Absolute stereochemistry of amphidinolide E

Article abstract of DOI:10.1021/jo016326n

The absolute configurations at eight chiral centers in amphidinolide E (1), a cytotoxic 19-membered macrolide isolated from a marine dinoflagellate Amphidinium sp., were determined to be 2R, 7R, 8R, 13S, 16S, 17R, 18R, and 19R on the basis of detailed analysis of NMR data and by chemical means.

Full text of DOI:10.1021/jo016326n

J . Org. Chem. 2002, 67, 1651-1656
1651
Absolu te Ster eoch em istr y of Am p h id in olid e E
Takaaki Kubota, Masashi Tsuda, and J un’ichi Kobayashi*
Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, J apan
jkobay@pharm.hokudai.ac.jp
Received November 27, 2001
The absolute configurations at eight chiral centers in amphidinolide E (1), a cytotoxic 19-membered
macrolide isolated from a marine dinoflagellate Amphidinium sp., were determined to be 2R, 7R,
8R, 13S, 16S, 17R, 18R, and 19R on the basis of detailed analysis of NMR data and by chemical
means.
Ch a r t 1
In tr od u ction
Our continuing search for bioactive metabolites from
symbiotic marine dinoflagellates of the genus Amphi-
dinium has resulted in the isolation of a series of
cytotoxic macrolides1 and two types of polyketides.2
Amphidinolide E (1) is a unique cytotoxic 19-membered
macrolide, which has been isolated from the Y-5′ strain
of a dinoflagellate Amphidinium sp.3 The gross structure
was elucidated by 2D NMR data, whereas the stereo-
chemisty remains unsolved because of the lack of sample.
Recently, the relative and absolute configurations at eight
chiral centers in 1 were elucidated on the basis of detailed
analysis of NMR data and by chemical means because a
substantial amount (2 mg) of 1 has been obtained through
repeated cultivation. This contribution describes the
determination of the relative and absolute configurations
of 1 (Chart 1).
C-8, (b) application of a modified Mosher method6 for a
hydroxyl group at C-17 in 1 and for that at C-1 of the
C-1-C-7 segment obtained by oxidative degradation, and
(c) comparison of NMR data for MTPA esters of the C-8-
C-17 segment obtained by oxidative degradation with
those of the corresponding synthetic compound.
Rela tive Ster eoch em istr y. The relative stereochem-
istry of H-13/H-16 on a tetrahydrofuran ring in 1 was
elucidated to be syn from the NOESY correlation for
H-13/H-16 (Figure 1a). On the other hand, the relative
stereochemistry of the 7,8-diol was assigned as threo by
NOESY correlations (Figure 1b) and the 3J (H-7, H-8)
value (8.8 Hz) of the 7,8-O-isopropylidene derivative (2)
of 1, which was obtained by treatment of 1 with 2,2-
dimethoxypropane (DMP) and pyridinium p-toluene-
sulfonate (PPTS).
Resu lts a n d Discu ssion
The relative stereochemistry of eight chiral centers (C-
2, C-7, C-8, C-13, C-16, C-17, C-18, and C-19) in amphi-
dinolide E (1) was elucidated by a combination of the
J -based configuration method4 and detailed analyses of
NOESY data, while the absolute stereochemistry was
determined by the following experiments: (a) application
of the exciton chirality method5 for a 1,2-diol at C-7 and
* Corresponding author. Tel: Int + 81 11 706 4985. Fax: Int + 81
11 706 4989.
(1) (a) Ishibashi, M.; Kobayashi, J . Heterocycles 1997, 44, 543-572
and references therein. (b) Kobayashi, J .; Kubota, T.; Endo, T.; Tsuda,
M. J . Org. Chem. 2001, 66, 134-142. (c) Kubota, T.; Endo, T.; Tsuda,
M.; Shiro, M.; Kobayashi, J . Tetrahedron 2001, 57, 6175-6179.
(2) (a) Kobayashi, J .; Kubota, T.; Takahashi, M.; Ishibashi, M.;
Tsuda, M.; Naoki, H. J . Org. Chem. 1999, 64, 1478-1482. (b) Kubota,
T.; Tsuda, M.; Takahashi, M.; Ishibashi, M.; Naoki, N.; Kobayashi, J .
J . Chem. Soc., Perkin Trans. 1 1999, 3483-3488. (c) Kubota, T.; Tsuda,
M.; Takahashi, M.; Ishibashi, M.; Oka, S.; Kobayashi, J . Chem. Pharm.
Bull. 2000, 48, 1447-1451. (d) Doi, Y.; Ishibashi, M.; Nakamichi, H.;
Kosaka, T.; Ishikawa, T.; Kobayashi, J . J . Org. Chem. 1997, 62, 3820-
3823. (e) Kubota, T.; Tsuda, M.; Doi, Y.; Takahashi, A.; Nakamichi,
H.; Ishibashi, M.; Fukushi, E.; Kawabata, J .; Kobayashi, J . Tetrahedron
1998, 54, 14455-14464.
(3) Kobayashi, J .; Ishibashi, M.; Murayama, T.; Takamatsu, M.;
Iwamura, M.; Ohizumi, Y.; Sasaki, T. J . Org. Chem. 1990, 55, 3421-
3423.
(4) Matsumori, N.; Kaneno, D.; Murata, M.; Nakamura, H.;
Tachibana, K. J . Org. Chem. 1999, 64, 866-876.
(5) Harada, N.; Nakanishi, K. Circular Dichroic Spectrometry:
Exciton Coupling in Organic Stereochemistry; University Science
Books: Mill Valley, CA, 1983.
3
For the C-16-C-17 bond, the J (H-16, H-17) value (7.5
Hz) was typical for an anti relationship (Figure 2a), while
the value for 2J (H-17, C-16) (-6.0 Hz), which was
obtained from the hetero half-filtered TOCSY (HETLOC)7
spectrum of 1, indicated that H-16 was gauche to 17-OH.
Gauche relations between C-15 and C-18 and between
C-15 and H-17 were implied by NOESY correlations for
H2-15/H-18 and H2-15/H-17, thus suggesting that the
relative configuration of C-16-C-17 was threo. An eryth-
ro relationship for C-18-C-19 was deduced from the
(6) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J . Am. Chem.
