Amphidinolides T2, T3, and T4, new 19-membered macrolides from the dinoflagellate Amphidinium sp. and the biosynthesis of amphidinolide T1

Article abstract of DOI:10.1021/jo005607c

Three new 19-membered macrolides, amphidinolides T2 (2), T3 (8), and T4 (4), structurally relater to amphidinolide T1 (1) have been isolated from two strains of marine dinoflagellates of the genus Amphidinium. The structures of 2-4 were elucidated on the basis of spectroscopic data. The absolute configurations at C-7, C-8, and C-10 of 1-4 were determined by comparison of NMR data of their C-1-C-12 segments with those of synthetic model compounds for the tetrahydrofuran portion. The biosynthetic origins of amphidinolide T1 (1) were investigated on the basis of ¹³C NMR data of a ¹³C enriched sample obtained by feeding experiments with [1-¹³C], [2-¹³C], and [1,2-¹³C₂] sodium acetates and ¹³C-labeled sodium bicarbonate in the cultures of the dinoflagellate. These incorporation patterns suggested that amphidinolide T1 (1) was generated from four successive polyketide chains, an isolated C₁ unit formed from C-2 of acetates, and three unusual C₂ units derived only from C-2 of acetates. Furthermore, it is noted that five oxygenated carbons of C-1, C-7, C-12, C-13, and C-18 were not derived from the C-1 carbonyl, but from the C-2 methyl of acetates.

Full text of DOI:10.1021/jo005607c

134
J . Org. Chem. 2001, 66, 134-142
Am p h id in olid es T2, T3, a n d T4, New 19-Mem ber ed Ma cr olid es
fr om th e Din ofla gella te Am ph id in iu m sp . a n d th e Biosyn th esis of
Am p h id in olid e T1
J un’ichi Kobayashi,* Takaaki Kubota, Tetsuya Endo, and Masashi Tsuda
Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, J apan
jkobay@pharm.hokudai.ac.jp
Received August 11, 2000
Three new 19-membered macrolides, amphidinolides T2 (2), T3 (3), and T4 (4), structurally related
to amphidinolide T1 (1) have been isolated from two strains of marine dinoflagellates of the genus
Amphidinium. The structures of 2-4 were elucidated on the basis of spectroscopic data. The absolute
configurations at C-7, C-8, and C-10 of 1-4 were determined by comparison of NMR data of their
C-1-C-12 segments with those of synthetic model compounds for the tetrahydrofuran portion. The
biosynthetic origins of amphidinolide T1 (1) were investigated on the basis of 13C NMR data of a
13C enriched sample obtained by feeding experiments with [1-13C], [2-13C], and [1,2-13C2] sodium
acetates and 13C-labeled sodium bicarbonate in the cultures of the dinoflagellate. These incorporation
patterns suggested that amphidinolide T1 (1) was generated from four successive polyketide chains,
an isolated C1 unit formed from C-2 of acetates, and three unusual C2 units derived only from C-2
of acetates. Furthermore, it is noted that five oxygenated carbons of C-1, C-7, C-12, C-13, and C-18
were not derived from the C-1 carbonyl, but from the C-2 methyl of acetates.
In tr od u ction
absolute configurations at C-7, C-8, and C-10 were
elucidated from comparison of 1H NMR data of 11a and
11b with those of bis-(S)-(-)- and bis-(R)-(+)-MTPA
esters (6a and 6b) derived from 1-4.
Amphidinolides are a series of unique cytotoxic mac-
rolides obtained from marine dinoflagellates of the genus
Amphidinium, which are symbionts of Okinawan marine
acoel flatworms Amphiscolops spp.1 Amphidinolide T1 (1)
is a 19-membered macrolide, which has been isolated
from the dinoflagellate Amphidinium sp. (Y-56), possess-
ing a tetrahydrofuran ring, an exo-methylene, three
branched methyls, a ketone, and a hydroxyl group.2,3
Absolute configurations at four (C-2, C-13, C-14, and
C-18) of seven chiral centers in amphidinolide T1 (1) were
elucidated on the basis of the NMR data of the MTPA
esters of 1 and the degradation products, and the relative
stereochemistry of the tetrahydrofuran ring was assigned
from NOESY data, whereas the absolute configurations
at C-7, C-8, and C-10 remain unsolved. Further investi-
gation of the extracts of two strains (Y-71 and Y-56) of
the dinoflagellates resulted in the isolation of three new
19-membered macrolides, amphidinolides T2 (2), T3 (3),
and T4 (4), together with amphidinolide T1 (1). The
structures of 2-4 were elucidated on the basis of the
spectroscopic data and those of their degradation prod-
ucts. To determine the absolute configurations at C-7,
C-8, and C-10 of 1-4, (S)-(-)- and (R)-(+)-MTPA esters
(11a and 11b, respectively) of a model compound pos-
sessing a tetrahydrofuran ring were synthsized, and the
The biosynthetic origins of amphidinolide T1 (1) were
investigated on the basis of 13C NMR data of 13C-enriched
sample obtained by feeding experiments with [1-13C],
[2-13C], and [1,2-13C2] sodium acetates in the cultures of
the dinoflagellate. These incorporation patterns sug-
gested that 1 was generated from an isolated C1 unit (C-
7) formed from C-2 of acetates and three unusual C2 units
(C-1-C-2, C-12-C-13, and C-18-C-19) derived only from
C-2 of acetates in addition to three successive diketides
and a monoketide. Furthermore, it was shown that five
(C-12, C-7, C-11, C-12, and C-18) of six oxygenated
carbons and four C1 branches (C-22, C-23, C-24, and
C-25) were derived from C-2 methyl of acetates.
Resu lts a n d Discu ssion
Isola tion of Am p h id in olid es T2 (2), T3 (3), a n d T4
(4). The dinoflagellate Amphidinium sp. (Y-71 strain)
was obtained from an acoel flatworm Amphiscolops sp.
collected off Sunabe, Okinawa. The mass cultured algal
cells (1200 g, wet weight) obtained from 1300 L of culture
were extracted with MeOH/toluene (3:1), and the extracts
were partitioned between toluene and water. The toluene
extracts were subjected to silica gel and then successive
C18 column chromatographies followed by C18 HPLC to
afford amphidinolide T2 (2, 0.0001%) together with
known macrolides, amphidinolides B,4 C,5 and T12 (1,
0.0009%) (Chart 1). On the other hand, the toluene-
soluble materials of the Y-56 strain of Amphidinium sp.
were subjected to silica gel column chromatography
(CHCl3/MeOH) followed by C18 HPLC (CH3CN/H2O) to
give amphidinolides T3 (3, 0.0006%, wet weight) and T4
* To whom correspondence should be addressed. Phone and Fax:
+81 11 706 4985. Fax: +81 11 706 4989.
(1) (a) Ishibashi, M.; Kobayashi, J . Heterocycles 1997, 44, 543-572.
(b) Tsuda, M.; Endo, T.; Kobayashi, J . Tetrahedron 1999, 55, 14565-
14570. (c) Kubota, T.; Tsuda, M.; Kobayashi, J . Tetrahedron Lett. 2000,
41, 713-716.2.
(2) Tsuda, M.; Endo, T.; Kobayashi, J . J . Org. Chem. 2000, 65, 1349-
1352.
(3) We use in this paper the following names for amphidinolide T
congeners: amphidinolide T initially reported in ref 2 is referred as
amphidinolide T1 (1), and the homologues reported here are amphi-
dinolides T2 (2), T3 (3), and T4 (4).
10.1021/jo005607c CCC: $20.00 © 2001 American Chemical Society
Published on Web 12/14/2000

Amphidinolides T2, T3, and T4
J . Org. Chem., Vol. 66, No. 1, 2001 135
Ta ble 1. 1H a n d 13C NMR Da ta of Am p h id in olid es T2 (2), T3 (3), a n d T4 (4) in C6D6
2
3
4
position
δC
δH (m, Hz)
δC
δH (m, Hz)
δC
δH (m, Hz)
1
2
3
175.0 s
41.2 d
34.6 t
174.9 s
41.2 d
34.6 t
175.4 s
41.0 d
33.8 t
2.47 m
1.63 m
2.46 m
1.62 m
2.46 m
1.72 m
1.38 m
1.37 m
1.29 m
4
5
6
26.5 t
26.5 t
29.7 t
1.51 m
1.32 m
1.50 m
1.39 m
26.5 t
26.5 t
29.7 t
1.53 m
1.31 m
1.51 m
1.42 m
26.6 t
26.0 t
28.7 t
1.48 m
1.32 m
1.47 m
1.43 m
1.50 m
1.49 m
1.59 m
1.21 m
1.21 m
1.21 m
7
8
9
79.2 d
36.3 d
39.9 t
3.80 ddd, 10.0, 4.4, 3.3
1.79 m
1.69 m
79.2 d
36.3 d
39.3 t
3.80 dt, 9.6, 3.4
1.79 m
1.69 dt, 15.0, 7.8
1.40 m
79.3 d
36.1 d
40.2 t
3.82 dt, 9.1, 4.4
1.93 m
1.65 m
1.39 m
1.50 m
10
11
76.1 d
39.3 t
4.06 m
2.01 ddd, 14.4, 3.3, 2.8
1.60 m
76.1 d
39.3 t
4.07 ddd, 10.6, 8.1, 2.8
2.00 dt, 14.5, 2.8
1.60 m
74.6 d
40.1 t
4.35 m
2.01 ddd, 14.2, 3.2, 7.7
1.66 m
12
12-OH
13
14
15
76.3 d
4.41 dt, 9.4, 2.8
4.58 d, 2.8
76.3 d
4.40 dt, 9.1, 2.8
4.57 d, 2.8
74.9 d
4.57 dt, 7.3, 3.1
4.05 d, 3.1
215.6 s
38.5 d
41.3 t
215.7 s
38.4 d
41.4 t
215.6 s
39.5 d
38.4 t
3.58 m
2.67 dd, 5.6, 13.3
1.91 dd, 8.9, 13.3
3.58 m
2.68 dd, 5.5, 13.5
1.89 dd, 8.7, 13.5
3.37 m
2.75 dd, 4.5, 14.1
2.07 dd, 9.9, 14.1
16
17
143.3 s
40.5 t
143.3 s
40.6 t
143.1 s
40.8 t
2.60 dd, 5.0, 13.9
2.17 dd, 8.9, 13.9
5.26 m
2.58 dd, 5.5, 13.5
2.17 dd, 8.5, 13.5
5.24 m
2.58 dd, 5.1, 13.6
2.15 dd, 8.6, 13.6
5.26 m
18
19
72.2 d
29.9 t
72.1 d
35.9 t
71.9 d
35.5 t
1.68b
m
1.58 m
1.56 m
1.51 m
1.53 m
20
34.7 t
67.2 q
1.46 m
1.38 m
3.55 m
18.8 t
14.1 q
1.40 m
18.7 t
13.9 q
1.40 m
1.35 m
1.35 m
21
0.88a t, 7.3
0.90a t, 7.4
21-OH
22
23
24
25
0.79 brd, 4.4
0.99a d, 6.1
1.16a d, 6.7
0.71a d, 7.0
1.17a d, 6.7
23.9 q
17.8 q
14.2 q
15.5 q
17.7 q
14.2 q
15.5 q
114.7 t
1.16a d, 6.9
0.71a d, 7.0
1.17a d, 6.6
4.92 s
18.0 q
14.1 q
15.9 q
115.4 t
1.15a d, 7.0
0.75a d, 7.1
1.02a d, 6.8
4.87a
s
4.89 s
26
114.7 t
4.93b
s
a
b
3H. 2H.
