Synthesis and algicidal activity of (+)-cyanobacterin and its stereoisomer

Article abstract of DOI:10.1271/bbb.69.391

(+)-Cyanobacterin, a photosynthesis inhibitor of freshwater cyanobacterium Schytonema hofmanni, was synthesized in 6 steps from a readily accessible chiral synthon 5R-5-(l-menthyloxy)-2(5H)furanone.

Full text of DOI:10.1271/bbb.69.391

Biosci. Biotechnol. Biochem., 69 (2), 391–396, 2005
Synthesis and Algicidal Activity of (+)-Cyanobacterin and Its Stereoisomer
y
2
Fumito ISHIBASHI,1; Suhwan PARK,1 Takako KUSANO,1 and Kazuyoshi KUWANO
1Faculty of Fisheries, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan
2Graduate School of Science and Technology, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan
Received October 7, 2004; Accepted November 11, 2004
(+)-Cyanobacterin, a photosynthesis inhibitor of
freshwater cyanobacterium Schytonema hofmanni, was
synthesized in 6 steps from a readily accessible chiral
synthon 5R-5-(l-menthyloxy)-2(5H)furanone.
reported an asymmetric synthesis of both enantiomers of
a dechlorinated analog of cyanobacterin and formal total
synthesis of (+)-1a by employing Evans’s asymmetric
alkylation as the key step.
In connection with our interest in the development of
algicide against red-tide phytoplankton, we synthesized
(+)-1a and its epimer 1b, and their algicidal activity was
evaluated against harmful algal blooms (HAB) micro-
algae.
Key words: cyanobacterin; photosynthesis inhibitor; al-
gicidal activity; allelopathy; short synthesis
Cyanobacterin (1a) is an allelopathic substance iso-
lated from the freshwater cyanobacterium Schytonema
hofmanni UTEX2349 by Mason et al.1) in 1982. Its
structure, involving the relative stereochemistry between
C-2 and C-3 positions, was determined on the basis
of spectroscopic informations in 1983 by Pignatello
et al.,2) and the proposed structure was confirmed by
total synthesis and X-ray diffraction analysis of the
racemate.3) Later, in 1987, the 2R,3R absolute config-
uration was assigned to the naturally occurring (+)-1a
on the basis of X-ray diffraction analysis by Gleason and
Porwoll.4) Cyanobacterin has been shown to be highly
toxic to other cyanobacteria and a variety of eukaryotic
algae by interrupting photosynthetic electron transport at
a site presumably close to the primary electron acceptor
in photosystem II.5,6) A basic structure-activity relation-
ship study was performed by Gleason’s group,7) in
which the halogen atom on the aromatic ring and the
hydroxyl group at C-3 position were shown to be
essential for the activity.
Results and Discussion
Feringa’s group developed a strategy for the synthesis
of optically active 2,3-disubstituted-ꢀ-butyrolactones
based on tandem conjugate addition-alkylation10) of
readily accessible chiral synthon 5R-5-(l-menthyloxy)-
2(5H)furanone (2),11) and they have demonstrated this
strategy to be profitable for the synthesis of chiral
butanediols12,13) and lignans.14,15) We envisaged (+)-1a
also to be synthesized using Feringa’s strategy as the key
step (Fig. 1).
The synthesis was commenced with stereoselective
1,4-addition of isopropyl anion (i-PrMgBr, TMSCl,
CuBr Me S, HMPA, THF)16) to (5R)-(l-menthyloxy)-
ꢀ
2
25
2(5H)furanone (2) [½ꢁꢁD ꢂ133.5ꢃ (c 1.02, EtOH),
lit.11) ½ꢁꢁD ꢂ136.4ꢃ (EtOH), 98% ee] prepared by
photooxidation of furfural followed by condensation
with l-menthol and optical resolution according to the
reported procedure,11) in which (4R,5R)-3 was obtained
exclusively in 51% yield. Alkylation of 3 (LDA, THF)
The first total synthesis of racemic 1a was achieved
by Jong et al.3) in 1984, and recently Haga et al.8,9)
Fig. 1. Strategy for the Synthesis of (+)-Cyanobacterin.
y
To whom correspondence should be addressed. Fax: +81-95-819-2799; E-mail: fumito@net.nagasaki-u.ac.jp

392
F. ISHIBASHI et al.
Scheme 1. Synthesis of (+)-Cyanobacterin (1a) and Its Epimer (1b).
