Allelochemicals of the tropical weed Sphenoclea zeylanica

Article abstract of DOI:10.1016/S0031-9422(00)00264-8

Nine plant growth inhibitors were isolated from the tropical weed Sphenoclea zeylanica, which shows allelopathic properties. Those compounds hitherto not reported from any plant source were the isomers of cyclic thiosulfinate, (1S,3R,4R)-(+)- and (1R,3R,4R)-(-)-4-hydroxy-3-hydroxymethyl-1,2-dithiolane-1-oxides, and (2R,3R,4R)-(-)- and (2S,3R,4R)-(+)-4-hydroxy-3-hydroxymethyl-1,2-dithiolane-2-oxides. These were named Zeylanoxide A, epi-zeylanoxide A, zeylanoxide B and epi-zeylanoxide B, respectively. The absolute configurations at C-3 and C-4 were elucidated by chemical synthesis of both enantiomers from L- and D-glucose. Two of the inhibitors were secologanic acid and secologanoside, and three other inhibitors were by known secoiridoid glucosides formed as artifacts during extraction with methanol. The cyclic thiosulfinates and secoiridoid glucosides completely inhibit the root growth of rice seedlings at 3.0 mM. While the specific activity of the inhibitors was not high, since they accumulated to circa 0.61% S. zeylanica by dry weight, this suggests that the inhibitors are nervertheless potent allelochemicals in this weed. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0031-9422(00)00264-8

Phytochemistry 55 (2000) 131±140
www.elsevier.com/locate/phytochem
Allelochemicals of the tropical weed Sphenoclea zeylanica
Nobuhiro Hirai a,*, Sou-ichi Sakashita a, Tadachika Sano a, Toshiki Inoue a,
Hajime Ohigashi a, Cha-um Premasthira b, Yukio Asakawa c,,, Jiro Harada c,
Yoshiharu Fujii c
aDivision of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
bNational Weed Science Research Institute, Bangkok 10900, Thailand
cNational Institute of Agro-environmental Sciences, Tsukuba 305-0856, Japan
Received 4 April 2000; received in revised form 27 June 2000
Abstract
Nine plant growth inhibitors were isolated from the tropical weed Sphenoclea zeylanica, which shows allelopathic properties.
Those compounds hitherto not reported from any plant source were the isomers of cyclic thiosul®nate, (1S,3R,4R)-(+)- and
(1R,3R,4R)-( )-4-hydroxy-3-hydroxymethyl-1,2-dithiolane-1-oxides, and (2R,3R,4R)-( )- and (2S,3R,4R)-(+)-4-hydroxy-3-
hydroxymethyl-1,2-dithiolane-2-oxides. These were named zeylanoxide A, epi-zeylanoxide A, zeylanoxide B and epi-zeylanoxide B,
respectively. The absolute con®gurations at C-3 and C-4 were elucidated by chemical synthesis of both enantiomers from l- and d-
glucose. Two of the inhibitors were secologanic acid and secologanoside, and three other inhibitors were by known secoiridoid
glucosides formed as artifacts during extraction with methanol. The cyclic thiosul®nates and secoiridoid glucosides completely
inhibit the root growth of rice seedlings at 3.0 mM. While the speci®c activity of the inhibitors was not high, since they accumulated
to circa 0.61% S. zeylanica by dry weight, this suggests that the inhibitors are nervertheless potent allelochemicals in this weed.
# 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Sphenoclea zeylanica; Sphenocleaceae; Gooseweed; Allelopathy; Plant growth inhibitor; Secoiridoid glucoside; Thiosul®nate; 4-Hydroxy-
3-hydroxymethyl-1,2-dithiolane-1-oxide; 4-Hydroxy-3-hydroxymethyl-1,2-dithiolane-2-oxide
1. Introduction
June. The seeds are only 0.5 mm in length, and remain
in dried soil until the next irrigation. When S. zeylanica
begins to invade paddy ®elds, it grows between rice
plants, but the size of the colony increases each year.
Thus, this weed has been designated as one of the most
serious weeds of rice (Holm et al., 1977).
Domination of rice ®elds by S. zeylanica may be
caused by its rapid growth and tolerance against 2,4-D
(Mercado et al., 1990). However, the growth of rice and
other weeds was depressed in soil where the weed had
grown (Premasthira and Zungsontiporn, 1996b), and
the methanol extracts from the weed inhibited growth of
rice seedlings (Premasthira and Zungsontiporn, 1996a).
These observations suggested the involvement of allelo-
pathy in this domination. Therefore, investigated plant
growth inhibitors of S. zeylanica, which might be alle-
lochemicals, as a ®rst step in an ecological study of the
weed, and found new inhibitors along with secoiridoid
glucosides. Here, we describe their identi®cation, che-
mical synthesis and contents in the weed.