Soc. 1991, 113, 4092-4095.
(7) (a) Otting, G.; Wu¨thrich, K. Q. 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.
10.1021/jo016326n CCC: $22.00 © 2002 American Chemical Society
Published on Web 02/08/2002

1652 J . Org. Chem., Vol. 67, No. 5, 2002
Kubota et al.
F igu r e 3. NOESY correlations and relative stereochemistry
of C-15-C-19 in the 7,8,17,18-di-O-isopropylidene derivative
(3b) of amphidinolide E (1). NOESY correlations are illustrated
by solid arrows.
F igu r e 1. NOESY correlations and relative stereochemistry
of (a) a tetrahydrofuran ring in amphidinolide E (1) and (b)
C-7-C-8 in the 7,8-O-isopropylidene derivative (2) of 1.
NOESY correlations are illustrated by solid arrows.
F igu r e 2. Rotation models for the (a) C-16-C-17, (b) C-18-
C-19, and (c) C-17-C-18 bonds of amphidinolide E (1). NOESY
correlations are illustrated by solid arrows.
Sch em e 1
F igu r e 4. CD spectrum of the 7,8-bis-O-p-methoxycinnamoyl
derivative (4) of amphidinolide E (1).
Rh-Al2O3 in MeOH caused considerable epimerization
(>20%) at the C-2 carbon of the carbonyl group, while
no epimerization was observed under acidic conditions
with 1% acetic acid. The pentaalcohol 3a was converted
into 3b through acetonidation. NOESY correlations from
an acetonide methyl signal (δH 1.60, s) to H-18 (δH 4.27)
and from the other methyl signal (δH 1.53, s) to H-17 (δH
3.90) indicated that the relative configuration of H-17/
H-18 in 3b was anti (Figure 3). Therefore, a threo relation
for the C-17-C-18 bond was established.
Absolu te Ster eoch em istr y. For application of the
exciton chirality method, the O-p-methoxycinnamoyl
group was chosen as an exciton chromophore of the
7,8-diol in amphidinolide E (1) because the UV spectrum
of 1 showed a strong absorption at 230 nm (ꢀ 26 000).
Treatment of 1 with p-methoxycinnamoyl chloride af-
forded the 7,8-bis-O-p-methoxycinnamate (4) and the
7,8,17-tris-O-p-methoxycinnamate in a ratio of 3:2, re-
spectively. In the 1H NMR spectrum of 4, the vicinal
coupling constant between H-7 and H-8 was 8.8 Hz,
suggesting that H-7 and H-8 had an anti relationship to
each other. The CD spectrum of 4 disclosed a negative
first Cotton effect (λext 324 nm, ∆ꢀ -14.3) and a positive
second Cotton effect (λext 289 nm, ∆ꢀ +18.9) (Figure 4),
indicating 7R and 8R configurations.8
3
3J (H-18, H-19) (9.7 Hz) and J (H-18/C-28) (3.0 Hz) values
and NOESY correlations, as shown in Figure 2b.
The 3J (H-17, H-18) (∼0 Hz) value and the NOESY
correlation for H-17/H-18 were attributed to a gauche
relation for H-17 and H-18 (Figure 2c). NOESY correla-
tions for H-16/H-18 and H-17/H3-28 were suggestive
of both gauche relations between C-16 and H-18 and
between H-17 and C-19. This threo relation for the
C-17-C-18 bond was supported by analysis of NOESY
correlations of the linear 7,8,17,18-di-O-isopropylidene
derivative (3b) of 1, obtained as follows (Scheme 1).
Reduction of six olefins with hydrogen gas and the Rh-
Al2O3 catalyst followed by reduction of the ester carbonyl
group at C-1 with LiAlH4 gave a crude pentaalcohol of
dodecahydroamphidinolide E (3a ). Hydrogenation using
(8) Lo, L.-C.; Berova, N.; Nakanishi, K.; Schlingmann, G.; Carter,
G. T.; Borders, D. B. J . Am. Chem. Soc. 1992, 114, 7371-7374.

Absolute Stereochemistry of Amphidinolide E
J . Org. Chem., Vol. 67, No. 5, 2002 1653
F igu r e 5. ∆δ values (∆δ (in ppm) ) δS - δR) obtained for the
7,8,17-tris-(S)- and (R)-MTPA esters (5a and 5b, respectively)
of amphidinolide E (1).
Sch em e 2
F igu r e 6. Proton signal patterns of H2-1 of the 1,7-bis-(S)-
and (R)-MTPA esters (6a and 6b, respectively) of C-1-C-7 in
amphidinolide E (1).
Sch em e 3
A modified Mosher method was used to elucidate the
absolute configuration at C-17. Amphidinolide E (1) was
treated with (R)-(-)- and (S)-(+)-2-methoxy-2-trifluoro-
methyl-2-phenylacetyl chloride (MTPACl) to afford the
7,8,17-tris-(S)- and (R)-MTPA esters (5a and 5b, respec-
1
tively).9 ∆δ values (δS - δR) obtained from H NMR data
of 5a and 5b are shown in Figure 5. The ∆δ values for
H-13, H2-14, H2-15, and H2-16 are positive, while negative
∆δ values are observed for H-18, H-19, H2-20, H-22, H-23,
H3-28, and H2-29, thus indicating a 17R configuration.
Therefore, the absolute configurations at C-13, C-16,
C-17, C-18, and C-19 were assigned as S, S, R, R, and R,
respectively, because the relative stereochemistry among
them was elucidated as described above.
