The structures of three new shishididemniols from a tunicate of the family Didemnidae

Article abstract of DOI:10.1016/j.tet.2007.04.081

Three new serinolipid derivatives, shishididemniols C (1), D (2), and E (3), were isolated as antibacterial constituents of a tunicate of the family Didemnidae. Their planar structures were elucidated by interpretation of NMR and MS data, whereas the abso

Full text of DOI:10.1016/j.tet.2007.04.081

Tetrahedron 63 (2007) 6748–6754
The structures of three new shishididemniols from
a tunicate of the family Didemnidae
Hirotsugu Kobayashi,a Yoshinari Miyata,a Kohtaro Okada,a Tsuyoshi Fujita,b
Takashi Iwashita,b Yoichi Nakao,a Nobuhiro Fusetania and Shigeki Matsunagaa,
*
aLaboratory of Aquatic Natural Products Chemistry, Graduate School of Agricultural and Life Sciences,
The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan
bSuntory Institute for Bioorganic Research, Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka 618-8503, Japan
Received 5 April 2007; revised 24 April 2007; accepted 24 April 2007
Available online 29 April 2007
Abstract—Three new serinolipid derivatives, shishididemniols C (1), D (2), and E (3), were isolated as antibacterial constituents of a tunicate
of the family Didemnidae. Their planar structures were elucidated by interpretation of NMR and MS data, whereas the absolute stereo-
chemistry was determined by chemical conversions. Shishididemniols C (3), D (4), and E (5) exhibited antibacterial activity against the
fish pathogenic bacterium Vibrio anguillarum.
Ó 2007 Published by Elsevier Ltd.
1. Introduction
octopamyl amide of the C28-polyoxygenated carboxylic acid
whose both termini are each etherified by a serinol moiety.4
Further separation of the antibacterial fraction of the extract
of the tunicate afforded three new congeners, shishididem-
niols C (3), D (4), and E (5) (Fig. 1). In the present paper we
report the isolation, structure elucidation, and antimicrobial
activity of these new metabolites.
Several polyoxygenated C28-carboxylic acid derivatives with
a serinol ether moiety, such as didemniserinolipids1,2 and
cyclodidemniserinol trisulfate,3 have been reported from tu-
nicates of the family Didemnidae. Recently, we reported the
structures of shishididemniols A (1) and B (2), which are
O
O
HN 12' R
HN 12' R
OH
5'
5'
6'
6'
O
OH
O
OH
OH
4'
3'
4'
3'
9'
11'
30
9'
11'
NH2
O
HO
HN
HO
HN
7'
7'
NH2
30
8'
8'
O
1'
1'
OH
OH
O
28
H
H
28
1
1
2
6
6
2
16
16
O
O
5
9
5
9
OH
OH
OH
OH Cl
2: R = Et
4: R = Me
OH
1: R = Et
3: R = Me
O
13'
HN 12'
16'
15'
5'
8'
O4'
6'
O
OH
OH
9'
11'
NH2
3'
O
7'
30
1'
OH
O
HN
O
H
H
28
1
6
2
16
O
5
9
OH
OH
5
Figure 1. Structures of shishididemniols A (1)—E (5).
Keywords: Antibacterial; Serinolipid; Tunicate; Vibrio anguillarum.
* Corresponding author. Fax: +81 3 5841 8166; e-mail: assmats@mail.ecc.u-tokyo.ac.jp
0040–4020/$ - see front matter Ó 2007 Published by Elsevier Ltd.
doi:10.1016/j.tet.2007.04.081

H. Kobayashi et al. / Tetrahedron 63 (2007) 6748–6754
6749
Table 1. 13C NMR data for shishididemniols C (3), D (4), and E (5) in
DMSO-d6
2. Results and discussion
a
ODS HPLC of an antibacterial fraction obtained by ODS
flash chromatography4 afforded two peaks in addition to
the peaks that contained 1 and 2: one of the peak contained
shishididemniol C (3), whereas another peak was a mixture
of two compounds. The latter HPLC peak was further sepa-
rated by ODS HPLC using MeCN as the organic component
of the mobile phase to give shishididemniols D (4) and E (5).