Sphenoclea zeylanica Gaertn. is an annual weed
belonging to the Family Sphenocleaceae. The weed is
distributed in the tropics, not only in Africa where the
weed is a native species, but also in Asia and America,
where it is called ``phak pot'' in Thailand and ``goose-
weed'' in the USA (Noda et al., 1994). It grows in sea-
sonal swamps or depressions, and thus paddy ®elds are
ideal for its growth (Holm et al., 1977). The life cycle of
the weed is coincident with that of rice plants. In Thai-
land, when rice ®elds are irrigated in March, seeds of the
weed germinate in the water under anaerobic conditions
similar to rice seeds (Pons, 1982). The weed is at a
¯owering stage in May, and the seeds are mature in
* Corresponding author. Tel.: +81-75-753-6282; fax: +81-75-753-
6284.
E-mail address: hirai@kais.kyoto-u.ac.jp (N. Hirai).
,
Deceased.
0031-9422/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(00)00264-8

132
N. Hirai et al. / Phytochemistry 55 (2000) 131±140
2. Results and discussion
Cc and He, and between Cd, and Ha,c and Hb,d. These co-
rrelations suggested that compound 1 had a partial
Materials extracted from dried S. zeylanica with 80%
aqueous methanol were partitioned between water and
ethyl acetate. The aqueous layer showed higher inhibi-
tory activity on rice seedling growth than the organic
layer. The aqueous materials were puri®ed with active
charcoal, and four inhibitors (1±4) were isolated from
fractions eluted with 10 and 20% aqueous acetone. The
fraction eluted with 50% aqueous acetone yielded ®ve
inhibitors (5±9) by silica gel chromatography. All inhi-
bitors were water-soluble.
structure of X-CbHb,d-CaHe(X)-CdHf(X)-CcHa,c-X where
1
X represent hydroxyl groups or sulfur atoms. The H-
NMR spectrum of 11 showed that Hb,d and Hf of 1 were
shifted to ꢀ 4.24 and 4.39 ppm, and to ꢀ 6.16 ppm,
respectively, by acetylation. This indicated that Cb and
Cd bonded to primary and secondary hydroxyl groups,
respectively. The partial structure, and the presence of
the cyclic thiosul®nyl group suggested that the plane
structure of 1 was 4-hydroxy-3-hydroxymethyl-1,2-
dithiolane-1-oxide or its 2-oxide. In the 13C-NMR
spectrum of 1, the chemical shift of C-5 (Cc) was lower
than that of C-3 (Ca), so the oxide bonded to S-1. The
relative con®guration at C-3 and C-4 could not be
determined by the coupling constant between H-3 and
1
H-4 in the H-NMR spectrum due to ¯exibility of the
®ve-membered ring. To ®x the ring, compound 1 was
converted to its 4,6-O-isopropylidene derivative (12). If
the relationship between H-3 and H-4 is cis, its coupling
constant should be less than 8 Hz, and if it is trans, the
coupling constant should be larger than 9 Hz. The
observed coupling constant was 2.8 Hz, indicating that
the relationship between H-3 and H-4 was cis.
Compounds 2±4 had a molecular weight of 168, and
showed 1H- and 13C-NMR spectra similar to those of 1,
but optical rotations di€erent from that of 1. The
1
HMQC and HMBC spectra of 2±4, and the H-NMR
spectra of their diacetates (13±15, respectively) showed
that these compounds were also 4-hydroxy-3-hydroxy-
methyl-1,2-dithiolane-1-oxide or its 2-oxide. In the
13C-NMR spectrum of 2, the chemical shift (69.2 ppm)
of C-5 was lower than that (66.5 ppm) of C-3, indicating
that compound 2 was 4-hydroxy-3-hydroxymethyl-1,2-
dithiolane-1-oxide. The 13C-NMR spectra of 3 and 4
showed that the chemical shifts (88.6 and 81.6 ppm,
respectively) of C-3 were lower than those (45.0 and
48.5 ppm, respectively) of C-5, so compounds 3 and 4
were identi®ed as 4-hydroxy-3-hydroxymethyl-1,2-
dithiolane-2-oxide. 4,6-O-Isopropylidene derivatives of
2±4 showed coupling constants of 4.5, 4.6 and 6.6 Hz
between H-3 and H-4, respectively, indicating that the
relationships between H-3 and H-4 of 2±4 were also cis.