Reagents: (a) i-PrMgBr, CuBr SMe2, TMSCl, HMPA, THF, ꢂ78 ꢃC (34%); (b) LDA, THF, HMPA, ꢂ78 ꢃC (69%); (c) KOH, aq. MeOH
ꢀ
(86%); (d) LDA, THF, ꢂ78 ꢃC, then p-TsOH, benzene, reflux; (e) aq. H2O2, CH2Cl2, rt (32%); (f) SeO2, dioxane, 80 ꢃC.
with 3-chloro-4,5-methylenedioxybenzyl bromide (4)3)
prepared in 5 steps from vaniline afforded 3,4-trans 5a
and 3,4-cis 5b in a ratio of 89:11 in 69% combined
yield. The chiral auxiliary was removed by alkaline
hydrolysis (1 M KOH, MeOH), giving lactol 6 in 86%
yield as a single stereoisomer, whereas an acid hydrol-
ysis gave inferior results (CF3COOH, CH2Cl2, rt,
24 h, 11%). The stereochemistry at the 5-position was
assigned as R (4,5-trans) due to a coupling constant
value (J ¼ 2:8 Hz) between H-4 and H-5.
moiety (ꢂ 0.77–0.88) were observed.
Finally, the synthesis was accomplished by allylic
hydroxylation of 9 with inversion of configuration at the
4-position. When Z-olefin 9 was treated with 3.6
equivalent of selenium dioxide in dioxane at 80 ꢃC,
(+)-1a and its epimer at 5-C (1b) were obtained in a
ratio of 1a:1b = 12:82 in 41% combined yield. A
mechanism for the selenium dioxide-mediated allylic
oxidation was first proposed by Sharpless et al.,17,18) in
which two consecutive pericyclic reactions, an initial
ene reaction followed by a [2,3]-sigmatropic shift, are
involved. Recently, the stereochemical aspects of the
initial ene reaction and the whole reaction were clarified
by a kinetic isotope effect study19) and an ab initio
study20) respectively. A proposed mechanism for the
reaction of Z- and E-9 with selenium dioxide is shown in
Fig. 2. The initial ene reaction of Z-9 might proceed on
the less hindered ꢃ-face of the molecule of 9 to form an
allylselenic acid intermediate (Z1/Z2). In the subse-
quent [2,3]-sigmatropic shift, the transition state Z2
leading to 1a might be destabilized by the steric
hindrance between the 4-methoxyphenyl moiety and
the substituents at the 3-C and 4-C positions. Conse-
quently, the reaction might proceed predominantly via
the transition state Z1, and afford 1b as the main
product. On the contrary, E-9 was expected to give 1a as
We then proceeded to incorporate the 4-methoxyben-
zylidene unit at the 5-position using selenide-selenoxide
chemistry. The addition of the lithium carbanion
generated from selenide 7 by treatment with LDA
(THF) to lactol 6 followed by lactonization (cat. p-
TsOH, benzene) afforded a diastereomeric mixture of
the adduct 8, which on treatment with hydrogen
peroxide (CH2Cl2–H2O) gave a separable mixture of
5Z- and 5E-benzylidenelactone Z-9 and E-9 in a ratio of
58:42 (based on NMR) in 32% combined yield. These
stereoisomers were separated by chromatography on
silica gel. The stereochemistry of these geometrical
isomers was assigned on the basis of NOESY spectrum
of Z-9, in which NOE correlation peaks between the
olefinic proton (ꢂ 5.42) and both methine protons at 4-C
(ꢂ 2.90–3.05) and methyl protons of the isopropyl

Synthesis of Cyanobacterin
393
Fig. 2. Proposed Mechanism for the Selenium Oxide-Mediated Allylic Oxidation of Z- and E-9.