Position
3
4
5
dC (mult.b J in Hz)
1
2
3
4
5
6
7
8
173.9 (s)
70.8 (d)
34.4 (t)
20.4 (t)
33.7 (t)
68.7 (d)
60.5 (d)
56.2 (d)
27.9 (t)
28.9–29.3
37.2 (t)
69.6 (d)
37.2 (t)
28.8–29.3
25.3 (t)
28.9 (t)
70.8 (t)
67.5 (t)
52.0 (d)
58.8 (t)
46.2 (t)
70.8 (d)
135.6 (s)
127.1 (d)
114.1 (d)
157.7 (s)
66.5 (t)
50.3 (d)
60.1 (t)
169.4 (s)
22.7 (q)
173.9 (s)
70.9 (d)
34.4 (t)
21.0 (t)
32.6 (t)
70.9 (d)
75.6 (d)
66.3 (d)
34.4 (t)
29.1–29.3
37.2 (t)
69.5 (d)
37.2 (t)
29.1–29.3
25.5 (t)
28.9 (t)
70.7 (t)
68.5 (t)
52.1 (d)
59.6 (t)
46.2 (t)
70.8 (d)
135.7 (s)
127.1 (d)
114.0 (d)
157.6 (s)
66.4 (t)
50.3 (d)
60.0 (t)
169.3 (s)
22.7 (q)
174.2 (s)
70.8 (d)
34.4 (t)
20.3 (t)
33.7 (t)
68.6 (d)
60.5 (d)
56.2 (d)
27.8 (t)
28.9–29.3
37.2 (t)
69.5 (d)
37.2 (t)
28.9–29.3
25.7 (t)
28.9 (t)
70.4 (t)
72.9 (t)
52.5 (d)
63.7 (t)
43.0 (t)
73.4 (d)
130.5 (s)
127.8 (d)
114.4 (d)
158.3 (s)
66.5 (t)
50.2 (d)
60.0 (t)
173.1 (s)
28.4 (t)
9.90 (q)
169.7 (s)
20.9 (q)
Shishididemniol C (3) had a molecular formula of
C44H79N3O11, which was smaller than that of shishididem-
9
1
niol A (1) by a CH2 unit. The H and 13C NMR spectra of
10–14
15
16
17
18–25
26
27
28
29
30
31
10
3 were almost superimposable on those of 1 except for the
replacement of the ethyl group by a methyl group (dH 1.82
and dC 22.7). Interpretation of the NMR and FABMS/MS
data of 3 led to its gross structure as the lower homolog of
shishididemniol A (Fig. 2).
The relative stereochemistry of the part from C-6 to C-8 in 3
was assigned as threo,cis (6S*,7R*,8S*) by comparison of
1H and 13C NMR data with those of 1 (Tables 1 and 2, and
Ref. 4). The absolute stereochemistry of C-2, C-6, C-30,
C-20, and C-100 in 3 was determined by application of the
modified Mosher’s method.5,6 Treatment of 3 with (S)- and
(R)-MTPACl yielded the (R)- and (S)-MTPA derivatives
(6a and 6b, respectively). Analysis of DdSꢀR values between
6a and 6b allowed the assignment of 6S and 30S.
20
30
40,80
50,70
60
90
100
110
120
130
140
150
160
The absolute stereochemistry of C-2, C-20, and C-100 in 3
was determined by comparison of H NMR spectra of 6a
1
and 6b with those of (R)- and (S)-MTPA derivatives of 1.
1H NMR chemical shift values of H2-3, H2-4, and H2-5 of
6a and 6b were in good agreement with those of the corre-
sponding signals of (R)- and (S)-MTPA derivatives of 1,
respectively, indicating that the absolute stereochemistry
of C-2 was S. Absolute stereochemistry of C-20 and C-100
was assigned as R and S, respectively, because the chemical
shift values of H2-10, H-20, H-40/80, H-50/70, H2-90, H-100, and
H2-110 were similar to those of the corresponding signals of
(R)- and (S)-MTPA derivatives of 1 (Fig. 3).4
13C NMR: 150 MHz.
Multiplicity from HSQC experiment.
a
b
The absolute stereochemistry of C-16 in 3 was assigned by
using the same method that was applied to 1. For that pur-
pose 3 was converted to its N-Cbz derivative 7, which was
treated with MgBr2$Et2O in THF to yield the bromohydrin
O
A
HN
O
12'OH
5'
6'
COSY, TOCSY
HMBC
4'
3'
9'
11'
7'
HO
8'
1'
OH
HN1
O
28
2
30
6
O
O
OH
16
OH
OH
NH2
O
B
HN
O
O
OH
HO
HN
244
428
372 344
288
216 188 160
OH
O
O
OH
OH
OH
NH2
458
358 330 302 258 230 202 174 146
Figure 2. (A) Key 2D NMR correlations of 3. (B) FABMS/MS analysis of 3.

6750
H. Kobayashi et al. / Tetrahedron 63 (2007) 6748–6754
Table 2. 1H NMR data for shishididemniols C (3), D (4), and E (5) in
DMSO-d6
a
A
HN
7.23
7.13
5'
1.33 1.62
1.15 1.54
1.58
1.44
3.62
3.55
O
OMTPA
11'
4.73
9'
3.88
Position
3
4
5
O
4.60
4.72
4'
5.07
5.07
1.45 1.84
1.26 1.74
1.75
1.62
4.92
5.00
6
3.68
3.55
6.03
4.62
3.58
6.55
6.00
4.15
3.98
4.53
3.74
dH (mult. J in Hz)
6.67
N
H
2'
2
8'
OMTPA
2
3.80 (br)
1.42 (m)
1.55 (m)
1.32 (m)
1.42 (m)
1.30 (m)
1.41 (m)
3.19 (br)
2.69 (dd, 4.6, 8.5)
3.80 (br)
1.43 (m)
1.56 (m)
1.31 (m)
1.43 (m)
1.30 (m)
1.37 (m)
3.45 (m)
3.27 (t, 4.4)
3.79 (br)
1.42 (m)
1.52 (m)
1.33 (m)
1.42 (m)
1.28 (m)
1.40 (m)
3.19 (br)
2.69 (dd,
4.4, 8.3)
2.87 (m)
OMTPA
OMTPA
3a
3b
4a
4b
5a
5b
6
B
HN
7.26
5'
1.58 1.31 1.65
3.63
3.56
O
11' OMTPA
4.73
7.14
9'
3.93
1.46 1.15 1.51
1.75 1.45 1.86
1.62 1.26 1.74
O
4.70
4.76
4'
4.92
5.00
6
3.67
3.56
5.00
6.04
4.54
3.74
6.61
5.08
6.01
4.59
3.86
4.11
3.97
(S)-MTPA
(R)-MTPA
6.67
N
2'
8'
2
H
OMTPA
OMTPA
OMTPA
7
Figure 3. (A) 1H NMR chemical shifts values for 6a (lower numbers in red)/
6b (upper numbers in blue). (B) 1H NMR chemical shift values for
(R)-MTPA derivative of 1 (lower numbers in red)/(S)-MTPA derivative of
1 (upper numbers in blue).