Reaction of 2±4 with p-thiocresol gave the same ( )-
bis(p-tolyldithio) derivative (10) as that of 1. This indi-
cated that C-3 and C-4 of compounds 2±4 had the same
absolute con®guration as of 1. The relative con®gura-
tions at the sul®nyl sulfurs of 1±4 were elucidated by the
1
The H-NMR spectrum of 1 showed signals of four
methylene protons at ꢀ 3.42 (Ha), 3.82 (Hb), 3.90 (Hc)
and 3.97 (Hd) ppm, and of two methine protons at ꢀ
4.34 (He) and 5.20 (Hf) ppm. This suggested that all
protons were bound to carbons bonding to heteroatoms
such as oxygen and sulfur. The presence of a cyclic
thiosul®nyl group in 1 was suggested by absorption
maxima at 210 and 248 nm in the UV spectrum, and at
1
1060 cm in the IR spectrum (Lindberg and Bergson,
1965). Reaction of 1 with p-thiocresol gave a ( )-bis(p-
tolyldithio) derivative (10), which con®rmed the pre-
sence of a cyclic thiosul®nyl group (Singh et al., 1988).
Acetylation of 1 gave a diacetate (11), indicating that 1
had two hydroxyl groups. The high-resolution mass
spectrum of 11 gave the molecular formula of 11
C8H12O5S2, and the molecular formula of 1 was deter-
mined to be C4H6O3S2.
The 13C-NMR spectrum of 1 showed four carbons at
ꢀ 61.0 (Ca), 61.4 (Cb), 70.4 (Cc) and 76.4 (Cd) ppm. The
HMQC spectrum of 1 showed that these carbons bon-
ded to He, Hb,d, Ha,c and Hf, respectively. In the HMBC
spectrum of 1, correlations were observed between Ca,
and Ha,c and Hb,d, between Cb, and He and Hf, between
1
chemical shifts of H-3 and H-4 in their H-NMR spec-
tra. In a cyclic thiosul®nate, a proton in the syn position
to a sul®nyl oxygen showed lower chemical shift than
that in the anti position (Wudl et al., 1969). This
deshielding e€ect was attributed to a proximity e€ect
and/or acetylenic-type anisotropy of a sul®nyl group
(Buck et al., 1966; Foster et al., 1967). The chemical
shift (4.34 ppm) of H-3 of 1 was lower than that (4.15

N. Hirai et al. / Phytochemistry 55 (2000) 131±140
133
ppm) of 2, so H-3 of 1 was in a syn position to the 1-S-
oxide, and H-3 of 2 was in an anti position. The chemi-
cal shift (5.20 ppm) of H-4 of 3 was lower than that
(5.14 ppm) of 4, showing that H-4 of 3 and 4 were in syn
and anti positions to the 2-S-oxides, respectively.
lysis and deprotection of 21 gave a dithiolane (23) via
22. Oxidation of 23 with hydrogen peroxide gave a
mixture of (3R,4R)-1±4 (Allen and Brook, 1962). The
mixture was separated into each isomer by chromato-
graphy. Enantiomeric isomers (3S,4S)-1±4 were synthe-
sized from d-glucose by the same method as used for
(3R,4R)-1±4. Speci®c optical rotations of natural 1±4
were in good accordance with those of synthetic (3R,4R)-
1±4, respectively. Thus, the absolute con®gurations at
C-3 and C-4 of natural 1±4 were identi®ed as R and R,
respectively, and the absolute con®gurations at S-1 of
natural 1±4 were determined to be 1S, 1R, 2R and 2S,
respectively, based on the relative con®guration between
the sul®nyl group and methine protons described above.
Compounds 1±4 were new cyclic thiosul®nates, and
were named zeylanoxide A, epi-zeylanoxide A, zeylan-
oxide B and epi-zeylanoxide B, respectively. Aspar-
agusic acid S-oxide (Yanagawa et al., 1973) and
brugierol (Kato and Numata, 1972) are other natural
cyclic thiosul®nates occurring in Asparagus ocinalis
and Brugiera conjugata, respectively.
Compounds 3 and 4 isomerized to each other in aqu-
eous solution and gave an equilibrium mixture in a ratio
of 2:3 by heating at 50ꢀC for 1 day, whereas 1 and 2 did
not. This indicated that inversion of the 2-sul®nyl sulfur
occurred, but inversion of the 1-sul®nyl sulfur, and
migration of the sul®nyl oxygen did not. The inversion
was not observed in methanol solution, suggesting that
the oxygen atom at S-2 was exchanged with an oxygen
atom from water. However, compound 3 incubated in
H218O solution did not show [MH+2]+ ions in its FAB
mass spectrum (data not shown). This result was in
agreement with the catalytic inversion at sul®nyl sulfur
by contaminating radical species (Ishii et al., 1997),
which probably attack sulfenyl sulfur. The more steri-
cally hindered sulfenyl sulfur of 1 and 2 as compared to
those of 3 and 4 might, therefore, depress inversion of
the 1-sul®nyl sulfur. Disul®de 23 is a potent biosynthetic
precursor of 1±4 in S. zeylanica. The presence of the
four isomers suggests that oxidation of the disul®de may
be non-stereoselective. It is unclear whether the oxida-
tion is catalyzed by an enzyme(s).
