Polycavernoside C and C2, the new analogs of the human lethal toxin polycavernoside A, from the red alga, Gracilaria edulis

Article abstract of DOI:10.1016/j.tetlet.2007.02.003

Two new analogs of the human lethal toxin polycavernoside A, polycavernoside C and C2 (0.1-0.2 mg for each), were isolated from the red alga, Gracilaria edulis. The relative stereostructure of polycavernoside C and the absolute structure of polycavernosid

Full text of DOI:10.1016/j.tetlet.2007.02.003

Tetrahedron Letters 48 (2007) 2255–2259
Polycavernoside C and C2, the new analogs of the human
lethal toxin polycavernoside A, from the red alga, Gracilaria edulis
Mari Yotsu-Yamashita,a,* Kazumi Abe,a Tetsuya Seki,a Kenshu Fujiwarab
and Takeshi Yasumotoc
aGraduate School of Agricultural Science, Tohoku University, Tsutsumidori-Amamiya, Aoba-ku, Sendai 981-8555, Japan
bDivision of Chemistry, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan
cJapan Food Research Laboratories, Tama Laboratory, 6-11-10 Nagayama, Tama-shi, Tokyo 206-0025, Japan
Received 21 January 2007; accepted 1 February 2007
Available online 7 February 2007
Abstract—Two new analogs of the human lethal toxin polycavernoside A, polycavernoside C and C2 (0.1–0.2 mg for each), were
isolated from the red alga, Gracilaria edulis. The relative stereostructure of polycavernoside C and the absolute structure of poly-
cavernoside C2 were determined by spectroscopic analysis and synthesis of the model of their aglycon.
Ó 2007 Elsevier Ltd. All rights reserved.
Polycavernoside A (PA, Fig. 1) and B (PB) were isolated
as the causative toxins of the human fatal poisoning (3
death/15 patients) which occurred in Guam in 1991,
resulting from ingestion of the red alga, Gracilaria edu-
lis.1,2 PA was also identified from the same alga and
Acanthophora specifera, which also caused fatal poison-
ing (8 death/36 patients) in Philippines in 2002–2003.3
We determined the planar structures of PA1 and the
analogs, PA2, PA3, PB, and PB2,4 which possess the
same macrolide aglycon structure. Structural variation
among these analogs are in the conjugated diene or tri-
ene side chain at C15, and in O-methylated or O-acetyl-
ated L-fucosyl-D-xylose sugar part at C5. The total
synthesis of (ꢀ)-PA was achieved by three groups,5–7
and the absolute structure of PA was established by
Fujiwara and Murai et al.5 Further synthetic efforts
have been continuously reported.8 The study of struc-
ture–activity relationship9 and pharmacological details
of PA are now progressing.10 We isolated two other
minor analogs, polycavernoside C (PC, 1) and C2
(PC2, 2) (0.1–0.2 mg for each), from G. edulis in 1992–
1994 collected in Guam. These were suggested to have
a common aglycon structure, and to be the first analogs
which have the different aglycon structure from that of
PA. Here we report the relative stereostructure of 1
and the absolute structure of 2 deduced by spectroscopic
analysis and synthesis of the model of their aglycon (3b).
Compound 1 (0.1–0.2 mg) was isolated together with PA
(0.2 mg) and PB (0.1 mg), from G. edulis (4 kg) collected
on June 25, 1991 in Guam, while 2 (0.1–0.2 mg) was iso-
lated from the same alga (2 kg, June 11, 1992) with PA
(0.4 mg), PA2 (0.1 mg), PA3 (0.4 mg), and PB2
(0.1 mg),4 by successive normal and reversed phase
chromatography as reported previously.1,4 Elution of 1
and 2 from the columns was monitored by diode array
UV detector. For final separation of 1 from PB,1 Capcell
pack CN (H2O–MeCN from 1:1 to 0:10) was used, and 1
was eluted before PB. Compound 2 was eluted after
PA2, PB2, and PA from the final column, Develosil
ODS-5 (H2O–MeCN 1:3)4 and further purified on the
same column. Compound 2 has been kept intact until
now after storage in CD3CN at ꢀ20 °C for more than
10 years, while 1 was decomposed in pyridine-d5. Thus,
2 was mainly used for the structural analysis of the com-
mon aglycon of 1 and 2.