8
2.87 (ddd, 4.6,
4.6, 8.5)
1.29 (m)
3.99 (m)
9a
9b
1.67 (m)
1.77 (m)
1.28 (m)
1.53 (m)
1.53 (m)
10–14
15
16
1.15–1.45 (m)
1.19–1.35 (m)
3.33 (m)
1.19–1.35 (m)
1.15–1.45 (m)
1.27 (m)
1.49 (m)
3.39 (m)
3.43 (m)
3.50 (m)
1.17–1.40 (m)
1.20–1.34 (m)
3.33 (m)
1.20–1.34 (m)
1.17–1.40 (m)
1.26 (m)
1.49 (m)
3.38 (m)
3.45 (m)
3.51 (m)
1.15–1.45 (m)
1.19–1.35 (m)
3.33 (m)
1.19–1.35 (m)
1.15–1.45 (m)
1.25 (m)
1.45 (m)
3.32 (m)
3.14 (m)
3.26 (m)
NMR spectra of 4 coincided well with those of 3 except
for the replacement of the epoxide group by an oxymethine
and a chlorinated methine, suggesting that 4 was a HCl ad-
duct of 3. The planar structure of 4 was determined on the
basis of analysis of NMR and FABMS/MS data (Tables 1
and 2).
17
18–25
26
27
28
29a
29b
30
31a
31b
3.22 (m)
3.48 (m)
3.56 (dd, ꢀ11.5, 4.6) 3.51 (m)
3.14 (m)
3.45 (m)
2.77 (m)
3.20 (m)
The relative stereochemistry from C-6 to C-8 in 4 was as-
1
signed as 6S*,7R*,8R* by comparison of the H and 13C
3.32 (dd,
ꢀ11.5, 4.6)
NMR data with those of 2 (Tables 1 and 2, and Ref. 4). In
order to determine the absolute stereochemistry of 4, its N-
Cbz derivative 12 was treated with potassium carbonate in
MeOH. The optical rotation and the NMR data for the prod-
uct ([a]1D9 ꢀ43.3 (c 0.10, MeOH)) were indistinguishable
from those of compound 7 derived from 3 ([a]1D9 ꢀ43.7 (c
0.10, MeOH)) (Scheme 2).
10a
3.11 (m)
3.31 (m)
4.55 (br)
7.22 (d, 8.7)
6.88 (d, 8.7)
3.93 (dd, ꢀ9.6, 6.0)
3.95 (dd, ꢀ9.6, 5.5)
4.02 (m)
3.12 (m)
3.31 (m)
4.55 (br)
7.22 (d, 8.6)
6.89 (d, 8.6)
3.93 (m)
3.95 (m)
4.02 (m)
3.46 (m)
3.50 (m)
1.82 (s)
3.32 (m)
3.49 (m)
5.72 (br)
10b
20
40,80
50,70
90a
7.23 (d, 8.0)
6.92 (d, 8.0)
3.94 (m)
3.95 (m)
4.02 (m)
3.45 (m)
3.49 (m)
2.10 (q, 7.6)
0.97 (t, 7.6)
1.99 (s)
90b
100
110a
110b
130
3.46 (m)
3.49 (m)
1.82 (s)
In order to confirm the stereochemistry of C-16, which was
remote from other stereogenic centers, 12 was converted to
the (R)- or (S)-2NMA esters (15a and 15b, respectively) in
three steps (Scheme 2).9 The positive DdRꢀS values observed
for H2-7 to H2-10 signals confirmed the 16R stereochemistry
(Fig. 4). Therefore, the absolute stereochemistry of 4 was
determined to be 2S,6S,7R,8R,16R,30S,20R,100S.
140
160
OH-2
OH-6
OH-7
OH-16
NH2-30 7.78 (br)
OH-31
NH-10
5.49 (d, 4.1)
5.22 (br)
5.48 (d, 4.1)
4.19b (br)
4.85b (br)
4.19b (br)
6.97 (br)
5.48 (d, 4.1)
4.88 (br)
4.17 (br)
4.20 (br)
7.77 (br)
4.46 (br)
5.22 (br)
7.52 (dd, 6.5, 5.5)
4.85b (br)
Shishididemniol E (5) had a molecular formula of
C47H83N3O12 as established by HRESIMS, indicating that
7.50 (dd, 6.1, 5.5) 7.76 (dd,
6.1, 5.8)
5.42 (br)
1
OH-20
NH-100
OH-110
5.40 (d, 3.2)
7.85 (d, 7.8)
4.83 (br)
a hydrogen in 1 was replaced by an acetyl group. The H
and 13C NMR spectra of 5 were almost superimposable on
those of 1 except for the presence of an additional singlet
methyl signal (dH 1.99 and dC 20.9) and deshielding of
H-20 signal (d 5.72). The structure of 5 was determined as
C-20 acetyl shishididemniol A (1), on the basis of 2D
NMR and FABMS/MS data (Tables 1 and 2).