To elucidate the absolute configuration at C-2, oxida-
tive degradation of two 1,2-diol units (C-7-C-8 and
C-17-C-18) of a reductive product (3a ) of amphidinolide
E (1) was performed as follows. The pentaalcohol 3a was
subjected to NaIO4 degradation of the two 1,2-diol units,
followed by NaBH4 reduction and esterification with (R)-
and (S)-MTPACl (Scheme 2). HPLC separation of the
reaction mixture furnished the 1,7-bis-(S)- and (R)-MTPA
esters of the C-1-C-7 segment (6a and 6b, respectively)
and the 8,17-bis-(S)- and (R)-MTPA esters of the C-8-
C-17 segment (7a and 7b, respectively).10 The absolute
configuration at C-2, where a methyl group was located,
was elucidated on the basis of chemical shift differences
and signal patterns of the two geminal protons10 at C-1
of 6a and b. The methylene protons at C-1 of 6a appeared
as two separated doublet signals at δH 4.12 and 4.17
(chemical shift difference (∆δ) 0.05) (Figure 6), while the
∆δ value (0.16) of H2-1 (δH 4.03 and 4.19) for 6b was
larger than that for 6a , indicating that the absolute
configuration at C-2 was R.
NMR data of 7a and b with that of the corresponding
synthetic segment (12a and b). 12a and b are enantio-
mers of 7b and a , respectively. The C-8-C-17 segment
was synthesized from the (13S,16R)-alcohol 8, which was
prepared from (R)-5-benzyloxymethyl-γ-butyrolactone. 8
was converted into the benzyl ether 9, which was
subjected to iodination followed by a coupling reaction
with 3-benzyloxypropyne to afford an acetylide 10 (Scheme
3). Reduction and deprotection of 10 gave a (13R,16R)-
(11) Mosher’s method has been applied for the determination of the
absolute stereochemistry of a methyl group at C-25 of steroids with a
primary hydroxyl group at C-26. In the 1H NMR spectra of the (+)-
(R)-MTPA esters, two methylene-26 protons of the 25-(S) isomer (A)
are much closer (∆δ ca. 0.04) to each other than are those (∆δ ca. 0.14)
of the 25-(R) isomer (B), whereas in the (-)-(S)-MTPA esters, the
mutual relation is reversed. (a) De Riccardis, F.; Minale, L.; Riccio,
R.; Giovannitti, B.; Iorizzi, M.; Debitus, C. Gazz. Chim. Ital. 1993, 123,
79-86. (b) Finamore, E.; Minale, L.; Riccio, R.; Rinaldo, G.; Zollo, F.
J . Org. Chem. 1991, 56, 1146-1153.
The absolute stereochemistry at C-13 and C-16 of the
C-8-C-17 segment was established by comparison of the
(9) Esterification of the 7,8-O-isopropylidene derivative (2) of amphi-
dinolide E (1) with MTPACl did not afford the 17-MTPA ester of 2.
(10) The (S)- and (R)-MTPA esters of C-18-C-26 segments were not
obtained.

1654 J . Org. Chem., Vol. 67, No. 5, 2002
Kubota et al.
2D NMR spectra were recorded on a 600 MHz spectrometer
at 300 K using 2.5 mm micro cells for CDCl3 (Shigemi Co. Ltd.).
HETLOC experiments were obtained with the pulse sequence
proposed by Woolborn and Leibfritz,12 with composite pulses
for broadband constant rotations (bandwidth (0.60).13 The
duration of the trim pulse, the delay in the BIRD pulse, and
the constant time for J CH evaluations were 2.5, 300, and 3.57
ms, respectively. The MLEV17 spin-lock period was set to 30
2,3
ms for
J
. For 256 t1 increments, 256 transients with 16
C,H
dummy scans were accumulated in 1K data points. Zero-filling
to 1K for F1 and multiplication with squared cosine-bell
windows shifted in both dimensions were performed prior to
2D Fourier transformation. The measuring time was ca. 48 h.
Cultivation conditions and isolation procedures were described
previously.3
7,8-O-Isop r op ylid en e Der iva tive (2) of Am p h id in olid e
E (1). Amphidinolide E (1, 0.4 mg) in acetone (30 µL) was
treated with DMP (10 µL) and PPTS (0.24 mg) at room
temperature for 10 min. After addition of Et3N (0.24 µL) and
evaporation of the solvent, the residue was subjected to a silica
gel column (hexane/EtOAc, 3:1) to afford the 7,8-O-isopropyl-
idene derivative (2, 0.3 mg) of 1 as a colorless oil: 1H NMR
(C6D6) δ 1.05 (3H, d, J ) 6.6 Hz, H3-28), 1.05 (1H, m, H-14),
1.21 (3H, d, J ) 6.7 Hz, H3-27), 1.26 (1H, m, H-15), 1.38 (1H,
m, H-12), 1.53 (1H, m, H-14), 1.53 (3H, s, CH3), 1.54 (3H, s,
CH3), 1.59 (1H, m, H-15), 1.69 (3H, s, H3-30), 1.73 (2H, m, H-11
and H-12), 2.04 (1H, dd, J ) 13.3 and 10.9 Hz, H-20), 2.10
(1H, m, H-11), 2.45 (1H, d, J ) 2.0 Hz, 17-OH), 2.74 (2H, m,
H2-24), 2.76 (1H, m, H-19), 2.82 (1H, dd, J ) 13.3 and 3.3 Hz,
H-20), 3.17 (1H, dq, J ) 9.0 and 6.7 Hz, H-2), 3.31 (1H, m,
H-13), 3.66 (1H, dt, J ) 6.4 and 8.6 Hz, H-16), 3.73 (1H, dd, J
) 8.6 and 2.0 Hz, H-17), 3.98 (1H, t, J ) 8.8 Hz, H-7), 4.09
(1H, t, J ) 8.8 Hz, H-8), 4.89 (1H, d, J ) 8.2 Hz, H-18), 4.86
(1H, brs, H-26), 4.89 (1H, brs, H-26), 4.97 (1H, brs, H-29), 5.05
(1H, brs, H-29), 5.30 (1H, ddd, J ) 15.2, 9.0, and 2.0 Hz, H-9),
5.45 (1H, dd, J ) 14.4 and 9.0 Hz, H-3), 5.47 (1H, dd, J ) 15.0
and 8.8 Hz, H-6), 5.49 (1H, m, H-10), 5.90 (1H, dd, J ) 15.0
and 10.8 Hz, H-5), 5.99 (1H, dt, J ) 15.9 and 7.4 Hz, H-23),
6.00 (1H, dd, J ) 14.4 and 10.8 Hz, H-4), and 6.14 (1H, d, J )
15.9 Hz, H-22); ESIMS m/z 563 (M + Na)+; HRFAB-MS m/z
541.3542 [calcd for C33H49O6 (M + H)+, 541.3529].