Compound 1 was found to have a molecular weight 856
as determined from FABMS [MꢀH2O+H]+ m/z 839,
[M+Na]+ m/z 879, [M+K]+ m/z 895. 2 was found to
have a molecular formula of C45H74O15 by HR-FAB
1
MS ([M+Na]+ 877.4930, D+0.5 mmu). H NMR spec-
1
tra and H–1H COSY of 1 and 2 (CD3CN), and UV
absorption (kmax 220 nm for 1, 259, 270, 280 nm for 2,
MeCN) suggested the presence of conjugated diene for
3
1 and conjugated triene for 2. The JHH values (16 Hz
for 1, 15.4 Hz for 2) indicated E, E and E, E, E geometry
for 1 and 2, respectively. The same disaccharide
*
Corresponding author. E-mail: myama@biochem.tohoku.ac.jp
0040-4039/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2007.02.003

2256
M. Yotsu-Yamashita et al. / Tetrahedron Letters 48 (2007) 2255–2259
Figure 1. Structures of polycavernoside A, C (1), C2 (2), and the models (3a,b) of the aglycon of 1 and 2.
structure as that in PB4 (O-2,3,4-tri-O-methylfucopyran-
oyl-(100-30)-O-4-acetyl-O-2-methylxylopyranosyl)
was
close 1H NMR chemical shifts of the signals correspond-
ing to 2-CH2 (2.22/2.62), 8-CH2 (2.19/2.85), and H15
(4.92) of 2 to those in PA, 2-CH2 (2.14/2.52), 8-CH2
(2.07/2.97) and H15 (5.00), respectively. All these data
led to the planar structures of 1 and 2 (Fig. 1) and
assignments of their NMR data (Table 1).
suggested in 1, while the same disaccharide structure
as that in PA34 (O-2,3,4-tri-O-methylfucopyranoyl-(100-
30)-O-2,4-dimethylxylopyranosyl) was suggested in 2,
1
by the agreement of the H NMR chemical shifts (the
3
difference was less than 0.03 ppm) and JHH values of
H10-H50 and H100-H600 signals of 1 and 2 (Table 1) with
those of the sugar parts of PB and PA3, respectively.
1H–1H COSY of 1 and 2 suggested that these com-
pounds have a common aglycon structure which was
distinctly different from the aglycon of PA and other
reported analogs. Partial structures of H2–H8, H10–H13,
H15–H23,28, H10–H50, and H100–H600 of 2 were deduced
The relative configurations of 1 and 2 between the tetra-
hydropyrane part (C2–C8) and the disaccharides were
determined based on the reported data by Fujiwara
et al.11 They synthesized both enantiomers of the
C2–C8 part and coupled with the disaccharide of PA.
Between these glycosides, the chemical shifts of H5
and H6a were most different (approximately 0.1 ppm),
and those of one enantiomer were well agreed with those
of PA. Thus, the good agreement of the chemical shifts
of H5 [1 (3.38), 2 (3.38), and PA (3.33)] and those of H6a
[1 (1.32), 2 (1.38) and PA (1.37)] suggested the same rel-
ative configurations in 1, 2, and PA. However, the com-
bination between the northern part (C10–15) and the
southern part (C2–C8) of the aglycon of 1 and 2, and
the stereochemistry of C10 and C13 were kept unknown,
since sufficient NOE data of 2 were not obtained.