7.86 (d, 7.7)
7.79 (d, 7.7)
4.90 (br)
5.10b (br)
1H NMR: 600 MHz.
These signals may be interchangeable.
a
b
8.7,8 Compound 8 was treated with NaIO4 followed by
reduction with NaBH4 to yield 9, whose two primary alco-
hols were protected by TBDPS groups to afford 10. The sec-
ondary alcohol in 10 was converted to (R)-2NMA ester 11
We sought to determine the stereochemistry of 5 by
converting both 1 and 5 to a common derivative. Com-
pound 1 was treated with Ac2O in the presence of triethyl-
amine to yield the peracetyl derivative 16. However,
acetylation of 5 under the same condition afforded a com-
plex mixture. Therefore, we took an alternative method of
acetylation. Compound 5 was reacted with acetic acid in
the presence of EDC, DMAP, and DMAP$HCl in CH2Cl2
to give 16 (Scheme 3): the optical rotation value of 16
derived from 5 and 1 were identical within experimental
(Scheme 1).9 The H NMR chemical shift values of 11
1
derived from 3 were indistinguishable from those of 11 de-
rived from 1 indicating that the absolute configuration at
C-16 was R.4 Therefore, the stereochemistry of 3 was
2S,6S,7R,8S,16R,30S,20R,100S.
Shishididemniol D (4) had a molecular formula of
C44H80ClN3O11 as established by HRESIMS. H and 13C
1

H. Kobayashi et al. / Tetrahedron 63 (2007) 6748–6754
6751
O
3
NH
NH
(a)
HO
HO
O
O
OH
OH
O
O
NHCbz
O
H
H
11'
2
O
O
OH
6
30
N
H
5
5
10
10
OH
OH
OH
OH
O
7
(b)
NHCbz
HO
6
Br
11'
2
OH
30
N
H
OH
OH
8
(c)
Br
OR2
NHCbz
OR1
7
R1O
O
30
16
5
10
9: R1 = R2 = H
(d)
(e)
10: R1 = TBDPS, R2 = H
11: R1 = TBDPS, R2 = (R)-2NMA
Scheme 1. Reagents and conditions: (a) Cbz–Cl, Et3N, CHCl3/MeOH (3:1), rt, 16 h; (b) MgBr2$OEt, THF, rt, 3 h; (c) NaIO4, MeOH/H2O (4:1), rt, 1 h then
NaBH4, rt, 15 min; (d) TBDPSCl, pyridine, rt, overnight; and (e) (R)-2NMA, EDC, DMAP$HCl, CH2Cl2, rt, overnight.
O
4
NH
(a)
HO
O
O
OH
11'
2
16
30
6
N
H
O
OH
5
10
OH
OH
OH
OH
Cl
(c)
NHCbz
12
(b)
7
Cl
OR2
NHCbz
OR1
7
R1O
O
16
31
5
10
13: R1 = R2= H
14: R1 = TBDPS, R2 = H
(d)
(e)
15a: R1 = TBDPS, R2 = (R)-2NMA
15b: R1 = TBDPS, R2 = (S)-2NMA
Scheme 2. Reagents and conditions: (a) Cbz–Cl, Et3N, CHCl3/MeOH (3:1), rt, 16 h; (b) K2CO3, MeOH, rt, 2.5 h; (c) NaIO4, MeOH/H2O (4:1), rt, 1 h then
NaBH4, rt, 15 min; (d) TBDPSCl, pyridine, rt, overnight; and (e) (R)- or (S)-2NMA, EDC, DMAP, DMAP$HCl, CH2Cl2, rt, overnight.
errors. Therefore, 5 was the 20-acetate of shishididemniol
A (1).
2NMA
Cl
O
+0.12
+0.10
Shishididemniols C (3), D (4), and E (5) exhibited antibac-
terial activity in disk agar diffusion assay against the fish
pathogenic bacterium Vibrio anguillarum (20 mg/6.5 mm f
disk, zone of inhibition; 7.5, 7.0, and 7.0 mm for 3, 4, and
5, respectively).
TBDPSO
7
16
+0.02
3
+0.01
+0.06
+0.06
+0.00
Figure 4. Distribution of DdRꢀS values for the 2NMA derivatives 15a/15b.
1
5
O
(a)
(b)
NH
AcO
O
Ac
O
OAc
HN
O
H
11'
H
2
O
OAc
6
N
H
31
5
10
OAc
OAc
OAc
16
Scheme 3. Reagents and conditions: (a) Ac2O, Et3N, CH2Cl2, rt, overnight and (b) AcOH, EDC, DMAP, DMAP$HCl, rt, 24 h.

