo0343634

Am p h id in olid e X, a Novel 16-Mem ber ed Ma cr od iolid e fr om
Din ofla gella te Am ph id in iu m sp .
Masashi Tsuda,† Naoko Izui,† Kazutaka Shimbo,† Masaaki Sato,† Eri Fukushi,‡
J un Kawabata,‡ Kazuhito Katsumata,§ Takeo Horiguchi,§ and J un’ichi Kobayashi*,†
Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, J apan, Graduate
School of Agriculture, Hokkaido University, Sapporo 060-8589, J apan, and Graduate School of Sciences,
Hokkaido University, Sapporo 060-0810, J apan
jkobay@pharm.hokudai.ac.jp
Received March 19, 2003
A novel cytotoxic 16-membered macrodiolide, amphidinolide X (1), has been isolated from a marine
dinoflagellate Amphidinium sp. (strain Y-42). The gross structure of 1 was elucidated on the basis
of spectroscopic data including one-bond and long-range 13C-13C correlations. The relative and
1
absolute stereochemistries were determined by combined analyses of NOESY data and H-1H and
1H-13C coupling constants of 1 and NMR data of the degradation products. Amphidinolide X (1) is
the first macrodiolide consisting of polyketide-derived diacid and diol units from natural sources.
The biosynthetic origins of 1 were investigated by means of feeding experiments with 13C-labeled
acetates.
In tr od u ction
cidated on the basis of spectroscopic data including one-
bond and long-range 13C-13C correlations. The relative
and absolute stereochemistries were determined by a
combination of analysis of NOESY data together with
1H-1H and 1H-13C coupling constants of 1 and NMR
data of the degradation products. The biosynthetic origins
of 1 were investigated by means of feeding experiments
with 13C-labeled acetates. This paper describes the isola-
tion, structure elucidation, and biosynthesis of 1.
Marine microorganisms have been proven to produce
a variety of chemically unique and biologically interesting
secondary metabolites1 such as long-chain polyketides
and polyethers from marine dinoflagellates.2 We have
isolated 25 macrolides, named amphidinolides, from
extracts of marine dinoflagellates of the genus Amphi-
dinium, which are symbionts of Okinawan marine acoel
flatworms, Amphiscolops spp.3,4 These macrolides have
a variety of backbone skeletons and different sizes of
macrocyclic lactone rings (12- to 29-membered rings), and
more than half (16) of the amphidinolides have odd-
numbered macrocyclic lactone rings. Most of the amphi-
dinolides contain vicinally located one-carbon branches
and at least one exo-methylene unit, and some of them
exhibit potent cytotoxicity and antitumor activity. There-
fore, amphidinolides have attracted great interest as
challenging targets for total synthesis5 and biosynthetic
studies.2
Resu lts a n d Discu ssion
Isola tion of Am p h id in olid e X (1). The dinoflagellate
Amphidinium sp. (strain Y-42)4 was mass cultured
unialgally at 25 °C for 14 days in a seawater medium
enriched with 1% Provasoli’s Ert-Schereiber (ES) supple-
ment. The harvested algal cells (363 g, wet weight, from
686 L of culture) were extracted with MeOH/toluene
(3:1), and the extracts were partitioned between toluene
In our continuing search for bioactive secondary me-
tabolites from laboratory-cultured marine dinoflagel-
lates,4,6 we encountered an unprecedented 16-membered
macrodiolide, amphidinolide X (1), consisting of polyketide-
derived diacid and diol units from the dinoflagellate
Amphidinium sp. (strain Y-42). The structure was elu-
(5) Note the following examples. (a) Amphidiolide A: Maleczeka,
R. E., J r.; Terrell, L. R.; Gang, F.; Ward, J . S., III Org. Lett. 2002, 4,
2841-2844. (b) Lam, H. W.; Pattenden, G. Angew. Chem., Int. Ed.
2002, 41, 508-811. (c) Trost, B. M.; Chisholm, J . D.; Wrobleski, S. T.;
J ung, M. J . Am. Chem. Soc. 2002, 124, 12420-12421. (d) Amphidi-
nolide J : Williams, D. R.; Kissel, W. S. J . Am. Chem. Soc. 1998, 120,
11198-11199. (e) Amphidinolide K: Williams, D. R.; Meyer, K. G. J .
Am. Chem. Soc. 2001, 123, 765-766. (f) Amphidinolide P: Williams,
D. R.; Myers, B. J . Org. Lett. 2000, 2, 945-948. (g) Amphidinolide T1:
Ghosh, A. K.; Liu, C. 2003, 125, 2374-2375. (h) Amphidinolide T4:
Fu¨rstner, A.; A¨ıssa, C.; Riveiros, R.; Ragot, J . Angew. Chem., Int. Ed.
2002, 41, 4763-4766.
(6) (a) Doi, Y.; Ishibashi, M.; Nakamichi, H.; Kosaka, T.; Ishikawa,
T.; Kobayashi, J . J . Org. Chem. 1997, 62, 3820-3823. (b) Kubota, T.;
Tsuda, M.; Doi, Y.; Takahashi, A.; Nakamichi, H.; Ishibashi, M.;
Fukushi, E.; Kawabata, J .; Kobayashi, J . Tetrahedron 1998, 54,
14455-14464. (c) Kobayashi, J .; Kubota, T.; Takahashi, M.; Ishibashi,
M.; Tsuda, M.; Naoki, H. J . Org. Chem. 1999, 64, 1478-1482. (d)
Kubota, T.; Tsuda, M.; Takahashi, M.; Ishibashi, M.; Naoki, N.;
Kobayashi, J . J . Chem. Soc., Perkin Trans. 1 1999, 3483-3488. (e)
Kubota, T.; Tsuda, M.; Takahashi, M.; Ishibashi, M.; Oka, S.; Koba-
yashi, J . Chem. Pharm. Bull. 2000 48, 1447-1451.
* Author to whom correspondence should be addressed. Fax: +81
11 706 4985 or +81 11 706 4989. Phone: +81 11 706 4985.
† Graduate School of Pharmaceutical Sciences.
‡ Graduate School of Agriculture.
§ Graduate School of Sciences.
(1) Dranas, A. H.; Norte, M.; Ferna´ndez, N. J . Toxicon 2001, 39,
1101-1132.
(2) Rein, K. S.; Borrone, J . Comp. Biochem. Physiol. 1999, B124,
117-131.
(3) (a) Kobayashi, J .; Ishibashi, M. Chem. Rev. 1993, 93, 1753-1769.
(b) Ishibashi, M.; Kobayashi, J . Heterocycles 1997, 44, 543-572.