(4, 0.0004%) together with known macrolides, amphidin-
olides C,5 F,6 T1 (1, 0.005%), and U.1b
Ch a r t 1
Str u ctu r e Elu cid a tion of Am p h id in olid es T2 (2),
T3 (3), a n d T4 (4). The FABMS spectrum of amphidin-
olide T3 (3) showed the pseudomolecular ion peak at m/z
423 (M + H)+, indicating that 3 had the same molecular
formula, C25H42O5, as that of amphidinolide T1 (1). The
1H and 13C NMR data (Table 1) of 3 were analogous to
those of 1 except for the resonances of C-12 (δH 4.40, δC
76.3), C-13 (δC 215.7), C-14 (δH 3.58, δC 38.4), and C-24
(δH 1.17, δC 15.5) of 3. Detailed analyses of 1H-1H COSY
and TOCSY data revealed the proton connectivities of
two structural units from H3-22 to 12-OH (δH 4.57) and
from H3-24 to H3-21 through an exo-methylene at C-16
(Figure 1). HMBC correlations from H-12, 12-OH, H-15
(δH 2.68), and H3-24 to a ketone carbonyl (C-13) suggested
that the carbonyl group was located at C-13. The chemi-
(4) (a) Ishibashi, M.; Ohizumi, Y.; Hamashima, M.; Nakamura, H.;
Hirata, Y.; Sasaki, T.; Kobayashi, J . J . Chem. Soc., Chem. Commun.
1987, 1127-1129. (b) Kobayashi, J .; Ishibashi, M.; Nakamura, H.;
Ohizumi, Y.; Yamasu, T.; Hirata, Y.; Sasaki, T.; Ohta, T.; Nozoe, S. J .
Nat. Prod. 1989, 52, 1036-1041. (c) Bauer, I.; Maranda, L.; Shimizu,
Y.; Peterson, R. W.; Cornell, L.; Steiner, J . R.; Clardy, J . J . Am. Chem.
Soc. 1994, 116, 2657-2658.
cal shift of H-18 (δH 5.24) and the HMBC correlation from
H3-22 (δH 1.16) to C-1 (δC 174.9) indicated that an ester
linkage was formed between C-2 and C-18 to construct a
lactone ring. The relative stereochemistry of H-7, H-8,
and H-10 were implied to be 7,8-syn and 7,10-anti by
NOESY correlations for H2-6/H3-23, H-7/H-11, and H-10/
H3-23. Thus, amphidinolide T3 (3) was assigned as the
12-hydroxy-13-keto form of amphidinolide T1 (1).
(5) Kobayashi, J .; Ishibashi, M.; Wa¨lchli, M. R.; Nakamura, H.;
Hirata, Y.; Sasaki, T.; Ohizumi, Y. J . Am. Chem. Soc. 1988, 110, 490-
494.
(6) Kobayashi, J .; Tsuda, M.; Ishibashi, M.; Shigemori, H.; Yamasu,
T.; Hirota, H.; Sasaki, T. J . Antibiot. 1991, 44, 1259-1261.

136 J . Org. Chem., Vol. 66, No. 1, 2001
Kobayashi et al.
F igu r e 3. ∆δ values [∆δ (in ppm) ) δS - δR] obtained for the
(S)-(-)- and (R)-(+)-MTPA esters (8a and 8b, respectively) of
amphidinolide T4 (4).
F igu r e 1. Selected 2D NMR correlations for amphidinolide
T3 (3).
S, R, and R, respectively, and the absolute configurations
at C-7, C-8, and C-10 of 3 were the same as those of
amphidinolide T1 (1).
Amphidinolide T4 (4) had the same molecular formula,
C25H42O5, as amphidinolide T1 (1) and amphidinolide T3
(3) as revealed by HRFABMS [m/z 423.3131 (M + H)+,
∆ +2.1 mmu]. Extensive 2D NMR data of 4 indicated that
the gross structure of 4, including the relative stereo-
chemistry of a tetrahydrofuran ring, was the same as that
of amphidinolide T3 (3). The absolute configuration at
C-12 in 4 was assigned as S by modified Mosher’s method
(Figure 3). Treatment of 4 with LiAlH4, NaIO4, NaBH4,
and (R)-(-)-MTPACl afforded the bis-(S)-(-)-MTPA es-
ters of C-1-C-12 (6a ) and C-13-C-21 segments (7a ), of
F igu r e 2. ∆δ values [∆δ (in ppm) ) δS - δR] obtained for the
(S)-(-)- and (R)-(+)-MTPA esters (5a and 5b, respectively) of
amphidinolide T3 (3).
1
which the H NMR data were identical with those of the
bis-(S)-(-)-MTPA esters of C-1-C-12 (6a ) and C-13-C-
21 segments (7a ), respectively, obtained from amphidin-
olides T1 (1) and T3 (3). Thus the absolute configurations
at C-2, C-14, and C-18 of amphidinolide T4 (4) were
assigned as S, R, and R, respectively.
Sch em e 1
The molecular formula of amphidinolide T2 (2) was
suggested to be C26H44O6 by HRFABMS data [m/z
453.3187 (M + H)+, ∆ -2.9 mmu]. 1H and 13C NMR data
(Table 1) of 2 were close to those of amphidinolide T3
(3), except for the presence of an additional oxymethine
carbon [δH 3.55 (1H, m); δC 67.2 (d)] and a hydroxyl group
1
[δH 0.79 (1H, brd, J ) 4.4 Hz)]. Analysis of the H-1H
COSY and TOCSY spectra revealed connectivities from
H3-23 to H-12 and from H3-25 to H3-22, indicating that
the hydroxyl group was located at C-21. The carbon
chemical shifts of three quaternary carbons (C-1, δC
175.0; C-13, δC 215.6; C-16, δC 143.3) of 2 corresponded
well to those (C-1, δC 174.9; C-13, δC 215.7; C-16, δC 143.3)
of 3. The relative stereochemistry of a tetrahydrofuran
ring was the same as that of 3. Thus the gross structure
of amphidinolide T2 was concluded to be 2.
The absolute configurations at C-12 and C-21 of
amphidinolide T2 (2) were elucidated to be R and S,
respectively, by modified Mosher’s method (Figure 4).
Treatment of 2 with LiAlH4, NaIO4, NaBH4, and (R)-
(-)- or (S)-(+)-MTPACl afforded the bis-(S)-(-)- and bis-
(R)-(+)-MTPA esters (6a and 6b, Scheme 2, respectively)
of the C-1-C-12 segment and the tris-(S)-(-)- and the
tris-(R)-(+)-MTPA esters (10a and 10b, respectively) of
the C-13-C-22 segment (Scheme 2). 1H NMR data of the
bis-(S)-(-)- and bis-(R)-(+)-MTPA esters (6a and 6b,
respectively) of the C-1-C-12 segment were identical
with those of the bis-(S)-(-)- and bis-(R)-(+)-MTPA esters
of the C-1-C-12 segment obtained from amphidinolide T1
The absolute configuration at C-12 of amphidinolide
T3 (3) was determined by modified Mosher’s method.
Treatment of 3 with (R)-(-)- and (S)-(+)-2-methoxy-2-
trifluoromethyl-2-phenylacetyl chloride (MTPACl) af-
forded the (S)-(-)- and (R)-(+)-MTPA esters (5a and 5b,
respectively). ∆δ values (δS - δR) of H2-15 (-0.01 and
-0.02), H2-17 (-0.03 and -0.01), H3-24 (-0.05), and H2-
25 (-0.01 and -0.04) showed negative values, while those
of H-8 (+0.17), H2-9 (+0.05 and +0.02), H-10 (+0.02), H2-
11 (+0.05 and +0.09), and H3-23 (+0.06) were positive
(Figure 2), thus indicating a 12R-configuration. Treat-
ment of 3 with LiAlH4, NaIO4, NaBH4, and (R)-(-)-
MTPACl afforded the bis-(S)-(-)-MTPA esters of C-1-
1
C-12 (6a ) and C-13-C-21 segments (7a ) (Scheme 1). H
NMR data of 6a and 7a corresponded well to those of
the bis-(S)-(-)-MTPA esters of C-1-C-12 and C-13-C-
21 segments prepared from amphidinolide T1 (1), respec-
tively. Therefore, the absolute configurations at C-2,
C-14, and C-18 of amphidinolide T3 (3) were assigned as

Amphidinolides T2, T3, and T4
J . Org. Chem., Vol. 66, No. 1, 2001 137
F igu r e 4. ∆δ values [∆δ (in ppm) ) δS - δR] obtained for the
bis-(S)-(-)- and bis-(R)-(+)-MTPA esters (9a and 9b, respec-
tively) of amphidinolide T2 (2).