6752
H. Kobayashi et al. / Tetrahedron 63 (2007) 6748–6754
3. Experimental
H-3a), 1.84 (m, 1H; H-3b), 1.26 (m, 1H; H-4a), 1.45 (m, 1H;
H-4b), 1.62 (m, 1H; H-5a), 1.75 (m, 1H; H-5b), 5.00 (m,
1H; H-6), 2.90 (dd, J¼8.9, 4.1, 1H; H-7), 2.98 (m, 1H;
H-8), 5.08 (m, 1H; H-16), 1.27 (m, 2H; H2-26), 1.46 (m, 2H;
H2-27), 3.32 (m, 2H; H2-28), 3.42 (m, 1H; H-29a), 3.38 (m,
1H; H2-29), 4.39 (m, 1H; H-30), 4.50 (m, 1H; H-31a), 4.38
(m, 1H; H-31b), 3.68 (m, 1H; H-10a), 3.55 (m, 1H; H-10b),
6.00 (m, 1H; H-20), 7.13 (d, J¼8.7, 2H; H-40/80), 6.67 (d,
J¼8.7, 2H; H-50/70), 4.15 (dd, J¼ꢀ9.9, 7.8, 1H; H-90a),
3.98 (dd, J¼ꢀ9.9, 4.2, 1H; H-90b), 4.72 (m, 1H; H-100),
4.53 (dd, J¼ꢀ11.4, 5.5, 1H; H-110a), 3.74 (m, 1H; H-110b),
3.1. Extraction and isolation
The antibacterial n-BuOH fraction was separated by ODS
flash chromatography as described previously.4 The fraction
eluted with 70% MeOH was separated by ODS HPLC (72%
MeOH containing 0.2 M NaClO4) to afford shishididem-
niols A (1), B (2), C (3, 165 mg), and a mixture of D (4)
and E (5). The mixture of 4 and 5 was purified by ODS
HPLC (45% MeCN containing 0.2 M NaClO4) to obtain
shishididemniols D (4, 40 mg) and E (5, 10 mg).
1
2.24 (s, 3H, H3-130). Compound 6b: H NMR (600 MHz,
CD3OD) 5.07 (m, 1H; H-2), 1.54 (m, 1H; H-3a), 1.62 (m,
1H; H-3b), 1.15 (m, 1H; H-4a), 1.33 (m, 1H; H-4b), 1.44
(m, 1H; H-5a), 1.58 (m, 1H; H-5b), 4.92 (m, 1H; H-6), 2.96
(dd, J¼9.0, 4.2, 1H; H-7), 3.03 (m, 1H; H-8), 5.08 (m, 1H;
H-16), 1.33 (m, 2H; H2-26), 1.52 (m, 2H; H2-27), 3.39 (m,
2H; H2-28), 3.47 (dd, J¼ꢀ9.6, 5.5, 1H; H-29a), 3.43 (dd,
J¼ꢀ9.6, 5.9, 1H; H-29b), 4.39 (m, 1H; H-30), 4.50 (dd,
J¼ꢀ11.0, 4.2, 1H; H-31a), 4.34 (dd, J¼ꢀ11.0, 6.4, 1H;
H-29a), 3.62 (m, 1H; H-10a), 3.55 (m, 1H; H-10b), 6.03 (m,
1H; H-20), 7.23 (d, J¼8.7, 2H; H-40/80), 6.55 (d, J¼8.7, 2H;
H-50/70), 3.88 (dd, J¼ꢀ9.2, 6.4, 1H; H-90a), 3.58 (br, 1H;
H-90b), 4.60 (m, 1H; H-100), 4.73 (dd, J¼ꢀ11.4, 5.5, 1H;
H-110a), 4.62 (m, 1H; H-110b), 2.22 (s, 3H, H3-130a).
3.1.1. Shishididemniol C (3). Colorless oil; [a]1D8 ꢀ19.0 (c
1.00, MeOH); UV (MeOH) lmax 275 nm (3 870), 281
(770); HRESIMS m/z 848.5542 (M+Na)+ (calcd for
C44H79N3NaO11, D ꢀ7.1 mmu). For 1H and 13C NMR
data, see Tables 1 and 2. HMBC correlations (DMSO-d6)
H-2/C-3; H-7/C-6; H-8/C-7, 9; H-27/C-26, 28; H-28/C-26,
27, 29; H-29a/C-28, 30, 31; H-29b/C-28, 30, 31; H-30/C-
29, 31; H-31/C-29, 30; H-10a/C-1, 20, 30; H-10b/C-1, 20, 30;
H-20/C-30, 40, 80; H-40, 80/C-20, 40, 50, 60, 70, 80; H-50, 70/C-
30, 50, 60, 70, 80; H-90a/C-60, 100, 110; H-90b/C-60, 100, 110;
H-100/C-90, 110, 120; H-130/C-120; NH-10/C-1, 10; NH-100/
C-100, 110, 120.
3.1.2. Shishididemniol D (4). Colorless oil; [a]1D9 ꢀ16.6 (c
1.00, MeOH); UV (MeOH) lmax 275 nm (3 1100), 281
(900); HRESIMS m/z 884.5379 (M+Na)+ (calcd for
3.3. Preparation of N-Cbz derivative 7
To a solution of 3 (30 mg, 0.036 mmol) in CHCl3/MeOH
(3:1, 0.4 mL) were added triethylamine (50 mL) and Cbz–
Cl (25.6 mL). After stirring for 16 h at rt, the reaction mixture
was concentrated, suspended in H2O, and extracted with
EtOAc. The organic layer was concentrated and the residue
was separated by ODS HPLC (gradient elution of 50%
MeCN to 60% MeCN) to give 7 (11.3 mg). Compound 7:
ESIMS m/z 960.4252 (M+H)+. 1H NMR (600 MHz,
CD3OD) 7.37–7.26 (m, 5H), 7.29 (d, J¼8.7, 2H), 6.93 (d,
J¼8.7, 2H), 5.08 (d, J¼–12.3, 1H), 5.05 (d, J¼–12.3, 1H),
4.71 (dd, J¼7.4, 4.6, 1H), 4.22 (m, 1H), 4.05 (m, 2H),
3.99 (m, 1H), 3.78 (m, 1H), 3.69 (d, J¼5.9, 2H), 3.57 (m,
2H), 3.51–3.40 (m, 6H), 3.39–3.33 (m, 2H), 2.98 (ddd,
J¼8.2, 4.1, 4.1, 1H), 2.82 (dd, J¼4.4, 8.2, 1H), 1.97 (s,
3H), 1.73–1.25 (m, 42H).