(4) (a) Shimbo, K.; Tsuda, M.; Izui, N.; Kobayashi, J . J . Org. Chem.
2002, 67, 1020-1023 and references therein. (b) Kobayashi, J .; Shimbo,
K.; Sato, M.; Tsuda, M. J . Org. Chem. 2002, 67, 6585-6582.
10.1021/jo0343634 CCC: $25.00 © 2003 American Chemical Society
Published on Web 06/03/2003
J . Org. Chem. 2003, 68, 5339-5345
5339

Tsuda et al.
TABLE 1. 1H a n d 13C NMR Da ta for Am p h id in olid e X (1) in CDCl3.
position
δC
δH (m, Hz)
HMBC (H)
LR 13C-13C
1
2
3
4
5
165.74 s
120.16 d
153.15 d
33.02 d
41.41 t
2, 3,
3
5a, 5b, 23
2, 3, 5a, 5b, 23
23
2, 4, 16, 17
5.79 d, 15.8
7.12 dd, 7.2, 15.8
2.79 m
2.58 dd, 3.7, 13.4
2.41 dd, 6.3, 13.4
3, 23
4
3
2
6
7
8
9
170.69 s
30.35 q
205.52 s
47.10 t
5a, 5b
5, 10
2.14b
s
7, 9a, 9b
7, 10
7, 9
10
2.69 dd, 6.0, 16.5
2.57 dd, 8.2, 16.5
5.21 m
2.69 m
4.95 d, 10.3
10
11
12
13
14
74.23 d
35.47 d
125.99 d
135.47 s
35.31 t
9a, 9b, 24
9
9a, 9b, 12, 24
10, 11, 14a, 14b, 24, 25
11, 14a, 14b, 25
12, 25
10, 12
11
14, 25
12, 15
2.18 m
2.11 brt, 9.4
1.95 tt, 2.9, 13.4
1.54 m
3.97 dt, 11.1, 3.6
5.19 m
2.16 m
1.75 dd, 2.4, 13.8
15
30.39 t
16
16
17
18
80.54 d
78.41 d
43.49 t
18a
15a, 18b
15, 17
18
17
19
20
21
22
23
24
25
26
82.92 s
44.17 t
17.81 t
14.61 q
17.48 q
18.09 q
15.37 q
24.52 q
17, 18a, 18b, 20, 26
18a, 18b, 21, 22
20, 22
18, 20, 26
1.50a
1.34a
m
m
0.92b t, 7.4
1.14b d, 6.8
0.92b d, 6.8
21
3, 5a
11, 12
12, 14b
18a, 18b
1.55b
1.30b
s
s
a
b
2H. 3H.
and water. The toluene-soluble materials were chromato-
graphed on a silica gel column (CHCl3/MeOH) to give a
lipophilic fraction including a small amount of mac-
rolides. To remove free fatty acids, the fraction was
treated with trimethylsilyl diazomethane and then puri-
fied by silica gel column chromatography to give a
macrolide-rich fraction, which was subjected to C18
column chromatography followed by C18 HPLC (CH3CN/
H2O) to afford amphidinolide X (1, 0.001%, wet weight)
together with known macrolides, amphidinolides G7
(0.0008%), H7 (0.0007%), and W (0.0012%).4a The 13C-
labeled sample of 1 was obtained from the algal cells
cultured in a medium enriched with 13C-labeled sodium
acetate.
attributed to carbonyl group(s). The UV absorption at 209
nm (ꢀ 6800) was suggestive of the presence of an enone
1
chromophore. H and 13C NMR data (Table 1) disclosed
the existence of a ketone, two ester carbonyls, an sp2
quaternary carbon, three sp2 methines, an sp3 quaternary
carbon, five sp3 methines including three oxygenated
ones, seven sp3 methylenes, and six methyl groups, three
of which resonated as singlet signals due to connection
to quaternary carbon(s). Since five out of seven unsat-
urations were accounted for, amphidinolide X (1) was
inferred to possess two rings.
1
Detailed analyses of the H-1H COSY, TOCSY, and
HSQC spectra revealed the following proton-proton
networks; (a ) from H-2 to H2-5 and H3-23, (b) from H2-9
to H-10, (c) from H-11 to H-12 and H3-24, (d ) from H2-
14 to H2-18, and (e) from H2-20 to H3-22 (Figure 1a). The
connection between partial structures b and c was
revealed by the HMBC correlation for H3-24 (δH 0.92) to
C-10 (δC 74.23), though the proton-proton connectivity
for H-10-H-11 was not assigned unambiguously by the
1H-1H COSY and TOCSY spectra due to its small
coupling constant [J (H-10/H-11) ) <1 Hz]. HMBC cor-
relations were observed for H2-9 (δH 2.69 and 2.57) and
H3-7 (δH 2.14, s) to C-8 (δC 205.52), implying the presence
of a methyl ketone group at C-9. HMBC correlations for
H-12 (δH 4.95)/C-14 (δC 35.31), H2-14 (δH 2.18 and 2.11)/
C-13, and H3-25 (δH 1.55)/C-13 suggested that the partial
structure c and an olefinic methyl (C-25) were attached
to an sp2 quaternary carbon (C-13) connected to C-14 in
the partial structure d . E-geometry for the trisubstituted
double bond at C-12-C-13 was assigned by NOESY
correlations for H-11 (δH 2.69)/H3-25 and H-12/H2-14. The
connection between partial structures d and e through
an oxygenated sp3 quaternary carbon (C-19) was deduced
Str u ctu r e Elu cid a tion of Am p h id in olid e X (1).
Amphidinolide X (1) showed molecular ion peaks at m/z
449 [(M + H)+] and 471 [(M + Na)+] in the ESIMS
spectrum, and the molecular formula C26H40O6 was
established by HRESIMS [m/z 471.2701, (M + Na)+, ∆
-2.2 mmu]. The broad IR absorption at 1718 cm-1 was
(7) (a) Kobayashi, J .; Shigemori, H.; Ishibashi, M.; Yamasu, T.;
Hirota, H.; Sasaki, T. J . Org. Chem. 1991, 56, 5221-5224. (b)
Kobayashi, J .; Shimbo, K.; Sato, M.; Shiro, M.; Tsuda, M. Org. Lett.
2000 2, 2805-2807.
5340 J . Org. Chem., Vol. 68, No. 13, 2003

Amphidinolide X, a Novel 16-Membered Macrodiolide
F IGURE 2. Rotation model for the C-10-C-11 bond of
amphidinolide X (1).