F igu r e 5. Proton signal patterns of H2-13 of the tris-(S)-(-)-
and tris-(R)-(+)-MTPA esters (10a and 10b, respectively) of
the C-13-C-22 segment of amphidinolide T2 (2).
Sch em e 2
F igu r e 6. ∆δ values [∆δ (in ppm) ) δS - δR] obtained for the
tris-(S)-(-)- and tris-(R)-(+)-MTPA esters (10a and 10b,
respectively) of the C-13-C-22 segment of amphidinolide T2
(2).
(1), indicating that the absolute stereochemistry at C-2,
C-7, C-8, and C-10 of 2 was the same as that of 1. The
chemical shift differences (Figure 5) of H2-13 for 10a (∆δ
0.03; δH 4.09 and 4.12) and 10b (∆δ 0.23; δH 4.03 and
4.26) suggested the 14R-configuration. The absolute
stereochemistry of C-18 was assigned as R by modified
Sch em e 3
1
Mosher’s method on the basis of the H NMR data of 10a
and 10b (Figure 6). Therefore, the absolute configura-
tions at C-2, C-12, C-14, C-18, and C-21 of amphidinolide
T2 (2) were concluded to be S, R, R, R, and S, respec-
tively.
To elucidate the absolute configurations at C-7, C-8,
and C-10, 1H NMR data of bis-(S)-(-)- and (R)-(+)-MTPA
esters (6a and 6b, respectively) of the C-1-C-12 segment
obtained from amphidinolides T1 (1), T2 (2), T3 (3), and
1
T4 (4) were analyzed. In the H NMR spectrum of 6a ,
the methylene protons at C-12 resonated at δH 4.41 (2H,
t, J ) 6.9 Hz), while in the 1H NMR spectrum of 6b, those
at C-12 were observed as a 2H multiplet signal at δH
4.36-4.45. To determine the absolute configurations at
C-7, C-8, and C-10 of amphidinolides T1 (1), T2 (2), T3
(3), and T4 (4), two model compounds (11a and 11b)
corresponding to the tetrahydrofuran ring portion in the
C-1-C-12 segments (6a and 6b) were synthesized from
an R,R-iodide 12 as shown in Scheme 3. Compound 12
was prepared from methyl (S)-(+)-3-hydroxy-2-methyl-
propionate according to the procedure reported previ-
ously.7 Four-carbon elongation of the iodide 12 with
cyanidation, reduction, Grignard reaction with allylmag-
nesium bromide, and then acetylation afforded a 1:1
mixture of the C-10 epimers of acetate 13. Three-step
conversion of 13 gave an alcohol 14, which was treated
with (R)-(-)-MTPACl to afford the (S)-(-)-MTPA ester.
Selective deprotection of the 2-(trimethylsilyl)ethoxy-
methyl (SEM) ether was achieved by treatment with
trifluoroacetic acid in CH2Cl2 at 0 °C. The secondary
hydroxyl group was converted into mesylate 15a. Deacety-
lation of 15a with K2CO3 followed by C18 HPLC separa-
tion afforded the desired the (S)-(-)-MTPA ester (11a )
and its stereoisomer (16a ). The relative streochemistry
of 11a was assigned as 7,8-syn and 7,10-anti by NOESY
correlations, while that of 16a was elucidated to be 7,8-
(7) Tsuda, M.; Hatakeyama, A.; Kobayashi, J . J . Chem. Soc., Perkin
Trans. 1 1998, 149-155.

138 J . Org. Chem., Vol. 66, No. 1, 2001
Kobayashi et al.
Ta ble 2. Isotop e In cor p or a tion Resu lts Ba sed on th e 13C
NMR Da ta of Am p h id in olid e T1 (1)a
intensity ratio
(labeled/unlabeled)b
assignment
position
δC
[1-13C]acetate [2-13C]acetate
c or mc
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
174.9 s
41.7 d
35.1 t
26.8 t
26.2 t
29.7 t
78.6 d
36.5 d
39.7 t
73.6 d
45.0 t
212.0 s
78.2 d
32.0 d
41.2 t
143.5 s
40.1 t
71.7 d
35.7 t
19.1 t
13.9 q
18.0 q
14.0 q
13.7 q
116.1 t
0.92
0.51
2.28
1.00
3.61
0.84
0.48
2.28
1.17
3.69
1.22
0.42
0.68
1.62
1.48
2.08
0.56
1.42
0.70
1.97
1.00
0.62
1.07
0.72
0.39
1.93
2.46
1.00
2.74
0.82
2.27
2.53
0.85
2.26
0.74
2.07
1.84
2.13
0.89
2.17
1.06
2.36
1.87
2.50
1.22
2.22
2.12
2.66
3.22
2.74
m
m
c
m
c
m
m
c
m
c
m
m
m
c
m
c
m
m
m
c
m
m
m
m
m
F igu r e 7. Proton signal patterns of H2-12 of the bis-(S)-(-)-
and bis-(R)-(+)-MTPA esters (6a and 6b, respectively) of the
C-1-C-12 segment and (S)-(-)- and (R)-(+)-MTPA esters (11a
and 11b, respectively) of the synthetic model compound for
the tetrahydrofuran portion
syn and 7,10-syn. Therefore, the absolute configurations
of 11a and 16a were concluded to be 7S, 8S, and 10R,
and 7S, 8S, and 10S, respectively. On the other hand,
the (R)-(+)-MTPA ester (11b) and its stereoisomer 16b
were prepared from 15b in two steps.
1
The H NMR spectra of 11a and 11b were compared
a
The 13C NMR spectra were recorded in C6D6 solution at 125
MHz with a sweep width of 35700 Hz using “zgpg30”. Numbers of
scans for unlabeled and labeled 1 were 24 000 and 8000, respec-
with those of 6a and 6b to elucidate the absolute
configuration of the latter compounds. Though the (S)-
(-)- and (R)-(+)-MTPA esters (11a and 11b) showed very
similar NMR profiles, significant differences were ob-
b
tively. Intensity of each peak in the labeled 1 divided by that of
the corresponding signal in the unlabeled 1, normalized to give a
ratio of 1 for unenriched peak (C-4 for [1-13C]acetate labeling and
C-3 for [2-13C]acetate labeling). c c denotes the carbon derived from
C-1 of acetate, while m indicates the carbon derived from C-2 of
acetate.
1
served in the signals of H2-12 (Figure 7). In the H NMR
spectrum of the (S)-(-)-MTPA ester (11a ), the methylene
protons at C-12 resonated at δH 4.42 (2H, t, J ) 6.9 Hz),
while in 1H NMR spectrum of 11b, those at C-12 were
observed as a 2H multiplet signal at δH 4.36-4.46. The
signal pattern of H2-12 of the bis-(S)-MTPA ester of the
C-1-C-12 segment (6a ) derived from natural amphidino-
lides T1 (1), T2 (2), T3 (3), and T4 (4) was close to that of
the (S)-MTPA ester (11a ), while the pattern of H2-12 of
the bis-(R)-MTPA ester (6b) derived from 1, 2, 3, and 4
corresponded to that of the (R)-MTPA ester (11b), thus
indicating that the absolute configurations of the tet-
rahydrofuran ring portion in amphidinolide T1 (1), T2
(2), T3 (3), and T4 (4) were 7S, 8S, and 10R-configura-
tions.
Biosyn th esis of Am p h id in olid e T1 (1). The dino-
flagellate Amphidinium sp. (strain Y-71) was cultured
in a 100 L nutrient-enriched seawater medium, and
feeding experiments were carried out with [1-13C]-,
[2-13C]-, and [1,2-13C2]sodium acetate and sodium 13C-
bicarbonate. In feeding experiments, the dinoflagellate
was supplemented with 610 µM of labeled precursors in
one portion at 4 days after inoculation, and then the
culture was harvested by centrifugation after 14 days.
In each case the extracts of the harvested cells were
purified by a silica gel column followed by C18 HPLC to
afford 13C-labeled amphidinolide T1 (1) in 0.0006% yield
from as an average from wet weight of the cells.
Assignments of 13C NMR signals and isotope incorpo-
ration results of 1 derived from 13C-labeled sodium
acetate were shown in Table 2 and Figure 8. The 13C
NMR spectrum (CDCl3) of 1 derived from [1-13C]sodium
acetate showed significant enrichment of seven carbons
(C-3, C-5, C-8, C-10, C-14, C-16, and C-20). On the other
hand, enrichment by [2-13C]sodium acetate was observed
F igu r e 8. Labeling patterns of amphidinolide T1 (1) resulting
from feeding experiments with 13C-labeled acetates.
for 18 carbons (C-1, C-2, C-4, C-6, C-7, C-9, C-11, C-12,
C-13, C-15, C-17, C-18, C-19, C-21, C-22, C-23, C-24, and
C-25). Thus, all 25 carbon signals contained in 1 were
shown to be labeled by acetates. The 13C NMR spectrum
of 1 labeled with [1,2-13C2]sodium acetate showed en-
riched carbon signals flanked by two strong satellite
signals. One-bond J CC coupling constants indicated the
definite incorporation of 10 acetate units for C-3/C-4 (34.5
Hz), C-5/C-6 (34.8 Hz), C-8/C-9 (32.4 Hz), C-10/C-11 (37.7
Hz), C-14/C-15 (34.4 Hz), C-16/C-17 (41.7 Hz), and C-20/
C-21 (34.9 Hz). This was also supported by one-bond 13C-
13C correlations observed in the INADEQUATE spectrum
of 1 labeled with [1,2-13C2]sodium acetate. These results
suggested that four parts from C-3 to C-6, from C-8 to
C-11, from C-14 to C-17, and C-20 to C-21 were likely to
be classical polyketide chains derived from two, two, two,

Amphidinolides T2, T3, and T4
J . Org. Chem., Vol. 66, No. 1, 2001 139
cells of the marine acoel flatworm Amphiscolops sp. collected
off Sunabe, Okinawa. The dinoflagellate was unialgally cul-
tured at 25 °C for two weeks in a seawater medium enriched
with 1% ES supplement. The harvested cells of the cultured
dinoflagellate (1200 g wet weight, from 1300 L of culture) were
extracted with MeOH/toluene (3:1, 600 mL × 5). After addition
of 1 M NaCl aq (500 mL), the mixture was extracted with
toluene (500 mL × 3). Parts (3.30 g) of the toluene-soluble
materials (6.63 g) were subjected to silica gel (CHCl3/MeOH,
98:2) and C18 column chromatographies (MeOH/H2O, 8:2) to
give a fraction (31.5 mg) containing some macrolides. The
fraction was separated by C18 HPLC (Develosil ODS-HG-5,
Nomura Chemical Co. Ltd., 10 × 250 mm; eluent, CH3CN/
H2O (65:35); flow rate, 2.5 mL/min; UV detection at 210 nm]
to afford amphidinolides T2 (2, 0.8 mg, 0.00013%, wet weight,
tR 22.4 min), B (0.0017%), C (0.0012%), and T1 (1, 0.00092%).