1
C44H8035ClN3NaO11, D ꢀ6.5 mmu). For H and 13C NMR
data, see Tables 1 and 2. HMBC correlations (DMSO-d6)
H-2/C-1, 3, 4; H-27/C-26, 28; H-28/C-27, 29; H-29a/C-28,
30, 31; H-29b/C-28, 30, 31; H-10a/C-1, 20, 30; H-10b/C-1,
20, 30; H-20/C-10, 30, 40, 80; H-40, 80/C-20, 40, 50, 60, 70, 80; H-
50, 70/C-30, 40, 50, 60, 70, 80; H-90a/C-60, 100; H-90b/C-60, 100;
H-100/C-90, 110, 120; H-130/C-120; NH-10/C-1, 10; NH-100/
C-100, 110, 120.
3.1.3. Shishididemniol E (5). Colorless oil; [a]1D9 ꢀ30.7
(c 0.50, MeOH); UV (MeOH) lmax 275 nm (3 760), 281
(650); HRESIMS m/z 904.5874 (M+H)+ (calcd for
C47H83N3NaO12, D ꢀ7.1 mmu). For 1H and 13C NMR
data, see Tables 1 and 2. HMBC correlations (DMSO-d6)
H-2/C-3; H-27/C-26, 28; H-28/C-26, 27, 29; H-29a/C-28,
30, 31; H-29b/C-28, 30, 31; H-30/C-29, 31; H-10a/C-1, 20,
30; H-10b/C-1, 20, 30; H-20/C-30, 40, 80, 150; H-40, 80/C-20, 40,
50, 60, 70, 80; H-50, 70/C-30, 40, 50, 60, 70, 80; H-90a/C-60, 100,
110; H-90b/C-60, 100, 110; H-100/C-90, 110; H-130/C-120, 140;
H-140/C-120, 130; H-160/C-150; NH-10/C-1, 10; NH-100/C-
100, 120.
3.4. Preparation of bromohydrin 8
To a solution of 7 (5 mg) in dry THF (0.5 mL) was added
MgBr2$OEt2 (9 mg). The solution was stirred at rt for 3 h
and was then concentrated. The residue was separated by
ODS HPLC (gradient elution of 50% MeCN to 60%
MeCN) to afford 8 (2.8 mg). Compound 8: ESIMS m/z
1
3.2. Preparation of (R)- and (S)-MTPA derivatives
of 3 (6a and 6b)
1062.1648 and 1064.1862 (M+Na)+. H NMR (600 MHz,
CD3OD) 7.37–7.26 (m, 5H), 7.30 (d, J¼8.7, 2H), 6.94 (d,
J¼8.7, 2H), 5.08 (d, J¼ꢀ12.9, 1H), 5.06 (d, J¼ꢀ12.9,
1H), 4.71 (dd, J¼7.3, 5.1, 1H), 4.22 (m, 1H), 4.12 (m,
1H), 4.05 (m, 2H), 4.00 (m, 1H), 3.78 (m, 1H), 3.69 (d,
J¼5.5, 2H), 3.67 (m, 1H), 3.57 (m, 2H), 3.52–3.40 (m,
6H), 3.38–3.32 (m, 2H), 1.97 (s, 3H), 1.94–1.25 (m, 42H).
To a solution of 3 (4 mg) in pyridine (50 mL) was added (S)-
MTPACl (60 mL). The reaction mixture was stirred at rt for
30 min and then concentrated. The residue was partitioned
between EtOAc and H2O with 0.1 M Na2CO3. The organic
layer was purified by ODS HPLC (gradient elution of
90% MeOH to MeOH) to yield (R)-MTPA derivative
6a (2.6 mg). The (S)-MTPA derivative 6b (2.2 mg) was
prepared in the same way. Compound 6a: 1H NMR
(600 MHz, CD3OD) 5.07 (m, 1H; H-2), 1.74 (m, 1H;
3.5. Preparation of (R)-2NMA derivative 11 from 8
A 2.8 mg portion of 8 was treated with NaIO4 (5 mg) in
MeOH/H2O (28:5, 0.33 mL) at rt for 1 h, followed by

H. Kobayashi et al. / Tetrahedron 63 (2007) 6748–6754
6753
reduction with NaBH4 (20 mg). The reaction mixture was
concentrated, suspended in H2O, and extracted with EtOAc.