F IGURE 3. NOESY correlations and relative stereochemistry
for the tetrahydrofuran ring in amphidinolide X (1).
were not obtained from the HETLOC11 spectrum, since
the J (H-10,H-11) value was quite small. Hence, the
J -IMPEACH-MBC12 experiment was employed to obtain
absolute values of nJ (C-H) values, and signs of coupling
constants were determined by analysis of the phase-
sensitive HMBC13 spectrum. Furthermore, NOE data
were obtained from the HSQC-NOESY14 spectrum in
addition to the NOESY spectrum, since signals due to
H-9a and H-11 were overlapped. The 3J (H-10,H-11) value
(<1 Hz) indicated that H-10 was gauche to H-11. The
relatively large 3J (C-12,H-10) (5.5 Hz) based on the value
[scaling factor 44 × J (C-H) ) 246.5 Hz] observed in the
J -IMPEACH-MBC spectrum suggested that H-10 was
anti to C-12, while the small 3J (C-24,H-10) value (<2 Hz)
indicated that H-10 was gauche to C-24. The anti-relation
for H-11 and 10-O and the gauche-relation for H-11 and
C-9 were deduced from 2J (C-10,H-11) (0 to ca. -2 Hz)
1
F IGURE 1. Selected 2D (a) 1H-1H and H-13C and (b) 13C-
13C correlations for amphidinolide X (1).
from 1H-13C long-range correlations for H2-18 (δH 2.16
and 1.75)/C-19 (δC 82.92) and H2-20 (δH 1.50, 2H)/C-19.
A singlet methyl signal (H3-26, δH 1.30) was correlated
to C-19, indicating the presence of a methyl group
(C-26) on C-19. The existence of a tetrahydrofuran ring
at C-16-C-19 was implied from the NOESY correlation
for H-16/H3-26. For the partial structure a , the relatively
large J (H-2/H-3) value (15.8 Hz) was suggestive of the
E-geometry for the C-2-C-3 double bond. H-2 (δH 5.79)
and H2-5 (δH 2.58 and 2.41) showed HMBC correlations
to two ester carbonyl carbons at δC 165.74 (C-1) and δC
170.69 (C-6), respectively. Although the relatively lower
field sp3 proton resonances at C-10 (δH 5.21) and C-17
(δH 5.19) suggested that these carbons were involved in
two ester linkages to C-1 or C-6, no HMBC correlations
for H-10 or H-17 to ester carbonyls were observed. To
elucidate the positions of the two ester linkages, 2D
DEPT C-C Relay8 and DEPT C-C Long-Range (LR)
Relay9 experiments were employed, using the 13C-labeled
sample of 1. Analyses of the DEPT C-C Relay spectrum
revealed three carbon-carbon networks as shown in
Figure 1b. Two-bond 13C-13C correlations for C-10 (δC
74.23)/C-6 and C-17 (δC 78.41)/C-1 and three-bond cor-
relation for C-16 (δC 80.54)/C-1 were observed in the 2D
DEPT C-C LR Relay spectrum, indicating that two ester
linkages existed between C-1 and C-17 and between C-6
and C-10. Therefore, the gross structure of amphidinolide
X was elucidated to be 1.
3
and J (C-9,H-11) (<2 Hz) values, respectively. Therefore,
the relative stereochemistry of the C-10-C-11 bond was
concluded to be erythro. This was supported by the
NOESY correlation for H-10/H3-24 and HSQC-NOESY
correlations for H3-24/C-10 and H-12/C-9. The relative
stereochemistry of three chiral centers (C-16, C-17, and
C-19) in the tetrahydrofuran ring was elucidated to be
anti for H-16 and H-17 and syn for H-16 and C-26 on
the basis of NOESY correlations for H-15b (δH 1.54)/
H-17, H-16/H3-26, H-17/H-18a (δH 2.16), and H-18b
(δH 1.75)/ H3-26 (Figure 3).
Absolu te Con figu r a tion s of Am p h id in olid e X (1).
Absolute configurations at six chiral centers were exam-
(10) Matsumori, N.; Kaneno, D.; Murata, M.; Nakamura, H.; Ta-
chibana, K. J . Org. Chem. 1999, 64, 866-876.
(11) (a) Otting, G.; Wu¨thrich, K. Quart. Rev. Biophys. 1990, 23, 39-
96. (b) Wollborn, U.; Leibfritz, D. J . Magn. Reson. 1992, 98, 142-146.
(c) Kurz, M.; Schmieder, P.; Kessler, H. Angew. Chem., Int. Ed. Engl.
1991, 30, 1329-1331.
(12) Williamson, R. T.; Marquez, B. L.; Gerwick, W. H.; Martin, G.
E.; Krishnamurthy, V. V. Magn. Reson. Chem. 2001, 39, 127-132.
(13) Zhu, G.; Live, D.; Bax, A. J . Am. Chem. Soc. 1994, 116, 8370-
8371.
(14) (a) Fukushi, E.; Tanabe, S.; Watanabe, M.; Kawabata, J . Magn.
Reson. Chem. 1998, 36, 741-746. (b) Shimbo, K.; Tsuda, M.; Fukushi,
E.; Kawabata, J .; Kobayashi, J . Tetrahedron 2000, 56, 7923-7926.
Rela tive Ster eoch em istr y of Am p h id in olid e X (1).
The relative stereochemistry of the C-10-C-11 bond was
elucidated by J -based configuration analysis10 (Figure 2).
n
However, J (C-H) values around the C-10-C-11 bond
(8) Kawabata, J .; Fukushi, E. J . Magn. Reson. A 1995, 117, 88-90.
(9) Fukushi, E.; Kawabata, J . In Symposium Papers of the 43rd
Symposium on The Chemistry of Natural Products in Osaka 2001, pp
341-346.
J . Org. Chem, Vol. 68, No. 13, 2003 5341

Tsuda et al.
SCHEME 1
SCHEME 2
ined by NMR data of degradation products of 1 as follows.