7,8,17,18-Di-O-isop r op ylid en e Der iva tive (3b) of Am -
p h id in olid e E (1). Amphidinolide E (1, 0.2 mg) in 1% AcOH/
MeOH (30 µL) was treated with 5% Rh-Al2O3 (0.2 mg) under
an H2 atmosphere at room temperature for 1 h. After filtration
and evaporation of the solvent, the residue in THF solution
(30 µL) was treated with LiAlH4 (0.5 mg) at room temperature
for 1 h. After addition of phosphate buffer (pH 6.85, 100 µL),
the mixture was extracted with EtOAc (200 µL × 3). The
organic phase was evaporated in vacuo to afford a crude
pentaalcohol (3a ). To a solution of 3a in acetone (15 µL) were
added DMP (7.5 µL) and PPTS (0.12 mg), and the mixture was
stirred at room temperature for 1 h. After addition of Et3N
(0.12 µL), the mixture was evaporated, and then the residue
was dissolved in CHCl3 (10 µL). After evaporation, the residue
was subjected to silica gel column chromatography (hexane/
acetone, 6:1) to afford the 7,8,17,18-di-O-isopropylidene deriva-
tive (3b , 0.12 mg) as a colorless oil: 1H NMR (C6D6) δ
0.85-1.00 (15H, m, H3-26, H3-27, H3-28, H3-29, and H3-30),
1.10-2.20 (32H, m), 1.49 (6H, s, CH3 × 2), 1.57 (3H, s, CH3),
1.60 (3H, s, CH3), 3.22 (1H, m, H-1), 3.28 (1H, m, H-1), 3.67
(2H, m, H-7 and H-8), 3.83 (1H, m, H-13), 3.90 (1H, m, H-17),
3.95 (1H, m, H-16), and 4.27 (1H, m, H-18); ESIMS m/z
619 (M + Na)+; HRFAB-MS m/z 597.5056 [calcd for C38H69O6
(M + H)+, 597.5094].
F igu r e 7. Proton signal patterns of H2-17 of the 8,17-bis-(S)-
and (R)-MTPA esters (7a and 7b, respectively) of C-8-C-17
in amphidinolide E (1) and the bis-(S)- and (R)-MTPA esters
(12a and 12b, respectively) of the synthetic C-8-C-17 segment.
C-8-C-17 segment (11), which was converted into the
8,17-bis-(S)- and (R)-MTPA esters (12a and 12b, respec-
tively). Though the bis-(S)- and (R)-MTPA esters (7a and
7b) had very similar NMR profiles, significant differences
were observed for the signal patterns of H2-17 (Figure
1
7). In the H NMR spectrum of the bis-(S)-MTPA ester
(7a ), the methylene protons at C-17 resonated at δH 3.93
(1H, dd, J ) 1.3 and 11.5 Hz, H-17) and 4.19 (1H, dd, J
1
) 6.3 and 11.5 Hz, H-17), while in the H NMR spectrum
of 7b, those at C-17 were observed as a 2H multiplet
signal at δH 4.07 (m) and 4.19 (dd, J ) 3.8 and 11.3 Hz).
The 1H NMR spectrum of the bis-(S)-MTPA ester (7a )
derived from a natural specimen was identical to that of
the bis-(R)-MTPA ester (12b) of the synthetic (13R,16R)-
C-8-C-17 segment, while that of 7b was identical to that
of the synthetic 12a . Thus, the absolute configurations
at C-13 and C-16 were determined to be S.
7,8-Bis-O-p-m eth oxycin n a m oyl Ester (4) of Am p h id i-
n olid e E (1). To a CH2Cl2 solution (15 µL) of amphidinolide
E (1, 0.1 mg) were added DMAP (30 µg), Et3N (1 µL), and
p-methoxycinnanmoyl chloride (0.6 mg) in CH2Cl2 (15 µL) at
room temperature, and stirring was continued for 3 h. After
addition of phosphate buffer (pH 6.85, 50 µL), the reaction
Therefore, the absolute configurations at eight chiral
centers in amphidinolide E (1) were found to be 2R, 7R,
8R, 13S, 16S, 17R, 18R, and 19R.
Exp er im en ta l Section
Gen er a l Meth od s. FABMS spectra were recorded in posi-
tive mode using p-nitrobenzyl alcohol as a matrix. 1H, 13C, and
(12) Wollborn, U.; Leibfritz, D. J . Magn. Reson. 1992, 98, 142-146.
(13) Shaka, A. J .; Pines, A. J . Magn. Reson. 1987, 71, 495-503.