We attempted to elucidate the absolute con®guration
at C-3 and C-4 using the Mosher method (Dale and
Mosher, 1973) and X-ray analysis, but could not obtain
a-methoxy-a-tri¯uoromethylphenylacetate monoesters
and good crystals of 1±4 for these experiments. The
absolute con®guration was ®nally identi®ed by chemical
synthesis of both enantiomers. The starting material for
the synthesis was determined by retrosynthesis (Fig. 1).
Cleavage of the disul®de bond of (3R,4R)-1±4 gives 1,3-
dithiothreitol. Substitution of sulfhydryl groups of 1,3-
dithiothreitol with hydroxyl groups gives l-erythritol,
accompanying inversion of the con®guration at C-1 by
an SN2 reaction. Oxidation at C-3 gives l-erythrose,
which can be converted from l-glucose by oxidation.
Compounds (3R,4R)-1±4 were synthesized from l-
glucose by the route shown in Fig. 2. Hydroxyl groups
at C-4 and C-6 of l-glucose were protected by an ethyl-
idene group to give derivative 16 (Hall and Stamm,
1970), and oxidative removal of C-2 and C-3 of 16 by
sodium periodate gave a mixture of an aldehyde (17)
and its formate (18) (Kiso et al., 1986). The mixture was
reduced with lithium borohydride, and the diol (19) thus
produced was tosylated. The ditosyl derivative (20) was
treated with potassium thiocyanate to give a dithiocya-
nate (21) (Corey and Mitra, 1962). In the 1H-NMR
spectra, the coupling constant between H-2 and H-3 of
21 was 1.8 Hz corresponding to a cis relationship, while
that of 20 was 9.4 Hz corresponding to a trans rela-
tionship. This con®rmed that the con®guration at C-3
of 20 was inverted by the SN2 reaction. Alkaline hydro-
Compound 5 was identi®ed as vogeloside (equals 7a-
methoxysweroside) by comparison of its spectral data
with those reported in the literature (Kawai et al., 1988).
Compounds 6±9 were also identi®ed as known secoir-
idoid glucosides, 7-epi-vogeloside (6) (Kawai et al.,
1988; Boros and Stermitz, 1991), secologanic acid (7)
(Damtoft et al., 1994), secologanin dimethyl acetal (8)
(Kawai et al., 1988) and secologanoside (9) (Calis and
Sticher, 1984). The methylated compounds 5 and 6 and
8 would be artifacts derived from 7 and secologanin,
respectively, during extraction with methanol (Tomas-
sini et al., 1995). In fact, compounds 5, 6 and 8 were not
detected in the acetone extracts from S. zeylanica, and
compound 7 easily formed 5 and 6 after dissolution in
Fig. 1. Retrosynthesis of (1S,3R,4R)-1.
Fig. 2. Synthesis of (3R,4R)-1±4.

134
N. Hirai et al. / Phytochemistry 55 (2000) 131±140
methanol. Steamed young plants of S. zeylanica are
used as a slightly bitter vegetable in Java (Soerjani et
al., 1987). The secoiridoid glucosides are probably
responsible for the bitter taste (Harborne et al., 1999).
In the privet tree, iridoid glucosides function as anti-
feedants against insects (Konno et al., 1999); the
secoirdoid glucosides might, therefore, protect S. zey-
lanica from insects.
e€ects on lettuce seed germination at 3 mM. These
secoiridoid glucosides may have selective activities
although their activities on rice seedlings were not high.
Compounds (3R,4R)-1±4 did not inhibit growth of fungi
or bacteria, whereas thiosul®nates such as allicin posses
antimicrobial activity (Cavallito and Bailey, 1944).
Compounds (3R,4R)-1±4 would not function in protec-
tion of the weed from microbial infection.
Compounds 1±7 showed higher inhibitory activities on
root growth of rice seedlings than on shoot growth. The
natural cyclic thiosul®nates, (3R,4R)-1±4 completely
inhibited root growth at 3 mM (Fig. 3a). Secoiridoid glu-
cosides 5±7 showed inhibitory activities similar to those
of the cyclic thiosul®nates (Fig. 3b). These compounds
elongated roots at low concentrations, but the root shape
was abnormal. Inhibitory activities of unnatural enan-
tiomers (3S,4S)-1±4 were similar to those of the natural
isomers (data not shown). The sul®nyl group seemed to
be more important for expression of activity than the
absolute con®guration at C-3 and C-4. In contrast to 1±4,
inhibitory activities of dithiolane 23 were di€erent
between the enantiomers. Dithiolane (3R,4R)-23 com-
pletely inhibited root growth of rice seedlings at 1mM,
but (3S,4S)-23, which has an unnatural con®guration at
C-3 and-4, required 3 mM for complete inhibition (Fig.