Two ester carbonyls at C-1 and C-6 of 1 were reduced
with LiAlH4 and then the reduction products were
treated with (R)-(-)-2-methoxy-2-trifluoromethyl-2-phe-
nylacetyl chloride (MTPACl). HPLC separation furnished
the 1,6-bis-(S)-MTPA ester of the C-1-C-6 segment (2a )
and the 8,10,17-tris-(S)-MTPA ester of the (8S)-C-7-
C-22 segment (3a ) (Scheme 1), though the 8R-isomer of
3a was not obtained. On the other hand, the 1,6-bis-(R)-
MTPA ester of the C-1-C-6 segment (2b) and the 8,10,-
17-tris-(R)-MTPA esters of the (8S)- and (8R)-C-7-C-22
segments (3b and 4, respectively) were obtained by the
same procedure as described above. Their structures were
∆δ values of H-11 (+0.11) and H3-24 (+0.15) and a
negative ∆δ value of one of H2-9 (-0.02, +0.03) were
interpreted as the 10S-configuration, although chemical
shifts of H2-9 were affected by both of the MTPA esters
at C-8 and C-10. A negative ∆δ sign for H-8 (-0.11) and
a positive one for H-10 (+0.06) corresponded to typical
∆δ patterns for diesters of S,S-1,3-diol with chiral aniso-
topic reagents reported by Riguera and co-workers,17 thus
indicating 8S- and 10S-configurations. Therefore, the
absolute configurations at C-10, C-11, C-16, C-17, and
C-19 were concluded to be S, R, S, R, and R, respectively.
To elucidate the absolute configuration at C-4, the
C-1-C-6 segment was prepared from methyl (2S)-4-
hydroxy-2-methylbutanoate18 (5), which was synthesized
from dimethyl (S)-(-)-methylsuccinate through regiose-
lective hydrolysis catalyzed by porcine pancreatic lipase18
and reduction of the carboxyl group with borane-dimethyl
sulfide.19 Compound 5 was treated with benzyloxymethyl
chloride (BOMCl), and then the ester carbonyl was
reduced with LiAlH4 to give the BOM alcohol (6), which
was subjected to oxidation with o-iodoxybenzoic acid
(IBX)20 and C2-elongation through a Wittig reaction to
afford the unsaturated ester (7). Reduction of the unsat-
urated ester (7) followed by catalytic hydrogenation gave
the (4R)-C-1-C-6 segment, which was converted into the
1,6-bis-(R)-MTPA ester of the C-1-C-6 segment (2b).
1
assigned on the basis of the H-1H COSY spectra and
FABMS data. The C-1-C-6 segments (2a and 2b) were
obtained as a 2,3-dihydro form, probably due to reduction
1
of the C-2-C-3 double bond by LiAlH4. In the H NMR
spectrum of 3b, methylene protons at C-9 appeared as
separated signals at δH 1.98 and 1.67, while those of 4
were observed as overlapped 2H signals at δH (1.77),
indicating that the relative stereochemistry of H-8 and
H-10 in 3b and 4 was syn and anti, respectively.15 Proton
signal patterns of H2-9 (δH 1.96 and 1.70) for 3a , which
were similar to those for 3b, implied a syn-relation for
H-8 and H-10.
1
The H NMR spectra of the 1,6-bis-(S)- and (R)-MTPA
ester (2a and 2b, respectively) of the C-1-C-6 segment
obtained from natural amphidinolide X (1) were com-
pared with those of the 1,6-bis-(R)-MTPA ester (2b) of
the synthetic C-1-C-6 segment (Figure 5). Though 2a
and 2b showed very similar NMR profiles, significant
differences were observed for signals due to methylene
protons at C-1 (2a , δH 4.33 and 4.22; 2b, δH 4.29 and 4.24)
F IGURE 4. ∆δ values [∆δ (in ppm) ) δS - δR] obtained for
the 8,10,17-tris-(S)- and (R)-MTPA esters (3a and 3b, respec-
tively) of the (8S)-C-7-C-22 segment of amphidinolide X (1).
1
and C-6 (2a , δH 4.31, 2H; 2b, δH 4.35 and 4.28). The H
The absolute configuration at C-17 was determined by
application of the modified Mosher’s method16 for 3a and
3b (Figure 4). The ∆δ value (δS - δR) of H-16 (-0.09)
showed the negative values, while those of H2-18 (+0.04
and +0.07), H2-20 (2H, +0.06), and H3-26 (+0.05) were
positive, thus suggesting the 17R-configuration. Positive
NMR data for the synthetic (R)-MTPA ester (2b) were
identical with those for the 1,6-bis-(R)-MTPA esters (2b)
derived from the natural specimen, respectively, thus
(17) Seco, J . M.; Martino, M.; Quin˜oa´, E.; Riguera, R. Org. Lett. 2000,
2, 3261-3264.
(18) Salaun, J .; Karkour, B.; Ollivier, J . Tetrahedron 1989, 45,
3151-4162.
(15) (a) Kobayashi, J .; Yonezawa, M.; Takeuchi, S.; Ishibashi, M.
Heterocycles 1998, 49, 39-42. (b) Ishiyama, H.; Ishibashi, M.; Ogawa,
A.; Yoshida, S.; Kobayashi, J . J . Org. Chem. 1997, 62, 3831-3836. (c)
Mynderse, J . S.; Moore, R. E. Phytochemistry 1979, 18, 1181-1184.
(16) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J . Am.
Chem. Soc. 1991, 113, 4092-4095.
(19) (a) Brown, H. C.; Krishnamurthy, S.; Stocky, T. P.; Yoon, N.
M.; Pak, C. S. J . Org. Chem. 1973, 38, 2786-2787. (b) Lane, C. F.;
Myatt, H. L.; Daniels, J .; Hopps, H. B. J . Org. Chem. 1974, 39, 3052-
3054.
(20) Frigerio, M.; Santagostino, M. Tetrahedron Lett. 1994, 35,
8019-8022.
5342 J . Org. Chem., Vol. 68, No. 13, 2003

Amphidinolide X, a Novel 16-Membered Macrodiolide
TABLE 2. Isotop e In cor p or a tion Resu lts Ba sed on 13C
NMR Da ta for Am p h id in olid e X (1) in CDCl3
assignment
position
[1-13C]-acetatea
[2-13C]-acetatea
c or mb
1
2
1.61
0.83
1
1.10
2.01
3.22
1
c
m
m
c
3
4
1.56
0.93
0.98
0.68
0.91
2.02
0.81
1.59
0.89
1.05
1.67
1.03
1.70
0.78
0.91
1.58
0.76
1.87
0.90
0.73
0.74
0.79
0.91
5
2.45
2.54
1.91
2.41
1.17
2.51
1.07
2.44
1.92
1.23
3.76
1.50
2.05
2.39
1.02
2.95
1.20
2.91
2.31
2.52
2.08
2.50
m
m
m
m
c
m
c
m
m
c
m
c
m
m
c
m
c
m
m
m
m
m
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
a
Intensity ratios of each peak in the labeled 1 divided by that
of the corresponding signal in the unlabeled 1, respectively,
normalized to give a ratio of 1 for the unenriched peak (C-3 for
b
[1-13C]-acetate labeling and C-4 for [2-13C]-acetate labeling). The
“c” denotes the carbon derived from the C-1 of acetate, while the
“m” indicates the carbon derived from the C-2 of acetate.