Absolute Stereochemistry of Amphidinolide E
J . Org. Chem., Vol. 67, No. 5, 2002 1655
mixture was extracted with CHCl3 (100 µL × 3), and then the
organic layer was evaporated in vacuo. The residue was
subjected to C18 HPLC (Develosil ODS-HG-5, Nomura Chemi-
cal Ltd., 10 × 250 mm; eluent CH3CN/H2O, 95:5; flow rate,
2.5 mL/min; UV detection at 311 nm) to afford the 7,8-bis-O-
p-methoxycinnamate (4, 0.03 mg, tR 15 min) and 7,8,17-tris-
O-p-methoxycinnamate (0.02 mg, tR 19 min) of 1. 4 (a colorless
oil): UV (MeOH) λmax 310 (ꢀ 60 000) and 228 nm (sh); 1H NMR
(CDCl3) δ 0.93 (3H, d, J ) 6.6 Hz, H3-28), 1.21 (3H, d, J ) 6.7
Hz, H3-27), 1.20-1.30 (2H, m, H-14 and H-15), 1.42-1.52 (2H,
m, H-14 and H-12), 1.62 (1H, m, H-15), 1.71 (3H, s, H3-30),
1.70-1.78 (2H, m, H-20 and H-12), 1.80 (1H, m, H-11), 2.25-
2.31 (2H, m, H-11 and H-19), 2.41 (1H, brd, J ) 13.7 Hz, H-20),
2.76 (1H, dd, J ) 7.2 and 15.4 Hz, H-24), 2.80 (1H, dd, J ) 6.2
and 15.4 Hz, H-24), 3.27 (1H, m, H-2), 3.47 (1H, m, H-13), 3.60
(1H, dt, J ) 7.5 and 7.0 Hz, H-16), 3.72 (1H, brd, J ) 6.6 Hz,
H-17), 3.81 (6H, s, MeO × 2), 4.68 (1H, d, J ) 9.7 Hz, H-18),
4.71 (1H, brs, H-26), 4.74 (1H, brs, H-26), 4.87 (1H, brs, H-28),
4.98 (1H, brs, H-28), 5.32 (1H, dd, J ) 7.9 and 15.4 Hz, H-9),
5.50-5.60 (3H, m, H-7, H-8, and H-6), 5.66-5.78 (2H, m, H-3
and H-23), 5.82 (1H, ddd, J ) 5.3, 8.3, and 14.7 Hz, H-10),
6.05 (1H, d, J ) 15.6 Hz, H-22), 6.22 (1H, d, J ) 15.9 Hz, H-2′),
6.24 (1H, d, J ) 15.9 Hz, H-2′), 6.24 (1H, dd, J ) 10.8 and
15.1 Hz, H-4), 6.38 (1H, dd, J ) 10.8 and 14.4 Hz, H-5), 6.85
(4H, d, J ) 8.9 Hz, H2-6′ and H2-8′), 7.41 (2H, J ) 8.9 Hz,
H-5′ and H-9′), 7.42 (2H, J ) 8.9 Hz, H-5′ and H-9′), 7.59
(1H, d, J ) 15.9 Hz, H-3′), and 7.60 (1H, d, J ) 15.9 Hz, H-3′);
ESIMS m/z 843 (M + Na)+; HRESIMS m/z 843.4062 [calcd
for C50H60O10Na (M + Na)+, 843.4084].
7,8,17-Tr is-(S)-MTP A E st er (5a ) of Am p h id in olid e E
(1). To a CH2Cl2 solution (20 µL) of amphidinolide E (1, 0.1
mg) were added DMAP (20 µg), triethylamine (1.2 µL), and
(R)-(-)-MTPACl (0.6 µL) at room temperature, and stirring
was continued for 6 h. N,N-Dimethyl-1,3-propanediamine (0.6
µL) was added, and the reaction mixture was stirred for 10
min. After addition of phosphate buffer (pH 6.85, 50 µL), the
reaction mixture was extracted with CHCl3 (100 µL × 3), and
then the organic layer was evaporated in vacuo. The residue
was subjected to C18 HPLC (Develosil ODS-HG-5, 10 × 250
mm; eluent CH3CN/H2O, 95:5; flow rate, 2.5 mL/min; UV
detection at 210 nm) to afford the 7,8,17-tris-(S)-MTPA ester
(5a , 0.12 mg) of 1. 5a (a colorless oil): 1H NMR (CDCl3) δ 0.86
(3H, d, J ) 6.7 Hz, H3-28), 1.25 (3H, d, J ) 6.7 Hz, H3-27),
1.35 (1H, m, H-14), 1.45 (1H, m, H-15), 1.56 (1H, m, H-12),
1.67 (1H, m, H-15), 1.70 (3H, s, H3-30), 1.72 (1H, m, H-12),
1.74 (1H, m, H-20), 1.79 (1H, m, H-14), 1.81 (1H, m, H-19),
2.00 (1H, m, H-11), 2.25 (1H, m, H-20), 2.28 (1H, m, H-11),
2.71 (2H, brt, J ) 7.1 Hz, H2-24), 3.24 (1H, m, H-2), 3.31 (1H,
m, H-13), 3.34 (3H, s, MeO), 3.39 (3H, s, MeO), 3.56 (3H, s,
MeO), 3.64 (1H, dt, J ) 6.7 and 9.3 Hz, H-16), 4.67 (1H, brs,
H-26), 4.72 (1H, brs, H-29), 4.74 (1H, brs, H-26), 4.78 (1H, d,
J ) 10.4 Hz, H-18), 4.93 (1H, brs, H-29), 5.21 (1H, dd, J ) 7.4
and 15.6 Hz, H-9), 5.38 (1H, d, J ) 9.3 Hz, H-17), 5.39 (1H,
dd, J ) 9.3 and 15.3 Hz, H-6), 5.54 (1H, dt, J ) 16.0 and 7.1
Hz, H-23), 5.60 (1H, brt, J ) 9.3 Hz, H-7), 5.65 (1H, m, H-8),
5.67 (1H, m, H-3), 5.75 (1H, dt, J ) 15.6 and 7.1 Hz, H-10),
5.96 (1H, d, J ) 16.0 Hz, H-22), 6.17 (1H, dd, J ) 10.4 and
15.3 Hz, H-4), 6.40 (1H, dd, J ) 10.8 and 15.3 Hz, H-5),
7.31-7.47 (14H, m, Ph), and 7.64 (1H, d, J ) 7.8 Hz, Ph);
ESIMS m/z 1171 (M + Na)+; HRFAB MS m/z 1171.4238 [calcd
for C60H65O12F9Na (M + Na)+, 1171.4230].
brs, H-29), 5.05 (1H, dd, J ) 7.1 and 15.6 Hz, H-9), 5.28 (1H,
dd, J ) 9.3 and 15.3 Hz, H-6), 5.36 (1H, d, J ) 9.3 Hz, H-17),
5.45 (1H, dt, J ) 15.6 and 7.1 Hz, H-10), 5.52 (1H, brt, J )
9.3 Hz, H-7), 5.58 (1H, m, H-8), 5.61 (1H, m, H-23), 5.64 (1H,
m, H-3), 6.03 (1H, d, J ) 15.6 Hz, H-22), 6.15 (1H, dd, J )
10.8 and 15.3 Hz, H-4), 6.35 (1H, dd, J ) 10.4 and 15.3 Hz,
H-5), 7.31-7.47 (14H, m, Ph), and 7.61 (1H, d, J ) 7.8 Hz,
Ph); ESIMS m/z 1171 (M + Na)+; HRFAB MS m/z 1171.4207
[calcd for C60H65O12F9Na (M + Na)+, 1171.4230].