Cultivation and separation of the dinoflagellate Amphidinium
sp. (strain number Y-56) was previously reported. The toluene-
soluble materials (3.67 g) obtained from the extract of the
mass-cultured dinoflagellate (420 g wet weight, from 580 L of
culture) were subjected to a silica gel column (CHCl3/MeOH,
98:2) and a Sep-Pak cartridge C18 (MeOH/H2O, 8:2) followed
by C18 HPLC [LUNA C18(2), 5 µm, Phenomenex, 10 × 250
mm; eluent, CH3CN/H2O (75:25); flow rate, 2.5 mL/min; UV
detection at 210 nm] to afford amphidinolides T3 (3, 1.5 mg,
0.0004%, wet weight, tR 34.5 min), T4 (4, 1.0 mg, 0.0002%, tR
27.5 min), C (0.0009%), F (0.0006%), T1 (1, 0.005%), and U
(0.0002%).
and one acetate units, respectively. Three irregular
labeling patterns (m -m ) derived only from C-2 of ace-
tates were observed for C-1/C-2, C-12/C-13, and C-18/C-
19, and an isolated C1 unit from C-2 of acetates was
observed for C-7. The C1 branches of C-22, C-23, C-24,
and C-25 were all derived from C-2 of acetates, in which
the carbonyl carbons were lost.
These incorporation patterns suggested that amphi-
dinolide T1 (1) was generated from four successive
polyketide chains, an isolated C1 unit from C-2 of
acetates, and three unusual “m -m ” units derived only
from C-2 of acetates. Furthermore, it is noted that five
oxygenated carbons of C-1, C-7, C-12, C-13, and C-18
were not derived from the C-1 carbonyl but from the C-2
methyl of acetates. The previous biosynthetic studies of
15- and 27-, and 26-membered macrolides, amphidino-
lides J , G, and H, have revealed that these are also
generated through nonsuccessive mixed polyketides.8,9
The labeling patterns “m -m (m )” of C-1-C-2(C-22) in
amphidinolide T1 (1) corresponded to that of C-1-C-2(C-
27) part in amphidinolides G and H. These unusual
labeling patterns have been reported for okadaic acid and
DTX-4,10 isolated from dinoflagellates of other genera.
On the other hand, all carbon atoms in amphidinolide
T1 (1) were labeled from 13C-labeled sodium bicarbonate
with relatively high incorporation ratio (ca. 33%), which
was estimated on the basis of the ratio of intensity of
proton signals and its satellite peaks, compared with
those of 13C-labeled acetates (2-3%). These results
indicated that a main carbon source of amphidinolide T1
(1) was derived from carbon dioxide, and acetates were
minor carbon sources. Sodium bicarbonate might be
converted into carbon dioxide, which might be utilized
via photosynthesis in chloroplast for biosynthsis of am-
phidinolide T1 (1). However, a small part of exogenously
applied acetate may be incorporated into chloroplast, and
converted into acetyl-CoA, which could be utilized for
biosynthesis of 1. These phenomena have been previously
reported for the biosynthesis of fatty acids in diatoms.11
Cytotoxicity. Amphidinolides T1 (1), T2 (2), T3 (3),
and T4 (4) exhibited cytotoxicity against murine lym-
phoma L1210 (IC50: 18, 10, 7.0, and 11 µg/mL, respec-
tively) and human epidermoid carcinoma KB cells (IC50:
35, 11.5, 10, and 18 µg/mL, respectively).
Am p h id in olid e T2 (2): a colorless oil; IR (KBr) νmax 3400,
2900, and 1720 cm-1; 1H and 13C NMR (Table 1); FABMS m/z
453 (M + H)+; HRFABMS m/z 453.3187 [calcd for C26H48O6
(M + H)+, 453.3206].
Am p h id in olid e T3 (3): a colorless oil; IR (KBr) νmax 3450,
2935, and 1720 cm-1; 1H and 13C NMR (Table 1); FABMS m/z
405 (M + H - H2O)+, 423 (M + H)+, and 445 (M + Na)+;
HRFABMS m/z 423.3110 [calcd for
423.3111].
C
25H43O5 (M + H)+,
Am p h id in olid e T4 (4): a colorless oil; IR (KBr) νmax 3450,
2935, and 1720 cm-1; 1H and 13C NMR (Table 1); FABMS m/z
405 (M + H - H2O)+, 423 (M + H)+, and 445 (M + Na)+;
HRFABMS m/z 423.3131 [calcd for
423.3111].
C
25H43O5 (M + H)+,
(S)-(-)-MTP A Ester (5a ) of Am p h id in olid e T3 (3). To a
CH2Cl2 solution (50 µL) of amphidinolide T3 (3, 0.3 mg) were
added 4-(dimethylamino)pyridine (10 µg), triethylamine (3 µL),
and (R)-(-)-MTPACl (0.5 µL) at room temperature, and
stirring was continued for 2 h. After addition of N,N-dimethyl-
1,3-propanediamine (2 µL) and evaporation of solvent, the
residue was passed through a silica gel column (hexane/EtOAc,
4:1) to afford the (S)-(-)-MTPA ester (5a , 0.2 mg) of 3. 5a : a
1
colorless oil; H NMR (CDCl3) δ 0.87 (3H, d, J ) 7.0 Hz, H3-
23), 0.90 (3H, t, J ) 7.3 Hz, H3-21), 0.93 (3H, d, J ) 6.6 Hz,
H3-24), 1.14 (3H, d, J ) 7.0 Hz, H3-22), 1.25-1.60 (12 H), 1.63
(1H, m, H-9), 1.72 (1H, dt, J ) 15.0, 7.8 Hz, H-9), 1.90 (1H,
dd, J ) 8.7, 13.5 Hz, H-15), 2.07 (1H, m, H-11), 2.11 (1H, m,
H-11), 2.14 (1H, dd, J ) 8.5, 13.5 Hz, H-17), 2.22 (1H, m, H-8),
2.37 (1H, dd, J ) 5.5, 13.5 Hz, H-17), 2.41 (1H, m, H-2), 2.61
(1H, brd, J ) 13.5 Hz, H-15), 3.13 (1H, m, H-14), 3.58 (3H, s,
OCH3), 3.86 (1H, m, H-7), 4.09 (1H, m, H-10), 4.83 (1H, s,
H-25), 4.91 (1H, s, H-25), 4.96 (1H, m, H-18), 5.51 (1H, t, J )
5.3 Hz, H-12), 7.40 (3H, m), and 7.58 (2H, m); FABMS m/z
639 (M + H)+; HRFABMS m/z 639.3488 [calcd for C35H49O7F3
(M + H)+, 639.3508].
(R)-(+)-MTP A Ester (5b) of Am p h id in olid e T3 (3).
Amphidinolide T3 (3, 0.3 mg) was treated with (S)-(+)-MTPACl
(0.5 µL) by the same procedure as described above to afford
the (R)-(+)-MTPA ester (5b, 0.2 mg) of 3. 5b: a colorless oil;
1H NMR (CDCl3) δ 0.81 (3H, d, J ) 7.0 Hz, H3-23), 0.92 (3H,
t, J ) 7.3 Hz, H3-21), 0.98 (3H, d, J ) 6.6 Hz, H3-24), 1.16
(3H, d, J ) 7.0 Hz, H3-22), 1.25-1.65 (14 H), 1.92 (1H, dd, J
) 8.7, 13.5 Hz, H-15), 1.98 (1H, m, H-11), 2.05 (1H, m, H-8),
2.08 (1H, m, H-11), 2.18 (1H, dd, J ) 7.5, 13.5 Hz, H-17), 2.40
(1H, m, H-17), 2.43 (1H, m, H-2), 2.71 (1H, brd, J ) 13.5 Hz,
H-15), 3.12 (1H, m, H-14), 3.68 (3H, s, OCH3), 3.81 (1H, m,
H-7), 4.07 (1H, m, H-10), 4.88 (1H, s, H-25), 4.92 (1H, s, H-25),
Exp er im en ta l Section
Gen er a l Meth od s. 1H and 2D NMR spectra were recorded
on a 600 MHz spectrometer, and 13C NMR spectra were
measured on a 500 MHz spectrometer. NMR spectra of all
MTPA esters were measured using 2.5 mm micro cells for
CDCl3 (Shigemi Co., Ltd., J apan). FABMS spectra were
recorded using diethanolamine (for 2-4 and their MTPA
esters) or p-nitrobenzyl alcohol (for degradation products and
synthetic products) as a matrix in positive mode. EIMS spectra
were measured at 70 eV. Positive mode electrosplay ionization
(ESI) mass spectra were measured using samples dissolved
in MeOH with flow rate of 0.1 mL/min.
Cu ltiva tion a n d Isola tion . The dinoflagellate Amphi-
dinium sp. (strain number Y-71) was isolated from the inside
(8) Kobayashi, J .; Takahashi, M.; Ishibashi, M. J . Chem. Soc., Chem.