F IGURE 5. 1H NMR spectra (partial) of (a) 1,6-bis-(S)- and
(b) 1,6-bis-(R)-MTPA esters (2a and 2b, respectively) of the
C-1-C-6 segment derived from amphidinolide X (1) and (c)
the 1,6-bis-(R)-MTPA ester (2b) of the synthetic C-1-C-6
segment.
indicating the 4R-configuration for the C-1-C-6 segment.
Therefore, the absolute configurations at six chiral
centers in amphidinolide X (1) were elucidated to be 4S,
10S, 11R, 16S, 17R, and 19R.
Biosyn th esis of Am p h id in olid e X (1). The feeding
experiments with [1-13C], [2-13C], and [1,2-13C2] sodium
acetates with the dinoflagellate Amphidinium sp. (strain
Y-42) were carried out under the following conditions:
the dinoflagellate was cultured in 50 L of 1% ES-
containing seawater treated with penicillin-streptomycin.
Each labeled precursor (610 µM) was added to the culture
solution in one portion 10 days after inoculation, and then
the culture was harvested by centrifugation after 14 days.
By the same separation procedure as described above,
13C-labeled amphidinolide X (1) was obtained. 13C-
Enrichments derived from labeled precursors were esti-
mated as ca. 5% on the basis of the relative intensity of
F IGURE 6. Labeling patterns of amphidinolide X (1) resulting
from feeding experiments with 13C-labeled acetates.
C-12 (44 Hz), C-14-C-15 (34 Hz), C-16-C-17 (39 Hz),
C-19-C-20 (39 Hz), and C-21-C-22 (34 Hz) as illustrated
in Figure 6.
The incorporation patterns for the C-7-C-22 part were
revealed to be “m -m -c-m -c(-m )-m -m (-m )-c-m -
c-m -m -c(-m )-m -c-m ”, in which three diacetate
units, five isolated C1 units (C-13, C-18, C-24, C-25, and
C-26) from the methyl carbon (C-2) of acetates, and a
“m -m ” unit (C-7-C-8) derived only from C-2 of acetates
were included. Thus, the C-7-C-22 part is suggested to
be a nonsuccessive mixed polyketide. A unique labeling
pattern, “c-m -m -c”, for the tetrahydrofuran ring por-
tion in 1 was observed, while those of amphidinolides C21
and T1,22 okadaic acid,23 and goniodomin A24 were “m -
c-m -m ”, “m -c-m -c”, “m -c-m -c”, and “c-m -c-
m ”, respectively.
1
satellite peaks in the H NMR spectra. Isotopic incorpo-
ration results of 1 are presented in Table 2. In the 13C
NMR spectrum of 1 enriched by [1-13C] sodium acetate,
significant enrichments of 8 out of 26 carbons were
observed as follows: C-1, C-4, C-9, C-11, C-14, C-16, C-19,
and C-21. Enrichments by [2-13C] sodium acetate were
observed for the remaining eighteen carbons (C-2, C-3,
C-5, C-6, C-7, C-8, C-10, C-12, C-13, C-15, C-17, C-18,
C-20, C-22, C-23, C-24, C-25, and C-26). J udging from
J (C-C) values as well as INADEQUATE correlations,
eight acetate units were directly incorporated for C-1-
C-2 (75 Hz), C-4-C-5 (32 Hz), C-9-C-10 (40 Hz), C-11-
(21) Kubota, T.; Tsuda, M.; Kobayashi, J . Tetrahedron 2001, 57,
5975-5977.
J . Org. Chem, Vol. 68, No. 13, 2003 5343

Tsuda et al.
Co., Inc., 10 × 250 mm; eluent, CH3CN/H2O (85:15); flow rate,
3 mL/min; UV detection at 230 nm] to give amphidinolide X
(1, 3.6 mg, 0.001%, wet weight, tR ) 17.0 min) together with
amphidinolide W (0.0012%, tR ) 15.4 min). Amphidinolides G
(0.0008%) and H (0.0007%) have been isolated from another
fraction.
Con clu sion
To our knowledge, all macrodiolides isolated from
marine and terrestrial sources consist of two molecules24
of homogeneous or heterogeneous hydroxy fatty acids,25
while amphidinolide X (1) is the first macrodiolide
natural product consisting of polyketide-derived diacid
and diol units. Furthermore, it is noted that amphidino-
lide X (1) is the second example of amphidinolides having
no exomethylene.4a Amphidinolide X (1) exhibited cyto-
toxicity against murine lymphoma L1210 and human
epidermoid carcinoma KB cells in vitro with IC50 values
of 0.6 and 7.5 µg/mL, respectively.
Am p h id in olid e X (1): colorless oil; [R]17 -12° (c 1.0,
D
CHCl3); UV (EtOH) λmax 209 nm (ꢀ 6800); IR (neat) νmax 2921
and 1718 cm-1 1H and 13C NMR (Table 1); ESIMS m/z 449
;
(M + H)+ and 471 (M + Na)+; HRESIMS m/z 471.2701 [calcd
for C26H40O6Na (M + Na)+, 471.2723].
Red u ction of Am p h id in olid e X (1) w ith LiAlH4. Am-
phidinolide X (1, 1.0 mg) was dissolved in THF (120 µL) and
treated with LiAlH4 (1.7 mg, 14 µmol) at 4 °C for 4 h. To the
reaction mixture was added 1 M phosphate buffer (100 µL)
and the solution was then extracted with EtOAc (3 × 200 µL×).