Oxid a tive Degr a d a tion of Am p h id in olid e E (1). To a
solution of 3a , which was prepared from amphidinolide E (1,
0.5 mg) by the same procedure as described above, in THF/1
M phosphate buffer (1:2, 180 µL) was added NaIO4 (1.5 mg),
and the mixture was stirred at room temperature for 1 h. After
addition of phosphate buffer (pH 6.85, 100 µL), the mixture
was extracted with EtOAc (200 µL × 3), and the organic layer
was evaporated in vacuo. To a solution of the residue in EtOH
(75 µL) was added NaBH4 (0.5 mg), and stirring was continued
at 0 °C for 30 min. The mixture was partitioned between
EtOAc (200 µL × 3) and phosphate buffer (100 µL). The organic
phase was evaporated, and the residue was dissolved in 1%
DMAP solution in CH2Cl2 (60 µL). To half of the mixture were
added Et3N (4 µL) and (R)-(-)-MTPACl (2 µL), and stirring
was continued at room temperature for 14 h. After addition
of N,N-dimethyl-1,3-propanediamine (2 µL), the solvent was
evaporated in vacuo. The residue was purified by C18 HPLC
(Develosil ODS-HG-5, 10 × 250 mm; eluent CH3CN/H2O, 95:
5; flow rate, 2.5 mL/min; UV detection at 210 nm) to give 6a
(0.08 mg, tR 22 min) and 7a (0.05 mg, tR 27 min) as colorless
oils. 6b (0.07 mg, tR 23 min) and 7b (0.04 mg, tR 27 min) were
prepared by treatment of the other half of the NaBH4 reduction
product with (S)-(+)-MTPACl, and then HPLC separation was
carried out as described above. 6a (a colorless oil): 1H NMR
(CDCl3) δ 0.90 (3H, d, J ) 6.7 Hz, H3-27), 1.12 (1H, m, H-3),
1.17-1.35 (5H, H-3, H2-4, and H2-5), 1.66 (2H, brt, J ) 6.6
Hz, H2-6), 1.80 (1H, m, H-2), 3.54 (6H, s, MeO), 4.11 (1H, dd,
J ) 5.7 and 10.7 Hz, H-1), 4.16 (1H, dd, J ) 6.5 and 10.7 Hz,
H-1), 4.27 (1H, m, H-7), 4.31(1H, m, H-7), 7.45-7.43 (6H, m,
Ph), and 7.46-7.55 (4H, m, Ph); ESIMS m/z 601 (M + Na)+;
HRESIMS m/z 601.2003 [calcd for C28H32O6F6Na (M + Na)+,
601.2001]. 6b (a colorless oil): 1H NMR (CDCl3) δ 0.88 (3H, d,
J ) 6.7 Hz, H3-27), 1.10 (1H, m, H-3), 1.18-1.33 (5H, m, H-3,
H2-4, and H2-5), 1.65 (2H, brt, J ) 6.9 Hz, H2-6), 1.79 (1H, m,
H-2), 3.52 (6H, s, MeO), 4.04 (1H, dd, J ) 6.5 and 10.7 Hz,
H-3), 4.19 (1H, dd, J ) 5.8 and 10.7 Hz, H-3), 4.25 (1H, dt, J
) 10.9 and 6.6 Hz, H-7), 4.30 (1H, dt, J ) 10.9 and 6.6 Hz,
H-7), 7.33-7.40 (6H, m, Ph), and 7.53-7.57 (4H, m, Ph);
ESIMS m/z 601 (M + Na)+; HRESIMS m/z 601.1991 [calcd
for C28H32O6F6Na (M + Na)+, 601.2001]. 7a (a colorless oil):
1H NMR (C6D6) δ 1.05-1.28 (6H, m), 1.30-1.52 (6H, m), 3.46
(3H, s, MeO), 3.52 (1H, m, H-13), 3.52 (3H, s, MeO), 3.81 (1H,
m, H-16), 3.93 (1H, dd, J ) 1.3 and 11.5 Hz, H-17), 4.01 (1H,
m, H-8), 4.05 (1H, m, H-8), 4.19 (1H, dd, J ) 6.3 and 11.5 Hz,
H-17), 7.05-7.17 (6H, m, Ph), 7.72 (2H, d, J ) 7.7 Hz, Ph),
and 7.78 (2H, d, J ) 7.5 Hz, Ph); ESIMS m/z 643 (M + Na)+;
HRESIMS m/z 643.2103 [calcd for C30H34O7F6Na (M + Na)+,
643.2107]. 7b (a colorless oil): 1H NMR (C6D6) δ 1.05-1.14
(4H, m), 1.18-1.25 (2H, m), 1.38-1.51 (6H, m), 3.46 (3H, s,
MeO), 3.51 (3H, s, MeO), 3.53 (1H, m, H-13), 3.83 (1H, m,
H-16), 4.01 (1H, m, H-8), 4.03-4.08 (2H, m, H-8 and H-17),
4.17 (1H, dd, J ) 3.8 and 11.3 Hz, H-17), 7.05-7.18 (6H, m,
Ph), 7.73 (2H, d, J ) 7.6 Hz, Ph), and 7.77 (2H, d, J ) 7.8 Hz,
Ph); ESIMS m/z 643 (M + Na)+; HRESIMS m/z 643.2120 [calcd
for C30H34O7F6Na (M + Na)+, 643.2107].