The organic layer was concentrated and the residue was sep-
arated by silica gel flash chromatography (CHCl3/MeOH¼
19:1) to yield 9 (1.4 mg). To a solution of 9 (1.4 mg) in
pyridine (40 mL) was added tert-butyldiphenylchlorosilane
(TBDPSCl) (10 mL). After stirring at rt overnight, the reac-
tion mixture was concentrated, suspended in H2O, and ex-
tracted with EtOAc. The organic layer was concentrated
and the residue was separated by silica gel flash chromato-
graphy (n-hexane/EtOAc¼4:1) to yield 10 (2.1 mg). To a
solution of 10 (2 mg) in CH2Cl2 (200 mL) were added (R)-
2-naphthylmethoxyacetic acid ((R)-2NMA) (1.5 mg), 1-ethyl-
(10 mL). After stirring at rt overnight, the reaction mixture
was concentrated, suspended in H2O, and extracted with
EtOAc. The organic layer was concentrated and the residue
was separated by silica gel flash chromatography (n-hexane/
EtOAc¼4:1) to yield 14 (9.3 mg). To a solution of 14 (4 mg)
in CH2Cl2 (200 mL) were added (R)-2NMA (2 mg), EDC
(5 mg), DMAP (1 mg), and DMAP$HCl (1 mg), and the
mixture was stirred at rt overnight. The reaction mixture
was concentrated and partitioned between EtOAc and
H2O. The organic layer was subjected to silica gel flash
chromatography (n-hexane/EtOAc¼9:1) to obtain the (R)-
2NMA derivative 15a (4.2 mg). (S)-2NMA derivative 15b
(4.2 mg) was prepared in the same way. Compound 13:
ESIMS m/z 600.2347 and 602.2281 (M+H)+. 1H NMR
(600 MHz, CDCl3) 7.36–7.27 (m, 5H), 5.45 (d, J¼6.6,
1H), 5.09 (s, 2H), 3.99 (m, 1H), 3.84–3.53 (m, 8H), 3.40
(t, J¼6.6, 2H), 1.88–1.18 (m, 36H). Compound 14: ESIMS
m/z 1076.2973 and 1078.3937 (M+H)+. 1H NMR (600 MHz,
benzene d6) 7.80–7.03 (m, 25H), 5.09 (s, 2H), 5.04 (d,
J¼8.7, 1H), 4.20 (m, 1H), 3.91–3.71 (m, 5H), 3.57 (dd,
J¼ꢀ9.4, 3.3, 1H), 3.47 (m, 1H), 3.37 (dd, J¼ꢀ9.4, 6.3,
1H), 3.20 (t, J¼6.6, 2H), 1.78–1.17 (m, 36H), 1.19 (s, 9H),
1.14 (s, 9H). Compound 15a: 1H NMR (600 MHz, benzene
d6) 8.67–7.00 (m, 32H, aromatic), 5.38 (s, 1H, –CH–O in
2NMR), 5.10 (s, 2H; –CH2– in Cbz), 5.08 (m, 1H; H-16),
5.02 (d, J¼8.6, 1H; NH-30), 4.21 (m, 1H; H-30), 3.89 (m,
1H; H-29a), 3.87 (m, 1H; H-8), 3.84 (dd, J¼ꢀ10.5, 5.5,
1H; H-7a), 3.78 (dd, J¼ꢀ10.5, 5.5, 1H; H-7b), 3.74 (dd,
J¼ꢀ9.6, 6.3, 1H; H-29b), 3.58 (br, 1H; H-31a), 3.37 (br,
1H; H-31b), 3.29 (s, 3H; OMe in 2NMA), 3.21 (br, 2H;
H2-28), 1.72 (m, 1H; H-9a), 1.61 (m, 1H; H-9b), 1.45 (m,
1H; H-10a), 1.27 (m, 1H; H-10b), 1.55–0.80 (m, 32H; H2-
11 to H2-15, H2-17 to H2-27), 1.20 (s, 9H; tert-butyl), 1.14
3-(3-dimethylaminopropyl)-carbodiimide
hydrochloride
(EDC) (5 mg), DMAP (1 mg), and DMAP$HCl (1 mg)
and the mixture was stirred at rt overnight. The reaction
mixture was concentrated and partitioned between EtOAc
and H2O. The organic layer was subjected to silica gel flash
chromatography (n-hexane/EtOAc¼9:1) to obtain the (R)-
1
2NMA derivative 11 (2.2 mg). H NMR spectra of 9, 10,
and 11 described above were indistinguishable from those
of 9, 10, and 11 prepared from 1 described in Ref. 4.
3.6. Preparation of N-Cbz derivative 12
To a solution of 4 (20 mg, 0.024 mmol) in CHCl3/MeOH
(3:1, 0.4 mL) were added triethylamine (25 mL) and Cbz–
Cl (17 mL). After stirring at rt for 16 h, the reaction mixture
was concentrated, suspended in H2O, and extracted with
EtOAc. The organic layer was purified by ODS HPLC (gra-
dient elution of 50% MeCN to 60% MeCN) to afford 12
(14.8 mg). Compound 12: ESIMS m/z 996.3154 and
998.2731 (M+H)+. 1H NMR (600 MHz, CD3OD) 7.37–
7.26 (m, 5H), 7.30 (d, J¼8.2, 2H), 6.93 (d, J¼8.2, 2H),
5.08 (d, J¼ꢀ12.8, 1H), 5.06 (d, J¼ꢀ12.8, 1H), 4.71 (dd,
J¼7.6, 4.9, 1H), 4.22 (m, 1H), 4.05 (m, 2H), 4.03–3.99
(m, 2H), 3.78 (m, 1H), 3.70 (d, J¼5.5, 2H), 3.67 (m, 1H),
3.58 (m, 2H), 3.52–3.33 (m, 8H), 1.97 (s, 3H), 1.89–1.25
(m, 42H).