the main product, since the transition state E2 leading
to 1a appeared to be more favorable than E1, which lead
to 1b. In fact, the reaction of E-9 with selenium dioxide
in dioxane at 80 ꢃC for 24 h gave a mixture of 1a and
1b (1a:1b = 89:11. based on HPLC) in 19% combined
yield with 71% recovery of E-9. Interestingly, no E-
isomer of 1a was formed in the reaction. The reaction of
E-9 required a longer reaction period than that of Z-9,
probably due to a structural disadvantage for the first
ene reaction, but the reaction at elevated temperature
(refluxing temperature) caused dehydration of the
alcohols to give an anhydro derivative of 1a as the
major product. The synthesized 1a was identical in all
respects with natural 1a, including the optical rotation
value [½ꢁꢁD +96ꢃ (CHCl3), lit ½ꢁꢁD +102ꢃ (CHCl3)].
Synthesized (+)-1a and its epimer (ꢂ)-1b were
briefly tested for algicidal activity against three harmful
algal blooms (HAB) microalgae (Table 1). Both (+)-1a
and (ꢂ)-1b showed algicidal activity as high as the
typical photosystem II inhibitor DCMU against Hetero-
capsa circularisquama (Raphidophyceae) and Chatto-
nella marina (Dinophyceae), while Heterosigma
akashiwo (Raphidophyceae) was insensitive to them.
In conclusion, although it is necessary to improve the
stereoselectivity and the chemical yield of individual
steps, we have accomplished a short synthesis of (+)-
cyanobacterin from the readily available chiral synthon.
Experimental
Melting points (mp) were uncorrected. 1H NMR
spectra were recorded in CDCl3 on a Varian Gemini
200, Gemini 300, or UNITY plus 500 instrument at
nearly 200, 300, or 500 MHz respectively. EIMS and
HREIMS were recorded on a JEOL JMS-DX303
spectrometer. 13C NMR Spectra were recorded in
CDCl3 on a JEOL JNM AL-400 spectrometer at
100 MHz. IR spectra were recorded on a Perkin Elmer
System 2000 spectrometer. Optical rotation was record-
ed on a Jasco DIP-370 polarimeter. Tetrahydrofuran
(THF) and dichloromethane were distilled from sodium
metal/benzophenone ketyl and phosphorous pentoxide
respectively under an atmosphere of dry nitrogen prior
to use.
Table 1. Toxicity of Cyanobacterin (1a), Its Epimer 1b and DCMU
to Harmful Algal Bloom (HAB) Microalgae at a Dose of 10 mg/ml
after 4 h, Mortality (%) to Control
(+)-1a
(ꢂ)-1b
7
45
97
DCMU
(4R,5R)-4-Isopropyl-5-menthyloxydihydro-2(3H)-fu-
ranone (3). To a solution of isopropylmagnesium
bromide prepared by slowly adding a solution of 2-
bromopropane (0.52 ml, 5.54 mmol) in dry THF (10 ml)
Heterosigma akashiwo
Heterocapsa circularisquama
Chattonella marina
9
30
64
14
63
84

394
F. ISHIBASHI et al.
to a stirred slurry of Mg powder (0.14 g, 5.94 mmol) in
dry THF (5 ml) followed by refluxing for 40 min, were
J ¼ 5:5, 14.0 Hz, 3-CH–Ar), 3.51 (1H, dt, J ¼ 4:0,
10.5 Hz, CH–O), 5.41 (1H, s, 5-H), 6.02 (2H, s, O–CH2–
O), 6.60 (1H, d, J ¼ 1:53 Hz, 20-H), 6.67 (1H, d, J ¼
1:53 Hz, 60-H). NMR ꢂC 15.5 (CH3), 20.4 (CH3), 20.9
(CH3), 21.2 (CH3), 22.3 (CH3), 23.1 (CH2–CH2–CH–
i-Pr), 25.5 (CH–i-Pr), 27.3 (CHMe2), 31.3 (CHMe2),
34.1 (CH2–Ar), 34.4 (CH–CH2–CH2), 39.2 (CH–CH2–
CH–O), 43.4 (CH–CH3), 47.8 (4-C), 48.6 (3-C), 75.7
(CH–O), 99.0 (5-C), 101.0 (OCH2O), 108.7 (20-C), 113.4
(50-C), 123.4 (60-C), 123.3 (10-C), 142.8 (40-C), 148.4
(30-C), 178.6 (2-C). IR ꢄmax (KBr) cmꢂ1: 2957 (m), 1778
(s), 1486 (s), 1428 (m), 1259 (s), 1048 (m), 940 (s).