7,8,17-Tr is-(R)-MTP A Ester (5b) of Am p h id in olid e E
(1). Amphidinolide E (1, 0.1 mg) was treated with (S)-(+)-
MTPACl (0.6 µL) by the same procedure as described above
to afford the bis-(R)-MTPA ester (5b, 0.15 mg) of 1. 5b
(colorless oil): 1H NMR (CDCl3) δ 0.90 (3H, d, J ) 6.7 Hz,
H3-28), 1.29 (3H, d, J ) 6.7 Hz, H3-27), 1.29 (1H, m, H-14),
1.41 (2H, m, H-12 and H-15), 1.58 (1H, m, H-12), 1.63 (1H, m,
H-15), 1.69 (3H, s, H3-30), 1.71 (1H, m, H-14), 1.80 (1H, dd, J
) 10.4 and 13.8 Hz, H-20), 1.82 (1H, m, H-11), 2.00 (1H, m,
H-19), 2.09 (1H, m, H-11), 2.33 (1H, dd, J ) 3.4 and 13.4 Hz,
H-20), 2.72 (2H, m, H2-24), 3.17 (1H, m, H-13), 3.26 (1H, m,
H-2), 3.43 (3H, s, MeO), 3.46 (3H, MeO), 3.46 (1H, m, H-16),
3.53 (3H, s, MeO), 4.67 (1H, brs, H-26), 4.73 (1H, brs, H-26),
4.81 (1H, d, J ) 10.4 Hz, H-18), 4.82 (1H, s, H-29), 4.99 (1H,
2-[(2′S,5′R)-5′-Ben zyloxym eth yltetr a h yd r ofu r a n -2′-yl]-
eth a n -1-ol (9). To a suspension of NaH (283 mg, 7.08 mmol,
60% oil dispersion) in DMF (1.6 mL) and THF (2.7 mL) was
added a solution of (2R,5S)-{5-[2′-(tert-butyldimethylsilyloxy)-
ethyl]tetrahydrofuran-2-yl}methanol (8, 1.00 g, 3.84 mmol) in
THF (0.7 mL) at 0 °C. After stirring at 0 °C for 30 min and
then at room temperature for 2 h, benzyl bromide (0.6 mL,
5.1 mmol) was added at 0 °C, and the mixture was stirred at
room temperature for 18 h. After addition of MeOH (0.4 mL)
and then H2O (11 mL), the mixture was extracted with ether
(20 mL × 3). The organic phase was dried with MgSO4 and

1656 J . Org. Chem., Vol. 67, No. 5, 2002
Kubota et al.
concentrated to give a crude benzyl ether (1.30 g). To the crude
product was added the mixture (60 mL) AcOH/H2O/THF (1:
1:1), and the solution was stirred at room temperature for 6
h. After extraction with CHCl3, the organic phase was dried
with MgSO4, evaporated, and purified by silica gel column
chromatography (hexane/EtOAc, 9:1) to yield benzyl alcohol
9 (809 mg, 3.43 mmol, 89% by two steps) as a colorless oil:
evaporation, the residue was subjected to silica gel column
chromatography (CHCl3/MeOH, 100:0 to 80:20) to afford 11
20
(46.5 mg, 247 µmol, 89.8%) as a colorless oil; [R]D -10.8° (c
1.00, CHCl3); IR (neat) ν˜max 3369, 2933, 2859, 1461, and 1044
cm-1; 1H NMR (CDCl3) δ 1.26-1.71 (10H, m), 1.81-1.98 (2H,
m), 3.45 (1H, dd, J ) 5.9 and 11.5 Hz), 3.58 (2H, t, J ) 6.5
Hz), 3.63 (1H, dd, J ) 3.1 and 11.5 Hz), 3.83 (1H, m), and
3.95 (1H, m); 13C NMR (CDCl3) δ 25.7 (t), 25.9 (t), 27.0 (t),
31.2 (t), 32.5 (t), 35.7 (t), 62.5 (t), 65.2 (t), 79.4 (d), and 80.0
(d); FAB-MS m/z 189 (M + H)+; HRFAB MS m/z 189.1487
[calcd for C10H21O3 (M + H)+, 189.1491].
22
[R]D +10.1° (c 1.00, CHCl3); IR (neat) ν˜max 3439, 3062, 3030,
2939, 2869, 1494, 1453, 1365, and 1078 cm-1; 1H NMR (CDCl3)
δ 1.55-2.05 (6H, m), 3.48 (2H, m), 3.78 (2H, t, J ) 5.6 Hz),
4.05-4.15 (2H, m), 4.55 (1H, d, J ) 12.5 Hz), 4.57 (1H, d, J )
12.5 Hz), and 7.25-7.37 (5H, m); 13C NMR (CDCl3) δ 27.8 (t),
31.0 (t), 37.5 (t), 61.2 (t), 72.8 (t), 73.3 (t), 78.4 (d), 79.8 (d),
127.6 (d), 127.7 (2C, d), 128.3 (2C, d), and 138.2 (s); FAB MS
m/z 237 (M + H)+; HRFAB-MS m/z 237.1473 [calcd for
Bis-(S)-MTP A Ester (12a ) of 11. To a CH2Cl2 solution (30
µL) of 11 (0.5 mg, 2.7 nmol) were added DMAP (30 µg), Et3N
(2 µL), and (R)-(-)-MTPACl (1 µL) at room temperature, and
stirring was continued for 6 h. N,N-Dimethyl-1,3-propanedi-
amine (1 µL) was added, and the reaction mixture was stirred
for 10 min. After addition of phosphate buffer (pH 6.85, 50
µL), the reaction mixture was extracted with CHCl3 (100 µL
× 3), and then the organic layer was evaporated in vacuo. The
residue was purified by C18 HPLC (Develosil ODS-HG-5, 10
× 250 mm; eluent CH3CN/H2O, 95:5; flow rate, 2.5 mL/min;
UV detection at 210 nm) to give 12a (1.5 mg, 2.4 nmol, 89%,
tR 27 min) as a colorless oil; 1H NMR (C6D6) δ 1.05-1.14 (4H,
m), 1.18-1.25 (2H, m), 1.38-1.41 (4H, m), 1.42-1.51 (2H, m),
3.46 (3H, s, MeO), 3.51 (3H, s, MeO), 3.53 (1H, m, H-13), 3.83
(1H, m, H-18), 4.01 (1H, m, H-8), 4.03-4.08 (2H, m, H-8 and
H-17), 4.17 (1H, dd, J ) 3.8 and 11.3 Hz, H-17), 7.05-7.18
(6H, m, Ph), 7.73 (2H, d, J ) 7.6 Hz, Ph), and 7.77 (2H, d, J
) 7.8 Hz, Ph); FAB-MS m/z 621 (M + H)+; HRFAB MS m/z
621.2274 [calcd for C30H35O7F6 (M + H)+, 621.2287].