3c). Inhibition of root growth by the dithiolanes might be
caused by a mechanism di€erent from that by the cyclic
thiosul®nates. Compounds (3R,4R)-1±4 almost com-
pletely inhibited germination of lettuce seeds at 3 mM
(Fig. 3d). This indicated that the cyclic thiosul®nates did
not have selective activities between monocotyledons or
dicotyledons. Compounds 5±7 did not show inhibitory
Contents of 1±7 in fresh and dried S. zeylanica at the
¯owering stage were analyzed using an ODS HPLC
approach. Compounds 8 and 9 were not analyzed due
to their small contents. Methanol was used to extract
the plant materials since this solvent showed higher
eciency for extracting the compounds than acetone.
Contents of 5 and 6, that were artifacts derived from 7
as described above, were included in those of 7. Com-
pounds 1 and 3, and 2 and 4 were not separated from
each other with the column, and contents of each pair
are shown in Table 1 along with the contents of 7. The
secoiridoid glucoside 7 showed higher contents than the
cyclic thiosul®nates 1±4 in fresh and dried plants. The
content of 7 in the dried plant corresponded to 23% of
that in the fresh plant considering the loss of water. The
secoiridoid glucoside seemed to be hydrolyzed by b-
glucosidase during drying, and the formed aglycone
may conjugate with amino acid and proteins through
the aldehyde group of the aglycone (Harborne et al.,
1999; Konno et al., 1999). Decreases in the contents of
1±4 by drying were less than that of 7, showing stability
of the cyclic thiosul®nates. The total content of the
cyclic thiosul®nates 1±4 and the secoiridoid glucoside 7
was 0.48% by weight in fresh plants, and 0.61% after
drying. Inhibitory activities of many allelochemicals on
plant growth are low, but their contents in plants
and amounts released from plants or residues into the
environment are high (Rice, 1984). The high contents
and low activities of 1±7 suggested that these inhibitors
are potent allelochemicals of S. zeylanica. The cyclic
thiosul®nates and secoiridoid glucosides may leach from
decomposed plant residues into the soil by rain and irri-
gation after withering of the weed, and a€ect the growth
of rice.
Table 1
Contents of compounds 1±4 and 7 in fresh and dried S. zeylanica
Compound
Fresh plant (mg/g)
Dried plant (mg/g)
1 and 3a
2 and 4a
7b
0.14
0.11
4.51
4.76
0.43 (0.09)c
0.42 (0.08)
5.26 (1.05)
6.11 (1.22)
Total
a
Compounds 1 and 3, and 2 and 4 were not separated from each
Fig. 3. Inhibitory activities of (3R,4R)-1±4 (a), of 5±7 (b), and of
(3R,4R)- and (3S,4S)-23 (c) on root weight of rice seedlings, and of
(3R,4R)-1±4 (d) on lettuce seed germination. (a) and (d) *: 1, *: 2, ~:
3, ~: 4; (b) *: 5, *: 6, ~: 7; (c) *: (3R,4R)-23, *: (3S,4S)-23.
other by HPLC.
b
Contents of 7 included those of 5 and 6.
Values in parentheses show the contents in fresh plants estimated
by considering that fresh plant lost 80% of its weight by drying.
c

N. Hirai et al. / Phytochemistry 55 (2000) 131±140
135
3. Experimental
ml. Fractions 60±62, 65±72 and 76±96 were con-
centrated separately to give compounds 8 (5 mg), 5 (160
mg) and 6 (150 mg) as colorless powders, respectively.
Materials (905 mg) eluted with 30% MeOH were sub-
jected to silica gel (80 g) chromatography using a mix-
ture of CHCl3, MeOH and HOAc (90 : 10 : 1) as the
eluant, and the eluate was collected in fractions of 16
ml. Fractions 59±76 were concentrated to give com-
pound 7 (242 mg) as a colorless powder. Materials
eluted in fractions 77±128 were puri®ed with an ODS
HPLC column (YMC AQ-311, 6 i. d.Â100 mm) by elu-
tion with 20% MeOH in 0.1% aqueous HOAc at 1.0 ml
min 1, with the eluate being monitored at 254 nm. A
material eluted at tR 10.0 min was collected, and con-
centrated to give compound 9 (4.8 mg).
3.1. General
1H- and 13C-NMR, and HMQC and HMBC spectra
were recorded with TMS as an internal standard using
1
Bruker AC 300 (300 MHz for H) and ARX 500 (500
MHz for 1H) spectrometers. Mass spectra were obtained
with JEOL DX-300 and JMS-600 mass spectrometers.