EI-MS m/z (%): 450 (Mþ, 49), 311 (59), 226 (40), 224
(43), 169 (72), 139 (58), 138 (100), 83 (87). Found: C,
66.91; H, 7.52. Calcd. for C25H35ClO5: C, 65.66; H, 8.04.
5b, NMR ꢂH (300 MHz) 0.79–0.98 (15H, m, CH3 ꢄ
5), 0.93–1.29 (4H, m, CH2 ꢄ 2), 1.34–1.45 (1H, m,
(CH3)2CH), 1.65–1.72 (2H, m, CH2), 1.87–1.91 (1H, m,
4-(CH3)2CH), 1.99–2.05 (1H, m, 4-H), 2.10–2.14 (1H,
m, Me–CH), 2.56 (1H, m, i-Pr–CH), 2.85–2.91 (1H, m,
3-CH–Ar), 3.07 (1H, dd, J ¼ 5:5, 13.5 Hz, 3-CH–Ar),
3.51 (1H, dd, J ¼ 4:52, 10.53 Hz, CH–O), 5.41 (1H, s,
5-H), 6.03 (2H, s, O–CH2–O), 6.58 (1H, d, J ¼ 1:53 Hz,
20-H), 6.65 (1H, d, J ¼ 1:53 Hz, 60-H).
added successively a solution of CuBr (CH ) S (0.41 g,
3 2
ꢀ
0.20 mmol) in HMPA (1.65 ml, 9.50 mmol), (CH3)3SiCl
(1.00 ml, 7.90 mmol), and a THF (10 ml) solution of
5R-2 (1.00 g, 3.96 mmol) at ꢂ78 ꢃC. After 3 h at that
temperature, the reaction was quenched by the addition
of triethylamine (1.0 ml), diluted with hexane, and
washed with water. The aqueous layer was extracted
with hexane. The combined organic layer was washed
with water, dried over Na2SO4, and concentrated. The
oily residue was chromatographed on silica gel eluted
with hexane:EtOAc ¼ 9:1 to give 3 (0.60 g, 2.02 mmol,
51%) as a colorless oil. ½ꢁꢁD ꢂ2.0ꢃ (c 6.86, CHCl3).
25
NMR ꢂH (300 MHz) 0.76–0.96 (15H, m, CH3 ꢄ 5),
0.77–1.30 (4H, m, CH2 ꢄ 2), 1.30–1.48 (1H, m,
CH3CH–), 1.59–1.83 (3H, m, CH2 and (CH3)2CH–),
2.02–2.19 (3H, m, (CH3)2CH– and i-Pr–CH{ ꢄ 2), 2.27
(1H, dd, J ¼ 5:4, 17.8 Hz, 3-H), 2.73 (1H, dd, J ¼ 8:8,
18.0 Hz, 3-H), 3.52 (1H, dt, J ¼ 4:2, 10.5 Hz, CH–O),
5.44 (1H, d, J ¼ 2:76 Hz, 5-H). NMR ꢂC 15.6 (CH3),
19.6 (CH3), 19.9 (CH3), 20.9 (CH3), 22.3 (CH3), 23.1
(CH2–CH2–CH), 25.4 (CH–i-Pr), 29.6 (CHMe2), 31.5
(CHMe2), 31.6 (3-C), 34.3 (CH2–CH2–CH), 39.8 (4-C),
42.9 (CH–CH2{CH–O), 47.7 (CH–CH3), 77.0 (CH–O),
103.9 (5-C), 176.2 (2-C). IR ꢄmax (KBr) cmꢂ1: 2960 (m),
1739 (s), 1268 (m), 1166 (s), 1116 (m), 1074 (m). Anal.
Calcd. for C17H30O3: C, 72.30; H, 10.71. Found: C,
72.00; H, 11.13%.