Commun. 1995, 1639-1640. (b) Ishibashi, M.; Takahashi, M.; Koba-
yashi, J . Tetrahedron 1997, 53, 7827-7832.
(9) Sato, M.; Shimbo, K.; Tsuda, M.; Kobayashi, J . Tetrahedron Lett.
2000, 41, 503-506.
(10) Wright, J . L. C.; Hu, T.; Mclachlan, J . L.; Needaham, J .; Walter,
J . A. J . Am. Chem. Soc. 1996, 118, 8757-8758.
(11) Cvejic, J . H.; Rohmer, M. Phytochemistry 2000, 53, 21-28.

140 J . Org. Chem., Vol. 66, No. 1, 2001
Kobayashi et al.
4.99 (1H, m, H-18), 5.46 (1H, t, J ) 5.3 Hz, H-12), 7.43 (3H,
m), and 7.68 (2H, m); FABMS m/z 639 (M+H)+; HRFABMS
m/z 639.3507 [calcd for C35H49O7F3 (M + H)+, 639.3508].
H-12), 7.39 (3H, m), and 7.57 (2H, m); FABMS m/z 639 (M +
H)+; HRFABMS m/z 639.3512 [calcd for C35H49O7F3 (M + H)+,
639.3508].
Oxid a tive Degr a d a tion of Am p h id in olid e T4 (4). Am-
phidinolide T4 (4, 0.2 mg) was treated with LiAlH4, NaIO4,
NaBH4, and (R)-(-)-MTPACl by the same procedure as
described above to afford compounds 6a (0.07 mg, tR 16.0 min)
and 7a (0.03 mg, tR 13.6 min). 6a : 1H NMR (CDCl3) δ 0.86
(3H, d, J ) 7.0 Hz, H3-23), 0.91 (3H, d, J ) 6.7 Hz, H3-22),
1.1-1.45 (8H, m, H2-3, H2-4, H2-5, and H2-6), 1.69 (2H, m, H2-
9), 1.78-1.93 (3H, m, H-2 and H2-11), 2.19 (1H, m, H-8), 3.54
(6H, s, 2 × OCH3), 3.79 (1H, m, H-7), 4.05 (1H, m, H-10), 4.06
(1H, dd, J ) 6.7, 10.7 Hz, H-1), 4.23 (1H, dd, J ) 5.6, 10.7 Hz,
H-1), 4.41 (2H, t, J ) 6.8 Hz, H2-12), 7.35-4.43 (6H, m, Ph),
and 7.48-7.54 (4H, m, Ph); FABMS m/z 699 (M + Na)+;
HRFABMS m/z 699.2754 [calcd for C34H42O7F6Na (M + Na)+,
699.2732]. 7a : 1H NMR (CDCl3) δ 0.88 (3H, d, J ) 6.3 Hz,
H3-24), 0.90 (3H, t, J ) 7.5 Hz, H3-21), 1.28-1.40 (2H, m, H2-
20), 1.56 (1H, m, H-19), 1.62 (1H, m, H-19), 1.84 (1H, m, H-15),
1.97-2.05 (1H, m, H-14, H-15), 2.17 (1H, dd, J ) 6.0, 14.2 Hz,
H-17), 2.28 (1H, dd, J ) 7.6, 14.2 Hz, H-17), 3.52 (3H, s, OCH3),
3.54 (3H, s, OCH3), 4.09 (1H, dd, J ) 5.8, 10.2 Hz, H-13), 4.13
(1H, dd, J ) 4.3, 10.2 Hz, H-13), 4.68 (1H, s, H-25), 4.75 (1H,
s, H-25), 5.20 (1H, m, H-18), 7.35-4.43 (6H, m, Ph), and 7.48-
7.54 (4H, m, Ph); FABMS m/z 641 (M + Na)+; HRFABMS m/z
641.2284 [calcd for C31H36O6F6Na (M + Na)+, 641.2314].
Bis-(S)-(-)-MTP A Ester (9a ) of Am p h id in olid e T2 (2).
Amphidinolide T2 (2, 0.1 mg) was treated with (R)-(-)-
MTPACl (0.3 µL) by the same procedure as described above
to afford the bis-(S)-(-)-MTPA ester (9a , 0.2 mg) of 4. 9a : a
Oxid a tive Degr a d a tion of Am p h id in olid e T3 (3). Am-
phidinolide T3 (3, 0.2 mg) was dissolved in THF (20 µL) and
treated with LiAlH4 (0.8 mg) at room temperature for 1 h. The
reaction mixture was partitioned between EtOAc (200 µL ×
3) and 1 M phosphate buffer (100 µL). The organic phase was
evaporated in vacuo to afford a crude residue. To a solution of
the residue in THF/1 M phosphate buffer (1:1, 50 µL) was
added NaIO4 (0.5 mg), and the mixture was stirred at room
temperature for 30 min. After evaporation, the reaction
mixture was extracted with EtOAc (200 µL), and the extract
was evaporated in vacuo. To a solution of the residue in EtOH
(50 µL) was added NaBH4 (0.3 mg) at 0 °C, and stirring was
continued for 30 min. The mixture was evaporated and then
partitioned between EtOAc (200 µL × 3) and 1 M phosphate
buffer (100 µL). The organic phase was evaporated, and the
residue was dissolved in 0.1% DMAP solution in CH2Cl2 (50
µL). To the mixture were added Et3N (2 µL) and (R)-(-)-
MTPACl (1 µL), and stirring was continued at room temper-
ature for 2 h. After addition of N,N-dimethyl-1,3-propanedi-
amine (3 µL), the solvent was evaporated in vacuo. The residue
was subjected to silica gel column chromatography (hexane/
EtOAc, 8:1) followed by C18 HPLC (Wakosil-II 5C18 RS, Wako
Pure Chemical Ind., Ltd., 4.6 × 250 mm; eluent CH3CN/H2O,
90:10; flow rate, 1.0 mL/min; UV detection at 230 nm) to give
compounds 6a (0.08 mg, tR 16.0 min) and 7a (0.05 mg, tR 13.6
min). 6a : 1H NMR (CDCl3) δ 0.86 (3H, d, J ) 7.0 Hz, H3-23),
0.91 (3H, d, J ) 6.7 Hz, H3-22), 1.1-1.45 (8H, m, H2-3, H2-4,
H2-5, and H2-6), 1.69 (2H, m, H2-9), 1.78-1.93 (3H, m, H-2
and H2-11), 2.19 (1H, m, H-8), 3.54 (6H, s, 2 × OCH3), 3.79
(1H, m, H-7), 4.05 (1H, m, H-10), 4.06 (1H, dd, J ) 6.7, 10.7
Hz, H-1), 4.23 (1H, dd, J ) 5.6, 10.7 Hz, H-1), 4.41 (2H, t, J )
6.9 Hz, H2-12), 7.35-4.43 (6H, m, Ph), and 7.48-7.54 (4H, m,
Ph); FABMS m/z 699 (M +Na)+; HRFABMS m/z 699.2720
[calcd for C34H42O7F6Na (M + Na)+, 699.2732]. 7a : 1H NMR
(CDCl3) δ 0.88 (3H, d, J ) 6.3 Hz, H3-24), 0.90 (3H, t, J ) 7.5
Hz, H3-21), 1.28-1.40 (2H, m, H2-20), 1.56 (1H, m, H-19), 1.62
(1H, m, H-19), 1.84 (1H, m, H-15), 1.97-2.05 (1H, m, H-14
and H-15), 2.17 (1H, dd, J ) 6.0, 14.2 Hz, H-17), 2.28 (1H, dd,
J ) 7.6, 14.2 Hz, H-17), 3.52 (3H, s, OCH3), 3.54 (3H, s, OCH3),
4.09 (1H, dd, J ) 5.8, 10.2 Hz, H-13), 4.13 (1H, dd, J ) 4.3,
10.2 Hz, H-13), 4.68 (1H, s, H-25), 4.75 (1H, s, H-25), 5.20 (1H,
m, H-18), 7.35-4.43 (6H, m, Ph), and 7.48-7.54 (4H, m, Ph);
FABMS m/z 641 (M + Na)+; HRFABMS m/z 641.2308 [calcd
for C31H36O6F6Na (M + Na)+, 641.2314].
1
colorless oil; H NMR (CDCl3) δ 0.87 (3H, d, J ) 7.0 Hz, H3-
24), 0.92 (3H, J ) 6.7 Hz, H3-25), 1.12 (3H, d, J ) 6.9 Hz,
H3-23), 1.26 (3H, d, J ) 6.1 Hz, H3-22), 1.25-1.75 (14 H), 1.88
(1H, dd, J ) 10.9, 14.5 Hz, H-15), 2.06 (1H, dt, 14.6 and 4.4
Hz, H-11), 2.10-2.15 (2H, m, H-11 and H-17), 2.22 (1H, m,
H-8), 2.36 (1H, dd, J ) 6.9, 13.9 Hz, H-17), 2.41 (1H, m, H-2),
2.58 (1H, br.d, J ) 14.5 Hz, H-15), 3.10 (1H, m, H-14), 3.53
(3H, s, OCH3), 3.56 (3H, s, OCH3), 3.85 (1H, m, H-7), 4.07 (1H,
m, H-10), 4.82 (1H, s, H-25), 4.88 (1H, s, H-25), 4.95 (1H, m,
H-18), 5.11 (1H, m, H-21), 5.50 (1H, t, J ) 5.2 Hz, H-12), 7.38-
7.43 (6H, m), 7.51 (2H, m), and 7.59 (2H, m); ESIMS m/z 907
(M + Na)+; HRESIMS m/z 907.3822 [calcd for C46H58O10F6Na
(M + Na)+, 907.3832].
Bis-(R)-(+)-MTP A Ester (9b) of Am p h id in olid e T2 (2).
Amphidinolide T2 (2, 0.1 mg) was treated with (S)-(+)-MTPACl
(0.3 µL) by the same procedure as described above to afford
the bis-(R)-(+)-MTPA ester (9b, 0.1 mg) of 4. 9b: a colorless
1
oil; H NMR (CDCl3) δ 0.80 (3H, d, J ) 7.0 Hz, H3-24), 0.97
(S)-(-)-MTP A Ester (8a ) of Am p h id in olid e T4 (4).