UV and IR spectra, and optical rotations were mea-
sured with Shimadzu UV-2200AI and FTIR-8100AI
spectrometers, and a Jasco DIP-1000 digital polari-
meter, respectively.
3.2. Extraction and isolation of compounds 1±9
Whole plants of S. zeylanica at the ¯owering stage
were collected from paddy®elds in Ang Thong, Thailand,
on 25±31 May 1995 and 1996. Voucher specimens of S.
zeylanica have been deposited at the Herbarium, Botany
Section, Botany and Weed Science Division, Department
of Agriculture, Bangkok 10900, Thailand. Isolation of
the compounds was guided by rice seedling assays.
3.3. Spectral data of compounds 1±4
Compound 1 [(1S,3R,4R)-(+)-4-hydroxy-3-hydroxy-
methyl-1,2-dithiolane-1-oxide; zeylanoxide A]. [ꢁ]D26
MeOH
max
+526ꢀ (MeOH; c 0.094); UV l
(3.26), 248 (2.95); IR n
nm (log ꢂ): 210
KBr
max
cm 1: 3350, 2920, 1660, 1400,
1300, 1210, 1170, 1120, 1060, 500, 470, 455; FABMS
1
Dried and pulverized S. zeylanica (700 g) was extrac-
ted with 2.81 of MeOH±H2O (80:20, v/v) at room tem-
(matrix: glycerol) m/z (rel. int.): 169 [MH]+ (100); H-
NMR (500 MHz, CD3OD): ꢀ 3.42 (1H, dd, J=13.2 and
4.4 Hz, H-5), 3.82 (1H, dd, J=11.6 and 7.1 Hz, H-6),
3.90 (1H, dd, J=13.2 and 6.7 Hz, H-5), 3.97 (1H, dd,
J=11.6 and 5.2 Hz, H-6), 4.34 (1H, ddd, J=7.1, 5.2 and
4.8 Hz, H-3), 5.20 (1H, ddd, J=6.7, 4.8 and 4.4 Hz, H-
4); 13C-NMR (125 MHz, CD3OD): ꢀ 61.0 (C-3), 61.4
(C-6), 70.4 (C-5), 76.4 (C-4).
perature for
6 days. The extract was ®ltered,
concentrated under reduced pressure, and partitioned
between H2O and EtOAc. Aqueous materials (49 g)
were subjected to charcoal (154 g) chromatography
using mixtures of H2O and various amounts of Me2CO
as eluant to give materials that eluted with 10, 20 and
50% Me2CO.
Compound 2 [(1R,3R,4R)-( )-4-hydroxy-3-hydroxy-
methyl-1,2-dithiolane-1-oxide; epi-zeylanoxide A]. [ꢁ]D29
Materials (9.0 g) eluted with 10 and 20% Me2CO were
combined, and subjected to silica gel (120 g) chromato-
graphy using a mixture of CHCl3 and MeOH containing
1% HOAc as eluant. Materials (405 mg) eluted with 20%
MeOH were subjected to silica gel (80 g) chromato-
graphy using a mixture of CHCl3±MeOH±HOAc (195: 5:
2). The eluate was collected in 15 ml fractions. Fractions
79±95 and 100±120 were concentrated to give compound
1 (104 mg) and compound 3 (55 mg) as colorless oils,
respectively. Materials of fractions 40±60 were subjected
to silica gel (30 g) chromatography using a mixture of
toluene±Me2CO±MeOH (20: 2: 1), with the eluate col-
lected in 15 ml fractions. Fractions 16±18 and 19±22 were
concentrated to give compound 4 (77 mg) as colorless
needles (mp 94ꢀC) and compound 2 (99 mg) as colorless
needles (mp 112ꢀC), respectively.
Materials (2.5 g) eluted with 50% Me2CO were sub-
jected to silica gel (30 g) chromatography using a mix-
ture of CHCl3 and MeOH containing 1% HOAc as the
eluant to give materials that eluted with 20 and 30%
MeOH. Materials (467 mg) eluted with 20% MeOH
were subjected to silica gel (80 g) chromatography using
a mixture of CHCl3, MeOH and HOAc (95 : 5 : 1) as the
eluant, and the eluate was collected in fractions of 16
MeOH
max
283ꢀ (MeOH; c 0.10); UV l
(3.13), 250 (2.99); IR n
nm (log ꢂ): 209
KBr
max
cm 1: 3440, 3320, 2920, 2870,
1630, 1470, 1410, 1380, 1310, 1280, 1250, 1170, 1080,
1060, 1020, 960, 890, 760, 610, 560, 460; FABMS
1
(matrix: glycerol) m/z (rel. int.): 169 [MH]+ (100); H-
NMR (500 MHz, CD3OD): ꢀ 3.39 (1H, dd, J=13.5 and
3.5 Hz, H-5), 3.88 (1H, dd, J=13.5 and 2.3 Hz, H-5),
3.99 (1H, dd, J=11.1 and 7.6 Hz, H-6), 4.15 (1H, ddd,
J=7.6, 6.1 and 4.0 Hz, H-3), 4.26 (1H, dd, J=11.1 and
6.1 Hz, H-6), 5.18 (1H, ddd, J=4.0, 3.5 and 2.3 Hz, H-
4); 13C-NMR (125 MHz, CD3OD): ꢀ 62.7 (C-6), 66.5
(C-3), 69.2 (C-5), 79.5 (C-4).