(3R,4R,5R)-5-Hydroxy-4-isopropyl-3[3-chloro-(4,5-
methylenedioxyphenyl)-methyl]dihydro-2(3H)-furanone
(6). To a solution of 5a (1.00 g, 2.38 mmol) in MeOH
(30 ml), 1 M KOH solution (5.0 ml, 5.0 mmol) was
added, and the mixture was allowed to stand for 3 h at
room temperature. The mixture was then acidified with
conc. HCl and extracted twice with CH2Cl2. The
combined organic extracts were washed with water,
dried (Na2SO4), and evaporated. The oily residue was
chromatographed on silica gel eluted with hexane:
EtOAc = 5:1 to give 6 (0.64 g, 2.05 mmol, 86% yield)
(3R,4R,5R)-4-Isopropyl-5-menthyloxy-3[3-chloro-(4,5-
methylenedioxyphenyl)-methyl]dihydro-2(3H)-furanone
(5a). To a cooled (ꢂ78 ꢃC) solution of 3 (4.3347 g,
15.35 mmol) in THF (70 ml) was added dropwise 2M
heptane/THF/ethylbenzene solution of LDA (Aldrich)
(10.00 ml, 20.00 mmol) and the mixture was stirred
for 1 h at ꢂ78 ꢃC. A solution of 3-chloro-4,5-methylen-
dioxybenzyl bromide (4) (4.2413 g, 17.00 mmol) in THF
(40 ml) was added dropwise. After 1 h, HMPA (5.30 ml,
30.00 mmol) was added and the whole was stirred
for another 1 h. The reaction was then quenched by 10%
NH4Cl solution (100 ml) and extracted twice with
t-BuOMe. The extracts were combined, dried over
Na2SO4, and concentrated. The oily residue was
chromatographed on silica gel eluted with hexane:
EtOAc = 9:1 to give a mixture of 5a and 5b (4.74 g,
10.53 mmol, 69%), which was separated by medium-
pressure liquid chromatography on silica gel (Merck,
LiChroprep Si60, benzene:EtOAc ¼ 50:1).
as a colorless viscous oil. ½ꢁꢁD ꢂ27.2 ꢃ (c 2.14,
25
CHCl3). NMR ꢂH (300 MHz) 0.82 (3H, d, J ¼ 6:0 Hz,
CH3), 0.84 (3H, d, J ¼ 6:8 Hz, CH3), 1.68 (1H, m, 4-
(CH3)2CH), 1.94 (1H, m, 4-H), 2.59 (1H, m, 3-H), 2.90
(1H, dd, J ¼ 8:8, 14.0 Hz, 2-CH–Ar), 3.07 (1H, dd,
J ¼ 3:6, 14.0 Hz, 2-CH–Ar), 3.98 (1H, br-s, OH), 5.58
[1H, br-s (d, J ¼ 2:8 Hz in CDCl3–D2O), 5-H], 6.01
(2H, s, OCH2O), 6.63 (1H, d, J ¼ 1:6 Hz, 20-H), 6.70
(1H, d, J ¼ 1:6 Hz, 60-H). NMR ꢂC 19.2 (CH3), 19.5
(CH3), 29.0 (CHMe2), 36.8 (CH2–Ar), 45.2 (4-C), 51.9
(3-C), 101.0 (5-C), 101.7 (OCH2O), 108.2 (20-C), 113.5
(50-C), 122.9 (60-C), 132.8 (10-C), 143.0 (40-C), 148.5
(30-C), 178.5 (2-C). IR ꢄmax (KBr) cmꢂ1: 3391 (br), 2964
(m), 1756 (s), 1618 (m), 1502 (m), 1486 (s), 1429 (s),
1261 (s), 1195 (m), 1048 (s), 939 (s). EI-MS m/z (%):
312 (Mþ, 40), 226 (100), 169 (72), 84 (14). HRMS m/z
(Mþ): Calcd. For C15H17ClO5: 312.0765, Found:
312.0740.