Amphidinolide T4 (4, 0.2 mg) was treated with (R)-(-)-
MTPACl (0.5 µL) by the same procedure as described above
(3H, d, J ) 6.6 Hz, H3-25), 1.12 (3H, d, J ) 6.9 Hz, H3-23),
1.34 (3H, d, J ) 6.3 Hz, H3-22), 1.23-1.60 (10 H), 1.67 (1H,
m, H-20), 1.87 (1H, m, H-15), 1.97 (1H, dt, J ) 15.5, 6.0 Hz,
H-11), 2.01-2.11 (3H, m, H-8, H-11, and H-17), 2.34 (1H, dd,
J ) 6.5, 14.0 Hz, H-17), 2.41 (1H, m, H-2), 2.65 (1H, br.d, J )
14.3 Hz, H-15), 3.07 (1H, m, H-14), 3.55 (3H, s, OCH3), 3.68
(3H, s, OCH3), 3.79 (1H, m, H-7), 4.06 (1H, m, H-10), 4.84 (1H,
s, H-25), 4.86 (1H, s, H-25), 4.93 (1H, m, H-18), 5.13 (1H, m,
H-21), 5.43 (1H, t, J ) 5.4 Hz, H-12), 7.36-7.45 (6H, m), 7.52
(2H, m), and 7.67 (2H, m); ESIMS m/z 907 (M + Na)+;
HRESIMS m/z 907.3821 [calcd for C46H58O10F6Na (M + Na)+,
907.3832].
to afford the (S)-(-)-MTPA ester (8a , 0.2 mg) of 4. 8a :
a
1
colorless oil; H NMR (CDCl3) δ 0.78 (3H, d, J ) 7.1 Hz, H3-
23), 0.90 (3H, t, J ) 7.3 Hz, H3-21), 1.17 (3H, d, J ) 7.0 Hz,
H3-22), 1.19 (3H, d, J ) 6.8 Hz, H3-24), 1.25-1.65 (14 H), 1.73
(1H, m, H-11), 1.92 (1H, m, H-11), 2.08 (1H, m, H-15), 2.12
(1H, m, H-8), 2.17 (1H, m, H-17), 2.37 (1H, dd, J ) 5.1, 13.5
Hz, H-17), 2.43 (1H, m, H-2), 2.47 (1H, m, H-15), 3.13 (1H, m,
H-14), 3.63 (3H, s, OCH3), 3.80 (1H, m, H-7), 3.96 (1H, m,
H-10), 4.90 (1H, s, H-25), 4.91 (1H, s, H-25), 4.92 (1H, m, H-18),
5.49 (1H, brd, J ) 8.6 Hz, H-12), 7.42 (3H, m), and 7.65 (2H,
m); FABMS m/z 639 (M + H)+; HRFABMS m/z 639.3523 [calcd
for C35H49O7F3 (M + H)+, 639.3508].
Oxid a tive Degr a d a tion of Am p h id in olid e T2 (2). Am-
phidinolide T2 (2, 0.2 mg) was dissolved in THF (30 µL) and
treated with LiAlH4 (1.0 mg) at room temperature for 1 h. The
reaction mixture was partitioned between EtOAc (200 µL ×
3) and 1M phosphate buffer (100 µL). The organic phase was
evaporated in vacuo to afford a crude residue. To a solution of
the residue in THF/1 M phosphate buffer (1:1, 30 µL) was
added NaIO4 (0.2 mg), and the mixture was stirred at room
temperature for 1 h. After evaporation, the reaction mixture
was extracted with EtOAc (200 µL) and the extract was
evaporated in vacuo. To a solution of the residue in EtOH (30
µL) was added NaBH4 (0.2 mg) at 0 °C, and stirring was
continued for 30 min. After addition of 1M phosphate buffer
(100 µL), the mixture was partitioned between EtOAc (200 µL
× 3). The organic phase was evaporated to give the residue
(R)-(+)-MTP A Ester (8b) of Am p h id in olid e T4 (4).
Amphidinolide T4 (4, 0.2 mg) was treated with (S)-(+)-MTPACl
(0.5 µL) by the same procedure as described above to afford
the (R)-(+)-MTPA ester (8b, 0.2 mg) of 4. 8b: a colorless oil;
1H NMR (CDCl3) δ 0.85 (3H, d, J ) 7.0 Hz, H3-23), 0.90 (3H,
t, J ) 7.3 Hz, H3-21), 1.17 (3H, d, J ) 7.0 Hz, H3-22), 1.17
(3H, d, J ) 6.8 Hz, H3-24), 1.25-1.65 (12 H), 1.70-1.82 (3H),
1.96 (1H, m, H-11), 2.06 (1H, dd, J ) 8.7, 13.5 Hz, H-15), 2.16
(1H, m, H-8), 2.17 (1H, m, H-17), 2.36 (1H, m, H-17), 2.43 (1H,
m, H-2), 2.46 (1H, brd, J ) 13.5 Hz, H-15), 3.12 (1H, m, H-14),
3.54 (3H, s, OCH3), 3.84 (1H, m, H-7), 4.19 (1H, m, H-10), 4.89
(2H, s, H2-25), 4.92 (1H, m, H-18), 5.52 (1H, t, J ) 5.3 Hz,

Amphidinolides T2, T3, and T4
J . Org. Chem., Vol. 66, No. 1, 2001 141
(0.5 mg), parts (0.25 mg) of which were dissolved in 0.1%
DMAP solution in CH2Cl2 (15 µL). To the mixture were added
Et3N (2 µL) and (R)-(-)-MTPACl (1 µL), and stirring was
continued at room temperature for 6 h. After addition of N,N-
dimethyl-1,3-propanediamine (1 µL), the reaction mixture was
partitioned between EtOAc (200 µL × 3) and 1 M phosphate
buffer (100 µL), and then the organic layer was evaporated in
vacuo. The residue was subjected to C18 HPLC (Wakosil-II
5C18 RS, 4.6 × 250 mm; eluent CH3CN/H2O, 83:17; flow rate,
1.0 mL/min; UV detection at 230 nm) to give compounds 6a
(0.05 mg, tR 15.6 min) and 10a (0.04 mg, tR 20 min). 6a : 1H
NMR (CDCl3) δ 0.86 (3H, d, J ) 7.0 Hz, H3-23), 0.91 (3H, d, J
) 6.7 Hz, H3-22), 1.1-1.45 (8H, m, H2-3, H2-4, H2-5, and H2-
6), 1.69 (2H, m, H2-9), 1.78-1.93 (3H, m, H-2 and H2-11), 2.19
(1H, m, H-8), 3.54 (6H, s, 2 × OCH3), 3.79 (1H, m, H-7), 4.05
(1H, m, H-10), 4.06 (1H, dd, J ) 6.7 and 10.7 Hz, H-1), 4.23
(1H, dd, J ) 5.6 and 10.7 Hz, H-1), 4.41 (2H, t, J ) 6.8 Hz,
H2-12), 7.35-7.43 (6H, m, Ph), and 7.48-7.54 (4H, m, Ph);
ESIMS m/z 699 (M + Na)+; HRESIMS m/z 699.2725 [calcd
for C34H42O7F6Na (M + Na)+, 699.2732]. 10a : 1H NMR (CDCl3)
δ 0.88 (3H, d, J ) 6.6 Hz, H3-25), 1.23 (3H, t, J ) 7.5 Hz, H3-
22), 1.56-1.72 (4H, m, H2-19 and H2-20), 1.84 (1H, dd, J )
6.3, 12.9 Hz, H-15), 1.96-2.04 (2H, m, H-14 and H-15), 2.14
(1H, dd, J ) 5.7, 14.7 Hz, H-17), 2.30 (1H, dd, J ) 7.5, 14.7
Hz, H-17), 3.47 (3H, s, OCH3), 3.50 (3H, s, OCH3), 3.53 (3H, s,
OCH3), 4.09 (1H, dd, J ) 6.0, 10.8 Hz, H-13), 4.13 (1H, dd, J
) 4.8, 10.8 Hz, H-13), 4.70 (1H, s, H-26), 4.75 (1H, s, H-26),
5.10 (1H, m, H-21), 5.20 (1H, m, H-18), 7.32-7.40 (9H, m, Ph),
and 7.44-7.54 (6H, m, Ph); ESIMS m/z 887 (M + Na)+;
HRESIMS m/z 887.2844 [calcd for C42H45O9F9Na (M + Na)+,
887.2818]. Crude products (0.25 mg) obtained after reactions
with LiAlH4, NaIO4, and NaBH4 of 2 described above were
treated with DMAP (15 ng), Et3N (2 µL), and (S)-(+)-MTPACl
(1 µL) in CH2Cl2 at room temperature for 6 h. Workup of the
reaction mixture was carried out by the same procedure
described above to afford compounds 6b (0.18 mg, tR 16.4 min)
and 10b (0.12 mg, tR 20.6 min). 6b: 1H NMR (CDCl3) δ 0.86
(3H, d, J ) 7.0 Hz, H3-24), 0.91 (3H, d, J ) 6.8 Hz, H3-23),
1.1-1.45 (8H, m, H2-3, H2-4, H2-5, and H2-6), 1.69 (2H, m,
H2-9), 1.76-1.91 (3H, m, H-2 and H2-11), 2.19 (1H, m, H-8),
3.52 (6H, s, 2 × OCH3), 3.79 (1H, m, H-7), 4.05 (1H, m, H-10),
4.15 (2H, m, H2-1), 4.39 (1H, m, H-12), 4.44 (1H, m, H-12),
7.35-7.43 (6H, m, Ph), and 7.48-7.54 (4H, m, Ph); ESIMS
m/z 699 (M + Na)+; HRESIMS m/z 699.2739 [calcd for
with MgSO4, evaporated in vacuo to give a crude aldehyde (336
mg). To a stirred solution of the crude aldehyde (336 mg) in
Et2O (10 mL) was added a 1 M Et2O solution of allylmagne-
sium bromide (5.7 mL, 5.7 mmol) at -10 °C. After be stirred
at room temperature for 14 h, saturated aqueous NH4Cl and
Et2O were added to the mixture, which was extracted with
Et2O. The extract was washed with H2O and brine, dried with
MgSO4, and evaporated in vacuo to afford a crude alcohol (315
mg). The crude alcohol (315 mg) was treated with Ac2O (2 mL)
and pyridine (2 mL) at room temperature for 6 h. After
evaporation of the solvent, the resulting residue was subjected
to silica gel column chromatography (hexane/EtOAc, 20:1) to
yield compound 13 (273 mg, 826 µmol, 56% by four steps) as
a colorless oil: [R]D -24° (c 1.0, CHCl3); IR (neat) νmax 3079,
2954, 1739, 1375, 1238, 1103, 1031, 936, and 919 cm-1 1H
;
NMR (CDCl3) δ 0.01 (9H, s), 0.87-0.96 (5H, m), 1.06 (1.5 H,
d, J ) 6.6 Hz), 1.08 (1.5H, d, J ) 6.6 Hz), 1.24 (0.5H, m), 1.37
(0.5H, m), 1.62-1.78 (1H, m), 2.01 (3H, s), 2.21-2.37 (3H, m),
3.50-3.65 (3H, m), 4.63 (1H, m), 4.67 (0.5H, d, J ) 16.5 Hz),
4.69 (0.5H, d, J ) 16.5 Hz), 4.97-5.07 (3H, m), and 5.73 (1H,
m); 13C NMR (CDCl3) δ -1.5 (3C, q), 14.7 (0.5C, q), 15.1 (0.5C,
q), 15.8 (0.5C, q), 16.3 (0.5C, q), 18.1 (1C, t), 21.1 (0.5C, q),
21.2 (0.5C, q), 34.4 (0.5C, t), 34.9 (0.5C, t), 36.6 (0.5C, d), 36.6
(0.5C, d), 38.5 (0.5C, t), 39.6 (0.5C, t), 65.0 (1C, t), 71.1 (0.5C,
d), 72.2 (0.5C, d), 76.2 (0.5C, d), 76.6 (0.5C, d), 93.28 (0.5C, t),
93.34 (0.5C, t), 117.7 (1C, t), 133.6 (0.5C, d), 133.7 (0.5C, d),
and 170.7 (1C, s); FABMS m/z 331 (M + H)+; HRFABMS m/z
331.2312 [calcd for C17H34O4Si (M + H)+, 331.2305].