Compound 3 [(2R,3R,4R)-( )-4-hydroxy-3-hydroxy-
methyl-1,2-dithiolane-2-oxide; zeylanoxide B]. [ꢁ]D29
MeOH
max
253ꢀ (MeOH; c 0.12); UV l
(3.25), 247 (2.83); IR n
nm (log ꢂ): 210
KBr
max
cm 1: 3350, 2900, 1720, 1570,
1410, 1320, 1260, 1240, 1160, 1040, 880, 760, 490, 460;
FABMS (matrix: glycerol) m/z (rel. int.): 169 [MH]+
1
(100); H-NMR (500 MHz, CD3OD): ꢀ 3.44 (1H, dd,
J=11.3 and 5.3 Hz, H-5), 3.67 (1H, ddd, J=9.5, 4.8 and
4.7 Hz, H-3), 3.85 (1H, dd, J=12.2 and 9.5 Hz, H-6),
3.96 (1H, dd, J=11.3 and 4.4 Hz, H-5), 4.17 (1H, dd,
J=12.2 and 4.8 Hz, H-6), 5.20 (1H, ddd, J=5.3, 4.7 and

136
N. Hirai et al. / Phytochemistry 55 (2000) 131±140
4.4 Hz, H-4); 13C-NMR (125 MHz, CD3OD): ꢀ 45.0 (C-
5), 57.6 (C-6), 76.7 (C-4), 88.6 (C-3).
Compound 4 [(2S,3R,4R)-(+)-4-hydroxy-3-hydroxy-
3.4.3. Diacetates (13±15) of 2±4
Compounds 2±4 (2 mg each) were treated by the same
method as used for 1 to give the diacetates (2 mg each).
Diacetate (13) of 2. EIMS (probe) 70 eV, m/z (rel. int.):
252 [M]+ (7), 192 (10), 172 (8), 150 (11), 132 (32), 112
(100), 70 (85); 1H-NMR (500 MHz, CDCl3): ꢀ 2.08 (3H,
s), 2.14 (3H, s), 3.41 (1H, dd, J=14.0 and 4.2 Hz, H-5),
3.92 (1H, dd, J=14.0 and 3.2 Hz, H-5), 4.29 (1H, ddd,
J=7.4, 6.8 and 4.7 Hz, H-3), 4.48 (1H, dd, J=11.4 and
6.8 Hz, H-6), 4.69 (1H, dd, J=11.4 and 7.4 Hz, H-6),
6.02 (1H, ddd, J=4.7, 4.2 and 3.2 Hz, H-4). Diacetate
(14) of 3. EIMS (probe) 70 eV, m/z (rel. int.): 252 [M]+
methyl-1,2-dithiolane-2-oxide; epi-zeylanoxide B]. [ꢁ]D29
MeOH
max
+487ꢀ (MeOH; c 0.12); UV l
(3.26), 248 (2.98); IR n
nm (log ꢂ): 209
KBr
max
cm 1: 3330, 3270, 2850, 1630,
1410, 1300, 1220, 1150, 1060, 1050, 1020, 990, 970, 910,
860, 750, 460; FABMS (matrix: glycerol) m/z (rel. int.):
169 [MH]+ (100); 1H-NMR (500 MHz, CD3OD): ꢀ 3.32
(1H, ddd, J=9.6, 4.7 and 1.4 Hz, H-3), 3.70 (1H, dd,
J=11.1 and 4.3 Hz, H-5), 3.83 (1H, dd, J=11.1 and 1.1
Hz, H-5), 4.22 (1H, dd, J=11.8 and 4.7 Hz, H-6), 4.34
(1H, dd, J=11.8 and 9.6 Hz, H-6), 5.14 (1H, ddd,
J=4.3, 1.4 and 1.1 Hz, H-4); 13C-NMR (125 MHz,
CD3OD): ꢀ 48.5 (C-5), 57.4 (C-6), 79.5 (C-4), 81.6 (C-3).