25
5a, ½ꢁꢁD ꢂ90.7 ꢃ (c 0.28, CHCl3). NMR ꢂH (500
MHz) 0.78 (3H, d, J ¼ 7:0 Hz, CH3), 0.81 (3H, d, J ¼
5:5 Hz, CH3), 0.82 (3H, d, J ¼ 5:5 Hz, CH3), 0.89 (3H, d,
J ¼ 7:0 Hz, CH3), 0.94 (3H, d, J ¼ 6:5 Hz, CH3), 0.93–
1.00 (4H, m, CH2 ꢄ 2), 1.23 (1H, m, (CH3)2CH), 1.38
(1H, m, i-Pr–CH), 1.61–1.69 (3H, m, 4-(CH3)2CH, and
CH2), 1.89 (1H, m, 4-H), 2.01 (1H, m, Me–CH), 2.10
(1H, m, i-Pr–CH), 2.54 (1H, qui, J ¼ 5:00, 4-H), 2.86
(1H, dd, J ¼ 9:0, 14.0 Hz, 3-CH–Ar), 3.03 (1H, dd,
1-Methoxy-4-(phenylselenomethy)lbenzene (7). Clean
cut sodium (0.40 g, 18 mmol) was added portionwise to
a solution of diphenyl diselenide (2.50 g, 8.01 mmol) in

Synthesis of Cyanobacterin
395
dry THF (15 ml) at room temperature under a nitrogen
atmosphere. The mixture was refluxed for 6 h, at which
time all of the sodium had been consumed and orange
crystals of sodium phenylselenoxide appeared. The
mixture was cooled to room temperature and a solution
of 4-methoxybenzyl chloride (2.60 g, 16.0 mmol) in
HMPA (3.07 ml, 17.6 mmol) was added. After refluxing
for 4.5 h, the reaction mixture was cooled again to room
temperature, poured into water, and extracted twice with
CH2Cl2. After drying (Na2SO4) and removing the
solvent, the oily residue was chromatographed on silica
gel eluted with benzene:CH2Cl2 ¼ 9:1 to give selenide 7
(3.55 g, 12.8 mmol, 80% yield) as white crystals. NMR
ꢂH (300 MHz) 3.78 (3H, s, ArOCH3), 4.08 (2H, s,
PhSeCH2Ar), 6.78 (2H, d, J ¼ 8:5 Hz, 30-H), 7.13 (2H,
d, J ¼ 8:5 Hz, 20-H), 7.22–7.30 (3H, m, Ph), 7.42–7.49
(2H, m, Ph).
200-H). Found: C, 66.58; H, 5.52. Calcd for C23H23ClO5:
C, 66.65; H, 5.59.
E-9: NMR ꢂH (300 MHz) 0.71 (6H, dd, J ¼ 6:90,
22.62 Hz, CH3 ꢄ 2), 1.90–2.04 (1H, m, 4-(CH3)2CH),
2.75–2.77 (2H, m, 3-CH2Ar), 2.95–3.09 (1H, m, 4-H),
3.16–3.27 (1H, m, 3-H), 3.80 (3H, s, ArOCH3), 6.01
(2H, s, OCH2O), 6.25 (1H, s, C¼CHAr), 6.65 (1H, d,
J ¼ 1:59 Hz, 20-H), 6.69 (1H, d, J ¼ 1:59 Hz, 60-H),
6.87 (2H, d, J ¼ 0:85 Hz, 300-H), 7.00 (2H, d, J ¼
8:85 Hz, 200-H). NMR ꢂC 16.4 (CH3), 19.3 (CH3), 29.4
(CHMe2), 37.3 (CH2–Ar), 43.8 (4-C), 47.3 (3-C), 55.3
(OCH3), 101.8 (OCH2O), 107.8 (20-C), 107.9 (C¼
CH{Ar), 113.8 (50-C), 114.2 (300-C and 500-C), 123.0
(60-C), 126.2 (100-C9, 128.9 (200-C and 600-C), 132.0 (10-
C), 144.9 (40-C), 148.8 (C¼CH{Ar), 152.2 (30-C), 158.3
(400-C), 177.8 (2-C). IR ꢄmax(KBr) cmꢂ1: 2961 (m), 1790
(s), 1674 (m), 1610 (m), 1514 (s), 1486 (s), 1429 (m),
1259 (s), 1123 (s), 1046 (s), 939 (m).