1
1
from analyses of H–1H COSY and TOCSY. A H sig-
nal at 2.96 ppm (d 10.2 Hz) coupling with a methyl
substituted methine 1H at 2.01 ppm (H11) showed corre-
lation with the 13C signal at 94 ppm in the HSQC spec-
1
trum. This H signal was assignable to H10, suggesting
that C10 hemiacetal in PA was reduced to oxymethine
in 2. The upfield shifts of the signals corresponding to
H11, H12a, H12b, and H13 of 2 (0.75, 0.72, 0.83, and
0.90 ppm, respectively) (Table 1) from those of PA1 sup-
ported that the five membered cyclohemiacetal ring
formed by C10–C13 in PA was opened and the hemiac-
etal was reduced in 2. A deuterium exchangeable proton
signal at 1.71 ppm (d 12.0 Hz) coupling with H13 was
assigned to 13-OH. Compared with the signals of PA,
the signals corresponding to H3, H7, and H8a of 2 were
downfield shifted (0.12, 0.32, 0.12 ppm, respectively),
and the signal corresponding to H8b was 0.12 ppm
upfield shifted. However, the same partial structure of
C2–C8 in 2 as that in PA including a tetrahydropyrane
ring was suggested by the similar coupling patterns of
H2–H8. Due to the small sample size of 2, the HSQC
To determine them, keto lactone 3a (10S, 13R) and C10-
epimeric 3b (10R,13R) (Fig. 1) were planned to be syn-
thesized as the models of aglycon of 1 and 2 from 4a and
4b (C10-epimer of 4a), respectively (Scheme 1). 4a and
4b are the synthetic intermediates of PA.5 The stereo-
chemistry of C13 in 1 and 2 was hypothesized in R as
same as in PA based on biosynthetic aspect. Selective
protection of the primary alcohol of diol 4a with
TBDPS, followed by methylation of secondary alcohol
5a and deprotection of TBDPS with TBAF afforded
alcohol 7a (Scheme 1). Swern oxidation of 7a with work-
up process under the acidic condition gave methyl acetal
hydrolyzed aldehyde 8a. By following the method used
for total synthesis of PA,5 secoic acid 10a obtained by
deprotection of PMB in 8a with DDQ and by subse-
quent NaClO2 oxidation was lactonized by modified
Yamaguchi’s method.12 NMR spectra of the product
in CDCl3 containing trace amount of HCl indicated
C10-epimerized keto lactone 3b, probably formed via
acetal lactone 11a. Detail analysis of stereochemistry
of C10 of this keto lactone (3b) was described below.
Similarly from 4b (10-epimer of 4a), corresponding sec-
oic acid was derived by the same way. In this case,
methyl acetal was not hydrolyzed by workup process
of Swern oxidation. After lactonization of the secoic
1
spectrum showed only partial H–13C correlations. For
the same reason, the HMBC spectrum clarified the con-
nectivities only around methyl groups by giving cross
2,3
peaks due to
J
between C10/10–OMe, C10/Me25,
CH
C11/Me25, C3/Me24, C4/Me24, C5/Me24, C14/Me26,
C14/Me27, C15/Me26, C15/Me27, C26/Me27, C27/
Me26, C400/Me600, C500/Me600, C20/20–OMe, C200/200–
OMe, C300/300–OMe, and C400/400–OMe. Among them,
the cross peak C10/Me25 supported the connectivity
of C10 and C11, and that C10/10–OMe indicated that
C10 bears a methoxy group. The presence of carbonyl
carbons at C1 and C9 and formation of lactone between
C1 and C15 were not evident from HMBC correlations.
However, these structural features were suggested by the

M. Yotsu-Yamashita et al. / Tetrahedron Letters 48 (2007) 2255–2259
Table 1. NMR spectral data of 1, 2 and 3b in CD3CN
2257
Pos
1
2
3b
J (Hz)
1H (ppm)
Mult.
J (Hz)
1H (ppm)
Mult.
J (Hz)
13C (ppm)
1H (ppm)
Mult.
13C (ppm)
1
2
.