The organic phase was evaporated in vacuo to afford a mixture
of reduction products of 1 (0.9 mg). To a solution of the mixture
(0.4 mg) in a 1% DMAP solution in CH2Cl2 (50 µL) were added
Et3N (5 µL) and (R)-(-)-MTPACl (1.5 µL), and the mixture
was stirred at 4 °C for 15 h. After addition of N,N-dimethyl-
1,3-propanediamine (5 µL), the solvent was evaporated in
vacuo. The residue was passed through a silica gel column
(hexane/acetone, 5:1) and purified by C18 HPLC (Wakosil-II
5C18 RS, Wako Pure Chemical Ind., Ltd., 4.6 × 250 mm;
eluent CH3CN/H2O, 92:8; flow rate, 1.0 mL/min; UV detection
at 230 nm) to give the 1,6-bis-(S)-MTPA ester of the C-1-C-6
segment (2a , 0.08 mg, tR ) 6.4 min) and the 8,10,17-tris-(S)-
Exp er im en ta l Section
Ma ter ia l. The dinoflagellate Amphidinium sp. (strain
number Y-42) was separated from the inside cells of the marine
acoel flatworm Amphiscolops sp., which was collected off
Sunabe, Okinawa. The culture was maintained in IMK
medium (Nippon Seiyaku, Osaka, J apan) at 20 °C under an
illumination of about 50 µmol photons‚m-2‚s-1 with a 16:8
light:dark cycle. Total genomic DNA was extracted from the
cultured cells, according to the method published by Horiguchi
et al.26 The small subunit ribosomal RNA gene (SSU rDNA)
was amplified by using the primer pairs described previously,26
and both coding and noncoding strands were sequenced with
use of a DNA Sequencer. The DNA sequence was compared
with those of the SSU rDNA in the databases by using BLAST
SEARCH, and the SSU rDNA of Amphidinium belauense
(accession No. L13719), which was originally described as a
symbiont of a flatworm, Haplodiscus sp.,27 was found to be
the closest relative (>99% identity). The sequence data of this
strain Y42 have been submitted to the DDBJ /EMBL/GenBank
under accession No. AB107845.
MTPA ester of the (8S)-C-7-C-22 segment (3a , 0.12 mg, tR
)
24.8 min). The 1,6-bis-(R)-MTPA ester of the C-1-C-6 segment
(2b) and the 8,10,17-tris-(R)-MTPA esters of the (8S)- and (8R)-
C-7-C-22 segments (3b and 4, respectively) were prepared
from the another batch (0.4 mg) of reductive products through
the same procedure as described above.
1,6-Bis-(S)-MTP A ester of th e C-1-C-6 segm en t (2a ):
1
colorless oil; H NMR (CDCl3) δ 0.87 (3H, d, J ) 6.0 Hz, H3-
Cu ltiva tion a n d Isola tion . The dinoflagellate was unial-
gally cultured at 25 °C for two weeks in seawater medium
enriched with 1% ES supplement, 16 h light and 8 h dark.
The harvested cells (363 g, wet weight, from 686 L of culture)
were extracted with MeOH/toluene (3:1, 3 × 400 mL). After
addition of 1 M aq NaCl (500 L), the mixture was extracted
with toluene (3 × 400 mL). The toluene-soluble fractions (3.79
g) were subjected to a silica gel column (CHCl3/MeOH, 98:2)
to afford a macrolide-containing fraction (765 mg). Treatment
of this fraction with a 2 M solution of TMSCHN2 in hexane (1
mL) and then silica gel column chromatography (hexane/
EtOAc, 2:1) afforded a crude macrolide fraction, which was
separated with a Sep-Pak cartridge C18 (MeOH/H2O, 8:2)
followed by C18 HPLC [Mightysil RP-18, 5 µm, Kanto Chemical
23), 1.13 (1H, m, H-3), 1.30 (1H, m, H-3), 1.47 (1H, m, H-4),
1.61 (2H, m, H2-2), 1.67 (2H, m, H2-5), 3.53 (3H, s, OCH3), 3.54
(3H, s, OCH3), 4.24 (1H, m, H-1), 4.29 (1H, m, H-1), 4.33 (1H,
m, H-6), 4.38 (1H, m, H-6), 7.36-7.42 (6H, m, Ph), and 7.46-
7.55 (4H, m, Ph); FABMS m/z 565 (M + H)+; HRFABMS m/z
565.2001 [calcd for C27H31O6F6 (M + H)+, 565.2025].
1,6-Bis-(R)-MTP A ester of th e C-1-C-6 segm en t (2b):
1
colorless oil; H NMR (CDCl3) δ 0.86 (3H, d, J ) 6.0 Hz, H3-
23), 1.14 (1H, m, H-3), 1.30 (1H, m, H-3), 1.46 (1H, m, H-4),
1.61 (2H, m, H2-2), 1.63 (2H, m, H2-5), 3.539 (3H, s, OCH3),
3.543 (3H, s, OCH3), 4.22 (1H, m, H-1), 4.25 (1H, m, H-6), 4.27
(1H, m, H-1), 4.32 (1H, m, H-6), 7.35-7.42 (6H, m, Ph), and
7.47-7.55 (4H, m, Ph); FABMS m/z 565 (M + H)+; HRFABMS
m/z 565.2003 [calcd for C27H31O6F6 (M + H)+, 565.2025].
8,10,17-Tr is-(S)-MTP A ester of th e (8S)-C-7-C-22 seg-
1
(22) Kobayashi, J .; Kubota, T.; Endo, T.; Tsuda, M. J . Org. Chem.
2001, 66, 134-142.
m en t (3a ): colorless oil; H NMR (CDCl3) δ 0.91 (3H, d, J )
6.2 Hz, H3-24), 0.92 (3H, t, J ) 7.3 Hz, H3-22), 1.15 (3H, s,
H3-26), 1.19 (3H, d, J ) 6.2 Hz, H3-7), 1.34 (2H, m, H2-21),
1.53 (2H, m, H2-20), 1.60 (2H, m, H2-15), 1.60 (3H, s, H3-25),
1.70 (1H, m, H-9), 1.80 (1H, dd, J ) 2.5 and 14.0 Hz, H-18),
1.96 (1H, m, H-9), 1.97 (1H, m, H-14), 2.05 (1H, m, H-14), 2.18
(1H, dd, J ) 7.7 and 14.0 Hz, H-18), 2.74 (1H, m, H-11), 3.52
(6H, s, 2 × OCH3), 3.53 (3H, s, OCH3), 3.88 (1H, m, H-16),
4.93 (1H, m, H-12), 4.96 (1H, m, H-8), 5.01 (1H, m, H-10), 5.09
(2H, m, H-17), 7.36-7.42 (9H, m, Ph), and 7.46-7.55 (6H, m,
Ph); FABMS m/z 977 (M + H)+ and 999 (M + Na)+; HRFABMS
m/z 977.3887 [calcd for C49H58O10F9 (M + H)+, 977.3886].
(23) Yasumoto, T.; Torigoe, K. J . Nat. Prod. 1991, 54, 1987.
(24) Murakami, M.; Okita, Y.; Matsuda, H.; Okino, T.; Yamaguchi,
K. Phytochemistry 1998, 48, 85-88.
(25) Note the following examples from marine origins. (a) Swinholide
A: Kobayashi, M.; Katori, T.; Matsuura, M.; Kitagawa, I. Tetrahedron
Lett. 1989, 30, 2963-2966. (b) Misakinolide A: Kato, Y.; Fusetani, N.;
Matsunaga, S.; Hashimoto, K.; Sakai, R.; Higa, T.; Kashman, Y.