4-Isopropyl-5-[(4-methoxyphenyl)methylene]-3-[3-
chloro(4,5-methylenedioxy-phenyl)methyl]dihydro-2(3H)-
furanones (9). To a cooled (ꢂ78 ꢃC) solution of selenide
7 (1.30 g, 4.50 mmol) in dry THF (20 ml) was added a
2M heptane/THF/ethylbenzene solution of LDA (2.5
ml, 5.0 mmol), and the mixture was stirred for 1 h. A
solution of 6 (0.64 g, 2.10 mmol) in THF (10 ml) was
added dropwise and the whole was stirred for 1 h before
being quenched with a 1 M HCl solution (20 ml). The
organic layer was separated and the aqueous layer was
extracted with EtOAc. Combined organic extracts were
dried (Na2SO4) and concentrated. The residue was
dissolved in benzene (50 ml) containing a catalytic
amount of p-TsOH, and refluxed for 30 min. The
mixture was concentrated and the residue was chroma-
tographed on silica gel eluted with toluene to give a
diastereomeric mixture of 8, which was used for the next
step without further purification.
To a cooled (0 ꢃC) and stirred solution of the crude
selenide 8 in CH2Cl2 (30 ml), was added 30% aqueous
H2O2 (30 ml). The two phase system was gradually
warmed to room temperature over 2 h and the reaction
was quenched by the addition of a 10%Na2SO3 solution
(30 ml) and a 5%NaHCO3 solution (30 ml). The organic
layer was separated and the aqueous layer was extracted
with CH2Cl2. The combined extracts were dried
(Na2SO4) and concentrated. The oily residue was
chromatographed on silica gel eluted with hexane:
EtOAc = 5:1 to give an E=Z mixture of 9 [0.27 g,
6.65 mmol, 32% overall yield from 6, E:Z ¼ 42:58
(based on NMR)]. The geometrical isomers were
separated using silica gel TLC (Merck silica gel 60
4-Hydroxy-4-isopropyl-5-[(4-methoxyphenyl)methyl-
ene]-3-[3-chloro-(4,5-methylenedioxyphenyl)methyl]di-
hydro-2(3H)-furanone (1a, 1b). A solution of Z-9
(59 mg, 0.142 mmol) and SeO2 (57 mg, 0.510 mmol) in
dioxane (15 ml) was heated at 80 ꢃC for 1 h. The solution
was diluted with EtOAc, washed with water, dried
(Na2SO4), and concentrated. The residue was purified
using silica gel TLC (Merck silica gel 60F254) developed
with toluene:EtOAc ¼ 50:1 to give 1a (3.0 mg, 0.0023
mmol, 5%) and 1b (21.6 mg, 0.0523 mmol, 36%).
1a: ½ꢁꢁD +96.6ꢃ (c 0.058, CHCl3). NMR ꢂH (500
27
MHz) 0.91 (3H, d, J ¼ 5:83 Hz, CH3), 1.09 (3H, d, J ¼
5:83 Hz, CH3), 1.85 (1H, s, OH), 2.18 (1H, qui, J ¼
6:64 Hz, 4-(CH3)2CH), 2.89 (1H, dd, J ¼ 13:85,
6.06 Hz, 3-CHAr), 3.10–3.18 (2H, m, 3-CHAr and H-
3), 3.82 (3H, s, ArOCH3), 5.71 (1H, s, C¼CHAr), 6.02
(2H, s, OCH2O), 6.77 (1H, d, J ¼ 1:60 Hz, 20-H), 6.82
(1H, d, J ¼ 1:60 Hz, 60-H), 6.88 (2H, d, J ¼ 8:80 Hz, 300-
H), 7.53 (2H, d, J ¼ 8:80 Hz, 200-H). IR ꢄmax (KBr)
cmꢂ1: 3500 (br), 1807 (m), 1682 (m), 1505 (s), 1487 (s),
1251 (s), 1046 (s), 939 (m). HRMS m/z [ðM ꢂ H2OÞþ]:
Calcd. for C23H21ClO5: 412.1078, Found: 412.1086.