(2R,3S,5RS)-5-Acetyloxy-3-m eth yl-2-[2-(tr im eth ylsilyl)-
eth oxym eth oxy]h ep ta n -7-ol (14). Compound 13 (294 mg,
889 µmol) was dissolved in a 1:1:1 mixture of H2O, acetone,
and CH3CN (3 mL), and to this mixture were added microen-
capsulated OsO4 (220 mg) and 4-methymorpholine N-oxide
(270 mg, 2.32 mmol). After stirring at room temperature for
64 h, insoluble materials were filtered, and the filtrate was
evaporated in vacuo to give a residue. This was purified by a
silica gel column (hexane/acetone, 2:1) to afford a mixture of
1, 2-diols (248 mg, 680 µmol, 76%), and compound 13 (54 mg,
163 µmol, 18%) was recovered. To a stirring solution of the
mixture (247 mg, 678 µmol) in THF (1 mL) and 1M potassium
phosphate buffer (pH 7.0: 1 mL) was added NaIO4 (339 mg,
1.58 mmol) at 0 °C. After stirring at 0 °C for 1 h, insoluble
materials were filtered, and the filtrate was evaporated in
vacuo to give a residue (254 mg). To a solution of the residue
(254 mg) in EtOH (3 mL) was added NaBH4 (60 mg, 1.58
mmol) at 0 °C, and the mixture was stirred at 0 °C for 1 h.
After addition of Et2O and 1 M aqueous HCl, the mixture was
extracted with Et2O. The extract was washed with saturated
aqueous NaHCO3, H2O, and brine, dried with MgSO4, and
evaporated in vacuo to give a crude alcohol. This was subjected
to silica gel column chromatography (hexane/EtOAc, 2:1) to
afford compound 14 (135 mg, 404 µmol, 60% by two steps) as
colorless oil: [R]D +16° (c 1.0, CHCl3); IR (neat) νmax 3461, 2954,
C
34H42O7F6Na (M + Na)+, 699.2732]. 10b: 1H NMR (CDCl3)
δ 0.86 (3H, d, J ) 6.6 Hz, H3-25), 1.23 (3H, d, J ) 6.3 Hz,
H3-22), 1.33 (1H, m, H-20), 1.45-1.54 (3H, m, H2-19 and H-20),
1.88 (1H, m, H-15), 2.01-2.09 (2H, m, H-14 and H-15), 2.12
(1H, dd, J ) 5.7, 14.7 Hz, H-17), 2.32 (1H, dd, J ) 7.5, 14.5
Hz, H-17), 3.49 (3H, s, OCH3), 3.51 (3H, s, OCH3), 3.54 (3H, s,
OCH3), 4.02 (1H, dd, J ) 6.0, 10.8 Hz, H-13), 4.25 (1H, dd, J
) 4.8, 10.8 Hz, H-13), 4.80 (1H, s, H-26), 4.81 (1H, s, H-26),
5.03 (1H, m, H-21), 5.16 (1H, m, H-18), 7.35-7.43 (9H, m, Ph),
and 7.48-7.54 (6H, m, Ph); ESIMS m/z 887 (M + Na)+;
HRESIMS m/z 887.2820 [calcd for C42H45O9F9Na (M + Na)+,
887.2818].
(2R,3S,5RS)-5-Acetyloxy-3-m eth yl-2-[2-(tr im eth ylsilyl)-
eth oxym eth oxy]oct-7-en e (13). To a solution of (2R,3R)-1-
iodo-2-methyl-3-[2-(trimethylsilyl)ethoxymethoxy]butane (12,
512 mg, 1.48 mmol) in DMSO (10 mL) was added sodium
cyanide (214 mg, 4.44 mmol) at room temperature, and stirring
was continued at 70 °C for 4 h. After addition of Et2O and
H2O, the reaction mixture was extracted with Et2O. The
extract was washed with brine, dried with MgSO4, and
evaporated in vacuo to afford a crude cyanide (359 mg). To a
stirred solution of the crude cyanide (359 mg) was added
dropwise a 0.95 M solution of DIBAL in hexane (1.86 mL, 1.77
mmol) at -78 °C. After being stirred for 1 h, the reaction
mixture was treated with MeOH (300 µL), allowed to warm
to room temperature. After addition of saturated aqueous NH4-
Cl (10 mL), the mixture was vigorously stirred for 20 min. Et2O
and saturated aqueous potassium sodium tartrate were added
to the mixture, and stirring was continued at room tempera-
ture for 1 h. The reaction mixture was extracted with Et2O,
and the extract was washed with H2O and then brine, dried
1
1740, 1376, 1248, 1055, 860, and 837 cm-1; H NMR (CDCl3)
δ 0.01 (9H, s), 0.88-0.96 (5H, m), 1.11 (1.5 H, d, J ) 6.6 Hz),
1.13 (1.5 H, d, J ) 6.6 Hz), 1.22 (0.5H, m), 1.50 (1H, t, J ) 7.8
Hz), 1.55-1.88 (3.5H, m), 2.05 (3H, s), 3.48-3.55 (1H, m), 3.62
(2H, m), 3.71 (0.5H, m), 3.78 (0.5H, m), 4.16 (1H, m), 4.29 (1H,
m), 4.65 (1H, m), and 4.72 (1H, m); 13C NMR (CDCl3) δ -1.48
(3C, q), 15.3 (0.5C, q), 15.7 (0.5C, q), 16.0 (0.5C, q), 16.7 (0.5C,
q), 16.69 (0.5C, t), 16.81 (0.5C, t), 235.2 (0.5C, d), 35.4 (0.5C,
d), 36.5 (0.5C, t), 37.4 (0.5C, t), 40.2 (0.5C, t), 40.7 (0.5C, t),
61.8 (0.5C, t), 65.1 (0.5C, t), 65.2 (0.5C, t), 65.9 (0.5C, d), 66.63
(0.5C, d), 76.7 (0.5C, d), 77.2 (0.5C, d), 93.2 (0.5C, t), 93.4 (0.5C,
t), 171.3 (0.5C, s), and 171.4 (0.5C, s); FABMS m/z 335 (M +
H)+; HRFABMS m/z 335.2254 [calcd for C16H35O5Si (M + H)+,
335.2254].
Com p ou n d 15a . To a solution of compound 14 (21.5 mg,
64.3 µmol) in CH2Cl2 (500 µL) were added DMAP (0.5 mg, 4
µmol), Et3N (32 µL, 230 µmol), and (R)-(-)-MTPACl (22 µL,
114 µmol), and stirring was continued at room temperature
for 3 h. The reaction mixture was partitioned between Et2O
and H2O, and then the organic phase was washed with brine,
dried with MgSO4, and evaporated in vacuo. The residue was
purified by a silica gel column (hexane/EtOAc, 10:1) to afford

142 J . Org. Chem., Vol. 66, No. 1, 2001
Kobayashi et al.
an (S)-(-)-MTPA ester (27.3 mg, 49.6 µmol, 77%). A solution
of the (S)-(-)-MTPA ester (27.0 mg, 49.0 µmol) in CH2Cl2 (3
mL) was treated with trifluoroaceitic acid (1 mL) at 0 °C for 1
h. The reaction mixture was partitioned between CH2Cl2 and
H2O. The organic layer was washed with brine, dried with
MgSO4, and evaporated in vacuo to give a crude product (27
mg). To a solution of the crude product (27 mg) in CH2Cl2 (3
mL) were added DMAP (0.5 mg, 4 µmol), Et3N (50 µL, 358
µmol), methanesulfonyl chloride (20 µL, 25.8 µmol) at 0 °C.