Tetrahedron Lett. 1987, 28, 6225-6228. (c) Aplysiatoxin: Kato, Y.;
Scheuer, P. J . J . Am. Chem. Soc. 1974, 96, 2245-2246. (d) Pateamine
A: Northcote, P. T.; Blunt, J . W.; Munro, M. H. G. Tetrahedron Lett.
1991, 32, 6411-6414. (e) Amphilactams: Overnden, S. P. B.; Capon,
R. J .; Lacey, E.; Gill, J . H.; Friendel, T.; Wadsworth, D. J . Org. Chem.
1999, 64, 1140-1144. (f) Aplidites A-G: Murray, L.; Lim, T. K.; Currie,
G.; Capon, R. J . Aust. J . Chem. 1995, 48, 1253-1266. (g) Lobata-
mides: Galinis, D. L.; McKee, T. C.; Pannell, L. K.; Cardellina, J . H.,
II; Boyd, M. R. J . Org. Chem. 1997, 62, 8968-8969. (h) YM-75518:
Suzunuma, K.; Takahashi, I.; Matsumoto, H.; Nagai, K.; Setiawan,
B.; Rantiatmodjo, R. M.; Suzuki, K.; Nagano, N. Tetrahedron Lett.
1997, 38, 7573-7576. (i) Colletoketol: Ho¨ller, U.; Ko¨nig, G. M.; Wright,
A. D. Eur. J . Org. Chem. 1999, 2949-2955.
8,10,17-Tr is-(R)-MTP A ester of th e (8S)-C-7-C-22 seg-
1
m en t (3b): colorless oil; H NMR (CDCl3) δ 0.76 (3H, d, J )
6.8 Hz, H3-24), 0.91 (3H, t, J ) 7.3 Hz, H3-22), 1.10 (3H, s,
H3-26), 1.32 (3H, d, J ) 6.2 Hz, H3-7), 1.33 (2H, m, H2-21),
1.47 (2H, m, H2-20), 1.60 (2H, m, H2-15), 1.61 (3H, s, H3-25),
1.67 (1H, m, H-9), 1.73 (1H, dd, J ) 2.4 and 14.1 Hz, H-18),
1.98 (1H, m, H-9), 1.99 (1H, m, H-14), 2.03 (1H, m, H-14), 2.14
(1H, dd, J ) 7.6 and 14.1 Hz, H-18), 2.63 (1H, m, H-11), 3.53
(6H, s, 2 × OCH3×), 3.54 (3H, s, OCH3), 3.97 (1H, m, H-16),
(26) Horiguchi, T.; Yoshizawa-Ebata, J .; Nakayama, T. J . Phycol.
2000, 36, 960-971.
(27) Trench, R. K.; Winsor, H. Symbiosis 1987, 3, 1-22.
5344 J . Org. Chem., Vol. 68, No. 13, 2003

Amphidinolide X, a Novel 16-Membered Macrodiolide
4.92 (1H, d, J ) 9.5 Hz, H-12), 4.96 (1H, m, H-10), 5.07 (1H,
m, H-8), 5.09 (2H, m, H-17), 7.35-7.42 (9H, m, Ph), and 7.45-
7.54 (6H, m, Ph); ESIMS m/z 999 (M + Na)+; HRESIMS m/z
999.3713 [calcd for C49H57O10F9Na (M + Na)+, 999.3704].
8,10,17-Tr is-(R)-MTP A ester of th e (8R)-C-7-C-22 seg-
m en t (4): colorless oil; 1H NMR (CDCl3) δ 0.78 (3H, d, J )
6.9 Hz, H3-24), 0.91 (3H, t, J ) 7.3 Hz, H3-22), 1.10 (3H, s,
H3-26), 1.24 (3H, d, J ) 6.2 Hz, H3-7), 1.31 (2H, m, H2-21),
1.48 (2H, m, H2-20), 1.57 (2H, m, H2-15), 1.59 (3H, s, H3-25),
1.72 (1H, dd, J ) 2.5 and 14.0 Hz, H-18), 1.77 (2H, m, H2-9),
1.97 (1H, m, H-14), 2.02 (1H, m, H-14), 2.13 (1H, dd, J ) 7.3
and 14.0 Hz, H-18), 2.75 (1H, m, H-11), 3.52 (6H, s, 2 × OCH3),
3.53 (3H, s, OCH3), 3.95 (1H, m, H-16), 4.86 (1H, d, J ) 8.8
Hz, H-12), 5.00 (1H, m, H-8), 5.02 (1H, m, H-10), 5.08 (2H, m,
H-17), 7.35-7.41 (9H, m, Ph), and 7.46-7.53 (6H, m, Ph);
ESIMS m/z 999 (M + Na)+; HRESIMS m/z 999.3713 [calcd
for C49H57O10F9Na (M + Na)+, 999.3704].
14.26, 19.41, 33.43, 35.74, 60.21, 65.67, 69.45, 94.72, 120.13,
127.69, 127.86 (2C), 128.43 (2C), 137.85, 153.62, and 166.70;
FABMS m/z 293 (M + H)+ and 315 (M + Na)+; HRFABMS
m/z 293.1750 [calcd for C17H25O4 (M + H)+, 293.1752].
Syn th etic 1,6-Bis-(R)-MTP A Ester of th e C-1-C-6 Seg-
m en t (2b). To the solution of unsaturated ester (39.2 mg, 134
µmol) in ether (2 mL) was added LiAlH4 (10 mg, 263 µmol) at
0 °C, and stirring was continued for 3 h. After addition of H2O
and then 15% aqueous NaOH, the insoluble materials were
filtered, and the filtrate was evaporated to afford an crude
alcohol (26.8 mg, 107 µmol, 80%). To a solution of the alcohol
(12.6 mg, 50.4 µmol) in MeOH (0.5 mL) was added 10% Pd/C
(5 mg), and stirring was continued under H2 atmosphere at
room temperature for 14 h. After filtration of insoluble
materials, evaporation of the solvent gave the crude C-1-C-6
segment (5.6 mg, 47.5 µmol, 94%). To a solution of the segment
(1.4 mg, 11.9 µmol) in CH2Cl2 (100 µL) were added DMAP (0.1
mg), Et3N (10 µL), and (S)-(+)-MTPACl (7 µL), and stirring
was continued at room temperature for 14 h. After addition
of N,N-dimethyl-1,3-propanediamine (10 µL) and evaporation
of the solvent, a fourth of the residue was subjected to C18
HPLC (Wakosil-II 5C18 RS, Wako Pure Chemical Ind., Ltd.,
4.6 × 250 mm; eluent CH3CN/H2O, 92:8; flow rate, 1.0 mL/
min; UV detection at 230 nm) to give the 1,6-bis-(R)-MTPA
ester of the C-1-C-6 segment (2b, 0.5 mg, 0.89 µmol, 30%, tR
) 6.0 min) as a colorless oil; 1H NMR (CDCl3) δ 0.86 (3H, d, J
) 6.0 Hz, H3-23), 1.14 (1H, m, H-3), 1.30 (1H, m, H-3), 1.46
(1H, m, H-4), 1.61 (2H, m, H2-2), 1.63 (2H, m, H2-5), 3.539 (3H,
s, OCH3), 3.543 (3H, s, OCH3), 4.22 (1H, m, H-1), 4.25 (1H, m,
H-6), 4.27 (1H, m, H-1), 4.32 (1H, m, H-6), 7.35-7.42 (6H, m,
Ph), and 7.47-7.55 (4H, m, Ph); FABMS m/z 565 (M + H)+;
HRFABMS m/z 565.2012 [calcd for C27H31O6F6 (M + H)+,
565.2025].