27
1b: ½ꢁꢁD ꢂ22.8ꢃ (c 1.0, CHCl3); NMR ꢂH (300
MHz) 0.97 (6H, d, J ¼ 6:3 Hz, CH3 ꢄ 2), 1.95 (1H, s,
OH), 2.00 (1H, m, 4-(CH3)2CH), 2.82–2.88 (2H, m, 3-
CH2Ar), 3.10 (1H, m, H-3), 3.82 (3H, s, ArOCH3), 5.70
(1H, s, C¼CHAr), 5.96 (1H, d, J ¼ 1:2 Hz, OCHO),
5.99 (1H, d, J ¼ 1:2 Hz, OCHO), 6.69 (1H, d, J ¼
1:5 Hz, 20-H), 6.76 (1H, d, J ¼ 1:5 Hz, 60-H), 5.96 (1H,
d, J ¼ 1:6 Hz, 300-H), 7.52 (2H, d, J ¼ 8:7 Hz, 200-H).
Compounds 1a and 1b were also obtained in 19%
combined yield (1a:1b ¼ 89:11, based on HPLC) by
the reaction of E-9 with SeO2 (5 eq.) in dioxane at 70 ꢃC
for 5 h.
F
254) developed with benzene:EtOAc ¼ 50:1.
Z-9: NMR ꢂH (300 MHz) 0.78–0.88 (6H, m,
CH3 ꢄ 2), 1.79–1.89 (1H, m, 4-(CH3)2CH), 2.70–2.79
(2H, m, 3-CH2Ar), 2.90–3.05 (1H, m, 4-H), 3.12–3.25
(1H, m, 3-H), 3.81 (3H, s, ArOCH3), 5.42 (1H, s,
C¼CHAr), 6.02 (2H, s, OCH2O), 6.59 (1H, d, J ¼
1:44 Hz, 20-H), 6.67 (1H, d, J ¼ 1:44 Hz, 60-H), 6.85
(2H, d, J ¼ 2:01 Hz, 300-H), 7.49 (2H, d, J ¼ 2:01 Hz,
Toxicity assay on HAB microalgae. The bioassay was
performed according to Kakisawa’s procedure21) with
slight modifications. In brief, microalgae used for the
bioassay were cultivated in EV media at 20 ꢃC for 5 d in

396
F. ISHIBASHI et al.
9) Haga, Y., Okazaki, M., and Shuto, Y., Systematic
strategy for the synthesis of cyanobacterin and its
stereoisomers. 2. Asymmetric total synthesis of the 2-
and 3-epimers of dechloro-cyanobacterin. Biosci. Bio-
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10) Feringa, B. L., de Lange, B., Jansen, J. F. G. A., de Jong,
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approaches in asymmetric synthesis using ꢀ-alkoxy-
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Asymmetric diels-alder reactions with 5-menthyloxy-2-
(5H)-furanone. Tetrahedron: Asymmetry, 2, 1247–1262
(1991).
the dark for 12 h, and under light (37 mmol photons/
ꢂ2sꢂ1) for 12 h per day prior to the bioassay. Each
m
culture was diluted with sea water to a cell density of
0:11{0:22 ꢄ 105 cells/ml, and pipetted into a 26-well
plastic microplate (1 ml/well). 2 ml of the methanolic
sample solution of appropriate concentration was added
to each of the three wells and another three wells was
used as a control (2 ml of MeOH/well). After incubation
for 2 h at 20 ꢃC under illumination (37 mmol photons/
m
ꢂ2sꢂ1), cell viability was counted under microscope
(ꢄ20) observation.
12) Jansen, J. F. G. A., and Feringa, B. L., Asymmetric 1,4-
addition to ꢀ-menthyloxybutenolides. Enantiospecific
synthesis of chiral 1,4-butanediols. Tetrahedron Lett.,
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13) Jansen, J. F. G. A., and Feringa, B. L., Asymmetric 1,4-
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Acknowledgment
We thank Professor Tatsuya Oda, who kindly pro-
vided us the strains of HAB microalgae. This work was
supported by a grant-aid from the Japan Society for the
Promotion of Science (no. 13660202).
14) Jansen, J. F. G. A., and Feringa, B. L., Asymmetric 1,4-
additions to ꢀ-menthyloxybutenolides (part III): enan-
tioselective synthesis of (ꢂ)-eudesmin. Tetrahedron
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