—
—
170
40
2.18
2.62
3.40
1.32
3.38
1.32
1.95
3.97
2.14
2.85
t
12
12, 3
2.19
2.62
3.41
1.28
3.38
1.36
1.98
3.96
2.19
2.85
m
dd
m
m
m
m
m
t
2.20
2.62
3.41
1.24
3.23
1.22
2.08
3.97
2.20
2.89
m
dd
m
m
dt
m
m
t
dd
m
m
m
q
m
t
d
13.8, 3.0
13.8, 2.9
14.7, 4.1
3
4
5
6
81
43
82
—
80
42
80
36
11
7
8
11
14
14, 9
11.3
72
—
11.0
71
42
m
dd
m
dd
dd
13.6, 8.7
10.2
17.2, 10.9
10.6
9
—
94
32
—
210
94
32
10
11
12
2.97
2.00
0.70
1.17
3.10
d
9.4
2.96
2.01
0.84
1.15
3.15
d
m
t
q
m
2.97
1.99
0.85
1.15
3.11
d
m
t
t
t
m
m
dd
t
9.0
12.0
8.8
10.3
10.6
33
9.3
11
13
14
15
16
79
41
81
—
78
40
80
140
4.90
5.45
d
dd
7
4.92
5.55
d
dd
7.8
15.4, 9.8
4.90
5.70
d
ddd
6.5
17.3, 10.8
6.5
16, 8
17
6.10
dd
16, 10
6.18
dd
15.4, 9.8
—
5.09
5.17
0.87
0.98
d
d
d
d
17.3
10.5
6.5
118
18
19
20
21
22
23
24
25
26
27
28
10
5.98
5.69
2.25
0.95
0.92
0.98
0.40
1.03
0.95
dd
dd
m
d
d
d
s
s
d
16, 10
16, 7
6.12
6.22
6.05
5.73
2.29
0.95
0.91
0.97
0.70
1.02
0.95
4.31
2.84
3.43
3.11
3.05
3.91
5.18
3.38
3.43
3.47
4.1
dd
dd
ddd
dd
m
d
d
d
s
s
15.4, 9.8
15.4, 9.8
15.4, 9.8, 1.2
15.4, 6.4
—
—
12
15
6.5
7
7
7
—
—
22
13
16
25
20
22
107
86
80
79
64
6.7
6.7
6.7
7
0.74
1.01
s
s
23
20
d
d
t
6.7
7.2
7.8
4.38
2.97
3.68
4.60
3.13
3.83
5.24
3.37
3.50
3.48
3.88
1.09
3.19
3.36
3.38
3.43
3.48
d
t
t
m
t
dd
d
br s
d
br s
q
d
8
10
10
20
30
m
m
t
dd
d
m
m
br s
q
d
s
s
s
40
50
11
12, 5
2
10.8
11.3, 5.4
3.6
100
98
79
—
80
67
16
59
59
58
58
62
61
200
300
12
400
500
6
7
6.6
6.7
600
1.05
3.19
3.30
3.38
3.38
3.44
3.47
1.71
10-OMe
OMe
OMe
OMe
OMe
OMe
13-OH
4-OAc
Bn–CH2
s
s
s
s
3.20
1.72
s
58
s
s
s
d
s
1.72
1.97
d
s
11
12.0
d
12.0
4.36
4.58
7.31
d
d
s
11.5
11.5
70
Bn–arom
120, 134
1H NMR spectra: 1 (400 MHz), 2 (600 MHz).—Denotes not determined.
1H internal reference (CHD2CN) was set at 1.9 ppm.
13C internal reference (CD313CN) was set at 118 ppm.
13C chemical shifts of 2 and 3b were roughly determined based on HSQC and HMBC.
acid derived from 4b, crude NMR spectra measured in
(9-O-methyl C10-epimer of 11a). This methyl acetal lac-
CDCl3 suggested the formation of methyl acetal lactone
tone in CDCl3 was shaken with aqueous 0.05 M acetic

2258
M. Yotsu-Yamashita et al. / Tetrahedron Letters 48 (2007) 2255–2259
RO
RO
O
H
1
OMe
OH
OMe
O
O
d
O
O
O
O
b
PMBO
RO
PMBO
OH
10
OMe
OMe
OBn
OBn
OBn
8a R=PMB
9a R=H
4a R=H
5a R=TBDPS
6a R=TBDPS
7a R=H
e
a
c
O
OH
OH
10
O
O
O
1
g, h
O
OH
f
O
MeO
MeO
HO
O
O
O
HO
O
O
OMe
OBn
10a
OBn
OBn
11a
3b
Scheme 1. Synthesis of 3b from 4a. Reagents and conditions: (a) TBDPSCl, Et3N, DMAP, CH2Cl2, 20 °C, 96%. (b) MeI, NaH, CH2Cl2, 20 °C, 86%.