(2S)-4-Ben zyloxym eth yloxy-2-m eth ylbu ta n -1-ol (6). To
a solution of methyl (2S)-4-hydroxy-2-methylbutyrate (434.6
mg, 3.29 mmol) in CH2Cl2 (1.5 mL) were added diisopropyl-
ethylamine (920 µL, 6.58 mmol) and BOMCl (688 µL, 4.94
mmol) at 0 °C, and the reaction mixture was stirred at room
temperature for 3.5 h. After addition of 1 N aqueous HCl, the
mixture was extracted with CH2Cl2. The organic layer was
washed with saturated aqueous NaHCO3 and H2O, dried with
MgSO4, and evaporated to afford the crude BOM ether (1028
mg). To a solution of the crude BOM ether (1028 mg) in ether
(2 mL) was added a suspension of LiAlH4 (140 mg, 3.68 mmol)
in ether (2 mL) at 0 °C, and stirring was continued for 30 min.
After addition of H2O, 15% aqueous NaOH, and then H2O and
filtration of insoluble materials, the filtrate was evaporated,
and then subjected to silica gel column chromatography
(hexane/EtOAc, 90:10 f 60:40) to afford alcohol 6 (519 mg,
2.32 mmol, 71% by two steps) asa colorless oil: [R]24 -4° (c
F eed in g Exp er im en ts w ith 13C-La beled P r ecu r sor s.
D
2.0, CHCl3); IR (neat) νmax 3425, 3031, 2929, 2875, 1456, 1381,
The dinoflagellate cultured in
a 100-L nutrient-enriched
1
1109, and 1045 cm-1; H NMR (CDCl3) δ 0.95 (3H, d, J ) 6.8
seawater medium was supplemented with [1-13C], [2-13C], or
[1,2-13C2] sodium acetate (610 µM) in one portion 4 days after
inoculation, and then the culture was harvested by centrifuga-
tion after 14 days to obtain cells of the dinoflagellate (70 g as
an average, wet weight). Extraction and isolation of amphi-
dinolide X (1) from the harvested cells were carried out through
the same procedure as described above. The 13C-labeled
amphidinolide X (1) was obtained in 0.001% yield as an
average from the wet weight of the cells.
Hz), 1.53 (1H, m), 1.71 (1H, m), 1.82 (1H, m), 3.45-3.54 (2H,
m), 3.65 (1H, m), 3.71 (1H, m), 4.61 (2H, s), 4.76 (2H, s), 7.30
(1H, m), and 7.32-7.39 (5H, m); 13C NMR (CDCl3) δ 16.96,
33.58, 33.69, 66.24, 68.08, 69.54, 94.65, 127.72, 127.86 (2C),
128.44 (2C), and 137.77; FABMS m/z 225 (M + H)+; HR-
FABMS m/z 225.1471 [calcd for C13H21O3 (M + H)+, 225.1491].
E t h yl (4S)-6-Ben zyloxym et h yloxy-4-m et h yl-2-h exen -
oa te (7). A solution of alcohol 6 (118.5 mg, 530 µmol) in THF
(1.5 mL) was treated with IBX (225 mg, 795 µmol) in DMSO
(3 mL) at room temperature for 3.5 h. After addition of H2O
and filtration of precipitates, the filtrate was extracted with
ether. The extract was washed with brine, dried with Na2SO4,
and evaporated to afford a crude aldehyde (75.6 mg). To a
solution of the aldehyde (76.0 mg) in benzene (2.5 mL) was
added (ethoxycarbonylmethylene)triphenylphosphorane (300
mg, 900 µmol), and stirring was continued at room tempera-
ture for 14 h. After evaporation of the solvent, the residue was
subjected to silica gel column chromatography (hexane/EtOAc,
9:1) to afford compound 7 (77.0 mg, 264 mmol, 50% by two
Ack n ow led gm en t. We thank Prof. T. Yamasu,
University of the Ryukyus, and Prof. M. Ishibashi,
Chiba University, for help with dinoflagellate collection
and Ms. S. Oka and Miss M. Kiuchi, Center for
Instrumental Analysis, Hokkaido University, for ESIMS
and FABMS measurements. This work was partly
supported by a Grant-in-Aid from the Takeda Science
Foundation and a Grant-in-Aid for Scientific Research
from the Ministry of Education, Culture, Sports, Sci-
ence, and Technology of J apan.
steps) as a colorless oil; [R]24 +21° (c 2.0, CHCl3); IR (neat)
D
νmax 3030, 2932, 2875, 1718, 1651, 1455, 1371, 1271, 1217,
1181, 1112, and 1051 cm-1; 1H NMR (CDCl3) δ 1.09 (3H, d, J
) 6.8 Hz), 1.28 (3H, t, J ) 7.2 Hz), 1.68 (2H, m), 2.52 (1H, m),
3.58 (2H, m), 4.18 (2H, q, J ) 7.2 Hz), 4.59 (2H, s), 4.74 (2H,
s), 5.81 (1H, d, J ) 15.5 Hz), 6.87 (1H, dd, J ) 8.0 and 15.5
Hz), 7.30 (1H, m), and 7.32-7.39 (5H, m); 13C NMR (CDCl3) δ
Su p p or tin g In for m a tion Ava ila ble: Spectral data for 1.
This material is available free of charge via the Internet at
http://pubs.acs.org.
J O0343634
J . Org. Chem, Vol. 68, No. 13, 2003 5345