After stirring for 4 h, Et2O and 1N aqueous HCl were added
to the mixture, which was extracted with Et2O. The organic
phase was washed with saturated aqueous NaHCO3, H2O, and
brine, dried with MgSO4, and evaporated in vacuo. The residue
was subjected to silica gel column chromatography (hexane/
EtOAc, 6:4) to afford the mesylate 15a (11.7 mg, 23.5 µmol,
48% by two steps) as colorless oil: [R]D -36° (c 1.0, CHCl3);
IR (neat) νmax 2944, 1741, 1353, 1238, 1175, 1019, and 905
cm-1; 1H NMR (CDCl3) δ 0.94 (1.5H, d, J ) 6.7 Hz), 0.99 (1.5H,
d, J ) 6.7 Hz), 1.24 (1.5H, d, J ) 6.3 Hz), 1.32 (0.5H, m), 1.35
(1.5H, d, J ) 6.3 Hz), 1.49 (0.5H, m), 1.59 (0.5H, m), 1.75-
2.00 (3.5H, m), 2.02 (1.5H, s), 2.04 (1.5H, s), 2.93 (1.5H, s),
2.98 (1.5H, s), 3.53 (3H, s), 3.85 (0.5H, m), 3.97-4.13 (1.5H,
m), 4.56 (0.5H, m), 4.68 (0.5H, m), 5.27 (1H, m), 7.36-7.43
(3H, m), and 7.47-7.54 (2H, m); 13C NMR (CDCl3) δ 15.0 (0.5C,
q), 15.13 (0.5C), 17.1 (0.5C), 17.8 (0.5C), 20.8 (1C), 32.3 (0.5C),
33.7 (0.5C), 34.4 (0.5C), 35.0 (0.5C), 35.9 (0.5C), 36.8 (0.5C),
38.55 (0.5C), 38.57 (0.5C), 55.4 (1C), 59.9 (0.5C), 60.2 (0.5C),
71.6 (0.5C), 72.54 (0.5C), 82.4 (0.5C), 82.7 (0.5C), 127.2 (2C),
128.5 (2C), 129.7 (1C), 131.9 (1C), 166.3 (1C), 170.7 (0.5C), and
170.8 (0.5C); FABMS m/z 521 (M + Na)+; HRFABMS m/z
521.1415 [calcd for C21H29F3O8SNa (M + Na)+, 521.1433].
Com p ou n d 15b. Compound 15b (40.1 mg, 80.4 µmol, 46%
by three steps) was obtained from compound 14 (58.2 mg, 174
µmol) by the same procedure using (S)-(+)-MTPACl as de-
scribed above. 15b: colorless oil; [R]D +10° (c 1.0, CHCl3); IR
and 7.51 (2H, m, Ph); EIMS m/z 189, 289, 304, and 359 (M -
H)-; HREIMS m/z 359.1488 [calcd for C18H22F3O4 (M - H)-,
359.1470]. 16a : colorless oil; [R]D -16° (c 0.5, CHCl3); IR (neat)
νmax 2960, 1749, 1271, 1170, 1121, and 1023 cm-1 1H NMR
;
(CDCl3) δ 0.90 (3H, d, J ) 7.0 Hz, H3-23 or H3-24), 1.06 (3H,
d, J ) 6.4 Hz, H3-6), 1.15 (1H, dt, J ) 12.3, 8.5 Hz, H-9), 1.92
(2H, m, H-9 and H-11), 2.09 (1H, dt, J ) 12.3, 7.3 Hz, H-11),
2.24 (1H, m, H-8), 3.55 (3H, s, OCH3), 3.78 (1H, m, H-10), 3.95
(1H, quint, J ) 6.4 Hz, H-7), 4.36-4.48 (2H, m, H2-12), 7.40
(3H, m, Ph), and 7.51 (2H, m, Ph); EIMS m/z 189, 289, 304,
and 359 (M - H)-; HREIMS m/z 359.1482 [calcd for C18H22F3O4
(M - H)-, 359.1470].
Com p ou n d s 11b a n d 16b. Compounds 11b (2.6 mg, 7.2
µmol, 34%, tR 30 min) and 16b (2.3 mg, 6.4 µmol, 31%, tR 33
min) were obtained from compound 15b (10.4 mg, 20.9 µmol)
by the same procedure as described above. 11b: colorless oil;
[R]D +32° (c 0.5, CHCl3); IR (neat) νmax 2966, 1748, 1271, 1169,
1
1122, and 1024 cm-1; H NMR (CDCl3) δ 0.88 (3H, d, J ) 7.0
Hz, H3-23 or H3-24), 1.13 (3H, d, J ) 6.3 Hz, H3-6), 1.70 (2H,
t, J ) 6.8 Hz, H2-9), 1.81 (1H, m, H-11), 1.87 (1H, m, H-11),
2.20 (1H, m, H-8), 3.55 (3H, s, OCH3), 4.02-4.11 (2H, m, H-7
and H-10), 4.35-4.47 (2H, m, H2-12), 7.40 (3H, m, Ph), and
7.51 (2H, m, Ph); EIMS m/z 189, 289, 304, and 359 (M - H)-;
HREIMS m/z 359.1462 [calcd for
359.1470]. 16b: colorless oil; [R]D +49° (c 0.3, CHCl3); IR (neat)
νmax 2963, 1748, 1270, 1169, 1120, and 1022 cm-1 1H NMR
C
18H22F3O4 (M - H)-,
;
(CDCl3) δ 0.91 (3H, d, J ) 7.0 Hz, H3-23 or H3-24), 1.08 (3H,
d, J ) 6.5 Hz, H3-6), 1.16 (1H, dt, J ) 12.3, 8.4 Hz, H-9), 1.93
(2H, m, H-9 and H-11), 2.08 (1H, dt, J ) 12.3, 7.3 Hz, H-11),
2.24 (1H, m, H-8), 3.35 (3H, s, OCH3), 3.79 (1H, m, H-10), 3.95
(1H, quint, J ) 6.4 Hz, H-7), 4.42 (2H, t, J ) 6.8 Hz, H2-12),
7.40 (3H, m, Ph), and 7.52 (2H, m, Ph); EIMS m/z 189, 289,
304, and 359 (M - H)-; HREIMS m/z 359.1472 [calcd for
C
18H22F3O4 (M - H)-, 359.1470].
Gen er a l F eed in g Exp er im en ts of 13C-La beled P r ecu r -
(neat) νmax 2943, 1740, 1353, 1237, 1175, 1019, and 906 cm-1
;
sor s. The dinoflagellate cultured in a 100 L nutrient-enriched
seawater medium was supplemented with [1-13C]-, [2-13C]-, or
[1,2-13C2]sodium acetate (610 µM) in one portion at 7 days after
inoculation, and then the culture was harvested by centrifuga-
tion after 14 days. Sodium 13C-bicarbonate (1.2 mM) was added
to 100 L culture of the dinoflagellate at 10 days, and then the
culture was harvested by centrifugation after 14 days. Extrac-
tion and isolation of amphidinolide T1 (1) from the harvested
cells were carried out by the same procedure as described
above. The 13C-labeled amphidinolide T1 (1) was obtained in
0.0006% yield as an average from wet weight of the cells.
1H NMR (CDCl3) δ 0.92 (1.5H, d, J ) 6.8 Hz), 1.01 (1.5H, d, J
) 6.8 Hz), 1.30 (1.5H, d, J ) 6.9 Hz), 1.33 (1.5H, d, J ) 6.9
Hz), 1.52 (0.5H, m), 1.66-1.80 (2H, m), 1.89-2.00 (2.5H, m),
2.04 (3H, s), 2.98 (1.5H, s), 3.13 (1.5H, s), 3.53 (3H, s), 3.92
(0.5H, m), 4.04 (1H, m), 4.12 (0.5H, m), 4.64 (1H, m), 5.21-
5.32 (1H, m), 7.36-7.43 (3H, m), and 7.47-7.54 (2H, m);
FABMS m/z 521 (M + Na)+; HRFABMS m/z 521.1430 [calcd
for C21H29F3O8SNa (M + Na)+,521.1433].
Com p ou n d s 11a a n d 16a . A solution of compound 15a
(11.0 mg, 22.1 µmol) in EtOH (1 mL) was treated with K2CO3
(6.5 mg, 46 µmol) at room temperature for 72 h. After
evaporation of the solvent, the residue was subjected to a silica
gel column (hexane/EtOAc, 6:1) to afford a mixture of 11a and
16a (6.1 mg, 16.9 µmol). The mixture was purified by C18
HPLC [LUNA C18(2), 5 µm, 10 × 250 mm; eluent, CH3CN/
H2O (68:32); flow rate, 2.5 mL/min; UV detection at 230 nm]
to give compounds 11a (2.7 mg, 7.5 µmol, 34%, tR 30 min) and
16a (2.6 mg, 7.2 µmol, 33%, tR 33 min). 11a : colorless oil; [R]D
-32° (c 0.5, CHCl3); IR (neat) νmax 2963, 1748, 1270, 1168,
Ack n ow led gm en t. This work was partly supported
by a Grant-in-Aid for Scientific Research from the
Ministry of Education, Science, Sports, and Culture of
J apan.
Su p p or tin g In for m a tion Ava ila ble: NMR spectra of 2,
1
3, and 4, H NMR spectra of 6a ,b, 10a ,b, and 11a ,b, and 13C
1
1122, and 1025 cm-1; H NMR (CDCl3) δ 0.88 (3H d, J ) 7.0
NMR and INADEQUATE spectra of 13C-labeled 1. This
material is available free of charge via the Internet at
http://pubs.acs.org.
Hz, H3-23 or H3-24), 1.07 (3H, d, J ) 6.3 Hz, H3-6), 1.71 (2H,
t, J ) 6.8 Hz, H2-9), 1.81 (1H, m, H-11), 1.87 (1H, m, H-11),
2.20 (1H, m, H-8), 3.55 (3H, s, OCH3), 4.04-4.11 (2H, m, H-7
and H-10), 4.42 (2H, t, J ) 6.8 Hz, H2-12), 7.40 (3H, m, Ph),
J O005607C