(c) TBAF, THF, 0 °C, 93%. (d) (COCl)2, DMSO, CH2Cl2, Et3N, ꢀ75 to 0 °C, 83%. (e) DDQ, CH2Cl2, H2O, 20 °C, 87%. (f) NaClO2, NaH2PO4, 2-
methyl-2-butene, i-PrOH–H2O (3.5:1), 0 °C, 89%. (g) 2,4,6-trichlorobenzoyl chloride, DIPEA, THF, 20 °C, then DMAP, toluene, 60 °C and (h)
CDCl3 (+HCl), 20 °C, 50% in two steps.
acid and allowed to stand for one day at room temper-
ature. Finally, keto lactone 3b was obtained in 30% yield
from the corresponding secoic acid in two steps. The
yields of the other steps were almost same as those in
the case from 4a to 3b. To determine the stereochemistry
of C10 of keto lactone (3b) derived from both of 4a and
4b, NOE experiments were performed on this keto lac-
tone. Positive NOEs, H6a/H8b, H8b/H11, H10/H12b,
H10/Me19, H12a/10-OMe, H12b/Me19, and H13/
Me19 were observed by NOESY1D measured in
CD3CN at 20 °C, whereas NOEs, Me19/10-OMe and
H10/H11 were not observed (Fig. 2). By these NOEs
0.1 ppm, except H5 (0.15 ppm), H6a (0.14 ppm), and
H6b (0.10 ppm) which were probably due to difference
of the substituents at C5. The differences of the 13C
NMR chemical shifts of C2–C15, C24–C27, 10-OMe
of 2 from C2–C15, C18–C21, 10-OMe of 3b, respec-
tively, were not more than 1 ppm except at C5
3
(2 ppm). In addition, JH10/H11 value of 2 (10.2 Hz)
was close to that of 3b (10.6 Hz). According to these
data, the planar structure of the aglycon of 1 and 2
was confirmed, and the relative stereostructures of 1
and 2 (10R*,13R*) were determined as shown in Figure
1. To determine the absolute configuration of 2, CD
spectra of 2 and 3b were measured.13 Both of these
CD spectra showed a similar characteristic single Cotton
effect of negative sign in the region of the transition of
3
together with JH10/H11 (10.6 Hz), Me19, and 10-OMe,
and H10 and H11 were suggested to be in anti-confor-
mation for each, as shown in Figure 2. Based on these
data, the stereochemistry of C10 of the obtained keto
lactone was determined to be in R, therefore, C10 in
3a was suggested to be epimerized, probably via forma-
tion of 9,10-enolate under acidic condition. Steric repul-
sion between 10-OMe and Me19 in 3a was presumed to
induce this epimerization. The NMR data of 3b were
compared with those of 2 (Table 1). The difference of
ketone (n-p ) around 300 nm, which was also shown
*
in the CD spectrum of PA.5 Based on these data, the
absolute configuration of 2 was determined as same as
those of 3b and PA.
The mouse (ddY, male, 12 g) administrated 2 (2.4 lg,
i.p.) did not show any symptoms and survived, suggest-
ing the value of LD99 for 2 was more than 0.2 mg/kg.15
Compound 3b did not show cytotoxicity at 20 lM in
two cell lines, mouse neuroblastoma cells (Neuro-2a)
and human acute monocytic leukemia cells (THP-1),
by WST-8 assay (Dojindo, Japan).14 Cyanobacterium
was proposed for the origin of polycavernosides.3,16
The structures of 1 and 2 provide information for the
biosynthetic pathway or metabolism of polycaverno-
sides, which are necessary for monitoring the occurrence
of these human lethal toxins.
1
the H NMR chemical shifts of H2–H15, H24–27, 10-
OMe, 13-OH of 2 from those of H2–H15, H18–21, 10-
OMe, 13-OH of 3b, respectively, was less than
H
21
H
20
Me
Me
H
15
O
14
13
H
H
H
OH
H
O
H
a
12
19
H
H
b
H
Me
OBn
O
H
b
Me
H
11
10
Acknowledgements
a
H
8
9
6
We are grateful to Professor V. J. Paul, Smithsonian
Marine Station at Fort Pierce, for kind supply of G. edu-
lis, and to Mr. Akiyoshi Goto, Hokkaido University, for
preparation of 4a and 4b. This work was supported by
the Nagase Science and Technology Foundation, and by
H
H
H
H
H
a
OMe
O
b
Figure 2. Key NOEs observed in keto-lactone (3b) derived from 4a
and 4b.

M. Yotsu-Yamashita et al. / Tetrahedron Letters 48 (2007) 2255–2259
2259
Lett. 2005, 46, 4887–4890; (c) Barry, C. S.; Bushby, N.;
Harding, J. R.; Willis, C. L. Org. Lett. 2005, 7, 2683–
2686.