Bis-(S)-MTP A Ester (12b) of 11. 11 (0.5 mg, 2.7 nmol)
was treated with (S)-(+)-MTPACl (1 µL) by the same procedure
as described above to afford the bis-(R)-MTPA ester (12b, 1.6
mg, 2.6 nmol, 96%) of 11. 12b (a colorless oil): 1H NMR (C6D6)
δ 1.05-1.28 (6H, m), 1.30-1.40 (4H, m), 1.42-1.52 (2H, m),
3.46 (3H, s, MeO), 3.52 (1H, m, H-13), 3.52 (3H, s, MeO), 3.81
(1H, m, H-16), 3.93 (1H, dd, J ) 1.3 and 11.5 Hz, H-17), 4.01
(1H, m, H-8), 4.05 (1H, m, H-8), 4.19 (1H, dd, J ) 6.3 and
11.5 Hz, H-17), 7.05-7.17 (6H, m, Ph), 7.72 (2H, d, J ) 7.7
Hz, Ph), and 7.78 (2H, d, J ) 7.5 Hz, Ph); FAB-MS m/z 643
(M + H)+; HRFAB MS m/z 621.2275 [calcd for C30H35O7F6 (M
+ H)+, 621.2287].
C
14H21O3 (M + H)+, 237.1491].
(2R,5R)-2-Ben zyloxym eth yl-5-(5′-ben zyloxypen t-3′-yn yl)-
tetr a h yd r ofu r a n (10). To a solution of 9 (200 mg, 847 µmol)
in toluene (8 mL) were added imidazole (143 mg, 2.10 mmol),
triphenylphosphine (552 mg, 2.10 mmol), and iodine (407 mg,
1.60 mmol), and stirring was continued at room temperature
for 30 min. The mixture was washed with saturated aqueous
Na2SO3 and brine, dried with MgSO4, evaporated, and purified
by silica gel column chromatography (hexane/EtOAc, 10:1) to
yield an iodide (220 mg, 636 µmol, 75.1%) as a colorless oil:
1H NMR (CDCl3) δ 1.52 (1H, m), 1.70 (1H, m), 1.88-2.12 (4H,
m), 3.25 (2H, m), 3.47 (2H, m), 3.94 (1H, m), 4.10 (1H, m),
4.56 (1H, d, J ) 12.5 Hz), 4.59 (1H, 12.5 Hz), and 7.25-7.39
(5H, m); 13C NMR (CDCl3) δ 24.5 (t), 27.8 (t), 30.3 (t), 39.9 (t),
72.7 (t), 73.1 (t), 78.1 (d), 79.3 (d), 127.3 (d), 127.4 (2C, d), 128.1
(2C, d), and 138.2 (s). To a solution of propyn-2-ynyloxymeth-
ylbenzene (187 mg, 1.28 mmol) in THF (3.2 mL) was added a
2.46 M solution of n-BuLi in hexane (546 µL, 134 mmol) at
-78 °C, and the mixture was then allowed to warm to -20 °C
over 2.5 h. To this mixture was added a solution of the iodide
(220 mg, 636 µmol) in HMPA (1.12 mL) and THF (1 mL) at
-78 °C. The mixture was allowed to warm to room tempera-
ture, and stirring was continued for 16 h. The reaction was
quenched with the addition of saturated aqueous NH4Cl (5
mL), and the mixture was extracted with Et2O (10 mL × 3).
The extract was washed with H2O and brine, dried with
MgSO4, evaporated, and purified by silica gel column chro-
matography (hexane/EtOAc, 10:1 to 8:2) to yield the acetylene
compound 10 (150.4 mg, 413 µmol, 65.0%) as a colorless oil:
Ack n ow led gm en t. We thank Professor N. Harada
(Tohoku University) for his suggestion of a CD exciton
chromophore. This work was partly supported by a
Grant-in-Aid from the Uehara Memorial Foundation, a
Grant-in-Aid from the Fujisawa Foundation, and a
Grant-in-Aid for Scientific Research from the Ministry
of Education, Culture, Sports, Science, and Technology
of J apan. T. K. thanks Research Fellowships of the
J apan Society for the Promotion of Science for Young
Scientists.
22
[R]D +7.5° (c 1.00, CHCl3); IR (neat) ν˜max 3062, 3029, 2940,
2857, 2222, 1495, 1452, 1356, and 1073 cm-1; 1H NMR (CDCl3)
δ 1.55 (1H, m), 1.73 (2H, m), 1.83 (1H, m), 1.98 (2H, m), 2.39
(2H, m), 3.49 (2H, m), 4.00 (1H, m), 4.11 (1H, m), 4.17 (2H,
m), 4.53-4.65 (4H, m), and 7.26-7.43 (10H, m); 13C NMR
(CDCl3) δ 15.6 (t), 28.0 (t), 30.5 (t), 34.7 (t), 57.5 (t), 71.1 (t),
72.9 (t), 73.1 (t), 75.8 (s), 77.9 (d), 78.4 (d), 86.6 (s), 127.3 (d),
127.4 (2C, d), 127.5 (d), 127.8 (2C, d), 128.1 (2C, d), 128.2 (2C,
d), 137.5 (s), and 138.2 (s); FAB-MS m/z 365 (M + H)+;
HRFAB MS m/z 365.2097 [calcd for C24H29O3 (M + H)+,
365.2117].
5-[(2′R,5′R)-5′-H yd r oxym et h ylt et r a h yd r ofu r a n -2′-yl]-
p en ta n -1-ol (11). To a solution of 10 (100 mg, 275 µmol) in
EtOH (2.5 mL) was added 5% Pd/C (100 mg), and stirring was
continued under an H2 atmosphere at room temperature for
16 h. After filtration of insoluble materials followed by
Su p p or tin g In for m a tion Ava ila ble: 1H NMR spectra of
1, 2, 4, 5a and b, 6a and b, 7a and b, and 12a and b. This
material is available via the Internet at http://pubs.acs.org.
J O016326N