1
(s, 9H; tert-butyl). Compound 15b: H NMR (600 MHz,
benzene d6) 8.03–7.02 (m, 32H, aromatic), 5.13 (m, 1H;
H-16), 5.10 (s, 2H; –CH2– in Cbz), 5.02 (d, J¼8.8, 1H;
NH-30), 4.93 (s, 1H, –CH–O in 2NMR), 4.21 (m, 1H; H-
30), 3.89 (m, 1H; H-29a), 3.85 (m, 1H; H-8), 3.83 (dd,
J¼ꢀ10.2, 5.5, 1H; H-7a), 3.78 (dd, J¼ꢀ10.2, 5.3, 1H; H-
7b), 3.74 (dd, J¼ꢀ9.3, 7.3, 1H; H-29b), 3.58 (br, 1H; H-
31a), 3.37 (br, 1H; H-31b), 3.31 (s, 3H; OMe in 2NMA),
3.21 (br, 2H; H2-28), 1.66 (m, 1H; H-9a), 1.55 (m, 1H; H-
9b), 1.33 (m, 1H; H-10a), 1.17 (m, 1H; H-10b), 1.55–0.80
(m, 32H; H2-11 to H2-15, H2-17 to H2-27), 1.20 (s, 9H;
tert-butyl), 1.14 (s, 9H; tert-butyl).
3.7. Preparation of 7 from 12
To a solution of 12 (4 mg) in 400 mL of MeOH was added
potassium carbonate (6 mg). The reaction mixture was
stirred at rt for 2.5 h, quenched with acetic acid, and concen-
trated. The residue was purified by ODS HPLC (gradient
elution of 50% MeCN to 60% MeCN) to yield 7 (2.1 mg)
3.9. Preparation of 16 from 1
1
whose H NMR spectrum and [a]D value (7 from 4: [a]D20
ꢀ43.3 (c 0.10, MeOH), 7 from 3: [a]2D0 ꢀ43.7 (c 0.10,
MeOH)) were indistinguishable from those of 7 prepared
from 3.
To a solution of 1 (30 mg) in CH2Cl2 (1 mL) were added tri-
ethylamine (450 mL) and Ac2O (300 mL). The solution was
stirred at rt overnight and was then concentrated. The residue
was partitioned between EtOAc and H2O with 0.1 M
NaHCO3. The organic layer was separated by ODS HPLC
(gradient elution of 70% MeOH to MeOH) to afford 16
(18 mg). Compound 16: 1H NMR (600 MHz, CD3OD)
7.29 (d, J¼8.7, 2H), 6.94 (d, J¼8.7, 2H), 5.80 (dd, J¼8.5,
4.8, 1H), 4.90 (dd, J¼7.6, 4.8, 1H), 4.83 (m, 1H), 4.78 (dt,
J¼4.6, 8.2, 1H), 4.46 (m, 1H), 4.28 (dd, J¼ꢀ11.0, 5.0,
1H), 4.25–4.16 (m, 3H), 4.09–4.01 (m, 3H), 3.55 (dd,
J¼ꢀ13.7, 4.6, 1H), 3.51–3.40 (m, 5H), 3.04–2.98 (m, 2H),
2.23 (q, J¼7.3, 2H), 2.10 (s, 3H), 2.06 (s, 3H), 2.05 (s,
3H), 2.033 (s, 3H), 2.028 (s, 3H), 2.01 (s, 3H), 1.94 (s,
3H), 1.80–1.23 (m, 42H), 1.12 (t, J¼7.3, 3H).
3.8. Preparation of (R)- and (S)-2NMA derivatives (15a
and 15b) from 12
A 10 mg portion of 12 was treated with NaIO4 (20 mg) in
MeOH/H2O (50:1, 1.02 mL) at rt for 1 h, followed by reduc-
tion with NaBH4 (20 mg). The reaction mixture was con-
centrated, suspended in H2O, and extracted with EtOAc.
The organic layer was concentrated and the residue was
separated by silica gel flash chromatography (CHCl3/
MeOH¼19:1) to yield 13 (6 mg). To a solution of 13
(5.5 mg) in pyridine (50 mL) was added TBDPSCl

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H. Kobayashi et al. / Tetrahedron 63 (2007) 6748–6754
3.10. Preparation of 16 from 5
References and notes
To a solution of 5 (2 mg) in CH2Cl2 (200 mL) were added
acetic acid (10 mL), EDC (10 mg), DMAP (1 mg), and
DMAP$HCl (1 mg), and the mixture was stirred at rt for
24 h. The reaction mixture was concentrated and the residue
was separated by ODS HPLC (gradient elution of 70%
ꢀ
ꢀ
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1
MeOH to MeOH) to afford 16 (0.6 mg) whose H NMR
spectrum and [a]D value (16 from 5: [a]1D7 ꢀ44 (c 0.04,
MeOH), 16 from 1: [a]1D7 ꢀ47 (c 0.04, MeOH)) were indis-
tinguishable from those of 16 prepared from 1.
5. Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am. Chem.
Soc. 1991, 113, 4092–4096.
Acknowledgements
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Thanks are due to the crew of R/V Toyoshio-maru for assis-
tance in collecting the tunicate. We thank Professor Takenori
Kusumi, Tokushima University, for supplying both (R)- and
(S)-2NMA. We also thank Professor Hidenori Watanabe,
The University of Tokyo, for valuable discussion. This work
was partially supported by Grant-in-aids from MEXT (Prior-
ity Area 16073207) and JSPS (Area B 16380142).