Grants-in-Aid for Scientific Research on Priority Areas
from MEXT (18032014), and from JSPS (17580091).
9. Barriault, L.; Boulet, S. L.; Fujiwara, K.; Murai, A.;
Paquette, L. A.; Yotsu-Yamashita, M. Bioorg. Med.
Chem. Lett. 1999, 9, 2069–2072.
References and notes
10. (a) Louzao, M. C.; Vieytes, M. R.; Yasumoto, T.; Yotsu-
Yamashita, M.; Botana, L. M. Chem. Res. Toxicol. 2006,
19, 788–793; (b) Cagide, E.; Louzao, M. C.; Ares, I. R.;
Vieytes, M. R.; Yotsu-Yamashita, M.; Paquette, L. A.;
Yasumoto, T. Cell. Phys. Biochem. 2007, 19, 185–
194.
11. Fujiwara, K.; Amano, S.; Murai, A. Chem. Lett. 1995,
855–856.
12. (a) Inagawa, J.; Hirata, K.; Saeki, H.; Katsuki, T.;
Yamaguchi, M. Bull. Chem. Soc. Jpn. 1979, 52, 1989–
1993; (b) Hikota, M.; Tone, H.; Horita, K.; Yonemitsu, O.
Tetrahedron 1990, 46, 4613–4628.
1. Yotsu-Yamashita, M.; Haddock, R. L.; Yasumoto, T. J.
Am. Chem. Soc. 1993, 115, 1147–1148.
2. Haddock, R. L.; Cruz, O. L. Lancet 1991, 338, 195–196.
3. Yotsu-Yamashita, M.; Yasumoto, T.; Yamada, S.; Baja-
rias, F. F. A.; Formelozsa, M. A.; Romero, M. L.;
Fukuyo, Y. Chem. Res. Toxicol. 2004, 17, 1265–1271.
4. Yotsu-Yamashita, M.; Seki, T.; Paul, V. J.; Naoki, H.;
Yasumoto, T. Tetrahedron Lett. 1995, 36, 5563–5566.
5. Fujiwara, K.; Murai, A.; Yotsu-Yamashita, M.; Yasu-
moto, T. J. Am. Chem. Soc. 1998, 120, 10770–10771.
6. (a) Paquette, L. A.; Barriault, L.; Pissarnitski, D. J. Am.
Chem. Soc. 1999, 121, 4542–4543; (b) Paquette, L. A.;
Barriault, L.; Pissarnitski, D.; Johnston, J. N. J. Am.
Chem. Soc. 2000, 122, 619–631.
7. (a) White, J. D.; Blakemore, P. R.; Browder, C. C.; Hong,
J.; Christopher, C. M.; Nagornyy, P. A.; Robarge, L. A.;
Wardrop, D. J. J. Am. Chem. Soc. 2001, 123, 8593–8595;
(b) Blakemore, P. R.; Browder, C. C.; Hong, J.; Lincoln,
C. M.; Nagornyy, P. A.; Robarge, L. A.; Wardrop, D. J.;
White, J. D. J. Org. Chem. 2005, 70, 5449–5460.
13. CD spectral data of 2 and 3b (MeCN, 22 °C). Compound
2 (0.12 mM): kext 210.0 (De ꢀ1.22), 221.6 (ꢀ1.14), 251.0
(ꢀ0.47), 272.6 (ꢀ0.41), 301.6 (ꢀ0.53). 320.0 (ꢀ0.33), 3b
(0.4 mM): 226.6 (ꢀ0.23), 292.2 (ꢀ0.95), 301.0 (ꢀ1.13),
309.0 (ꢀ1.04), 321.2 (ꢀ0.57).
14. Ishiyama, M.; Tominaga, H.; Shiga, M.; Sasamoto, K.;
Ohkura, Y.; Ueno, K.; Watanabe, M. In Vitro Toxicol.
1995, 8, 187–190.
15. LD99 for PA was reported as 0.2–0.4 mg/kg.1
16. Yasumoto, T. Chemical Record 2001, 1, 228–242.
8. (a) Perez-Balado, C.; Marko, I. E. Tetrahedron 2006, 62,
2331–2349; (b) Perez-Balado, C.; Marko, I. E. Tetrahedron