1
(20), 172 (10), 150 (13), 112 (53), 84 (39), 70 (100); H-
NMR (500 MHz, CDCl3): ꢀ 2.12 (6H, s), 3.50 (1H, dd,
J=11.8 and 5.8 Hz, H-5), 4.01 (1H, ddd, J=9.3, 5.4 and
5.1 Hz, H-3), 4.11 (1H, dd, J=11.8 and 5.1 Hz, H-5),
4.25 (1H, dd, J=12.3 and 9.3 Hz, H-6), 4.57 (1H, dd,
J=12.3 and 5.4 Hz, H-6), 6.14 (1H, dt, J=5.8 and 5.1
Hz, H-4). Diacetate (15) of 4. EIMS (probe) 70 eV, m/z
(rel. int.): 252 [M]+ (32), 172 (15), 144 (10), 132 (4), 112
3.4. Derivatives of compounds 1±4
3.4.1. Bis(p-tolyldithio) derivative (10) of 1.
To a soln of 1 (10 mg) in MeOH (1 ml) was added
p-thiocresol (15 mg), and stirred for 1 h at room
temperature under N2 in the dark. The mixture was
concentrated and subjected to silica gel (2.4 g)
chromatography using a mixture of toluene and Me2CO
(9:1) as the eluant to give 10 (9 mg). [ꢁ]2D7 32ꢀ (MeOH;
c 0.10); FABMS (matrix: glycerol) m/z (rel. int.): 398
[M]+ (10); 1H-NMR (500 MHz, CDCl3): ꢀ 2.336 (3H, s,
40-CH3), 2.342 (3H, s, 400-CH3), 2.94 (2H, d, J=6.5 Hz,
H-1), 3.13 (1H, ddd, J=5.4, 4.7 and 3.1 Hz, H-3), 3.79
(1H, dd, J=11.7 and 4.7 Hz, H-4), 3.92 (1H, dd, J=11.7
and 5.4 Hz, H-4), 4.25 (1H, td, J=6.5 and 3.1 Hz, H-2),
7.13 (4H, d, J=8.0 Hz, H-30,-50,-300 and-500), 7.43 (2H, d,
J=8.0 Hz, H-20 and-200, or H-60 and-600), 7.46 (2H, d,
J=8.0 Hz, H-60 and-600, or H-20 and -200); 13C-NMR (75
MHz, CD3OD): ꢀ 21.0, 21.1, 44.3, 62.0, 62.9, 69.4,
129.9, 130.5, 130.9, 134.9, 135.4, 138.7, 139.0. Com-
pounds 2±4 (5 mg each) were treated by the same
method as used for compound 1 to give the bis(p-tolyl-
dithio) derivatives (6 mg each). The derivatives of 2±4
showed [ꢁ]2D7 31ꢀ, [ꢁ]D27 33ꢀ and [ꢁ]2D7 30ꢀ (MeOH; c
1
(70), 84 (45), 70 (100); H-NMR (500 MHz, CDCl3): ꢀ
2.12 (3H, s), 2.14 (3H, s), 3.52 (1H, dt, J=9.4 and 5.1
Hz, H-3), 3.79 (1H, dd, J=12.4 and 5.4 Hz, H-5), 3.88
(1H, dd, J=12.4 and 2.0 Hz, H-5), 4.59 (1H, dd, J=12.0
and 9.4 Hz, H-6), 4.70 (1H, dd, J=12.0 and 5.1 Hz, H-
6), 6.14 (1H, ddd, J=5.4, 5.1 and 2.0 Hz, H-4).
3.4.4. 4,6-O-Isopropylidene derivative (12) of 1
To a solution of 1 (3 mg) in DMF (0.5 ml) was added
Me2CO (1 ml) and p-toluenesulfonic acid monohydrate
(5 mg), and stirred for 5 h at room temperature. The
mixture was concentrated and subjected to silica gel (8 g)
chromatography using a mixture of toluene and Me2CO
(7:3) as the eluant to give 12 ( 2 mg, 56% yield). EIMS
(probe) 70 eV, m/z (rel. int.): 208 [M]+ (13), 193 (33), 150
(100), 126 (58); 1H-NMR (300 MHz, CDCl3): ꢀ 1.39 (3H,
s, CH3), 1.49 (3H, s, CH3), 3.47 (1H, dd, J=13.9 and 4.8
Hz, H-5), 3.97 (1H, dd, J=13.9 and 1.8 Hz, H-5), 4.04
(1H, dd, J=15.2 and 4.3 Hz, H-6), 4.45 (1H, ddd, J=4.3,
3.4 and 2.8 Hz, H-3), 4.47 (1H, dd, J=15.2 and 3.4 Hz,
H-6), 5.18 (1H, ddd, J=4.8, 2.8 and 1.8 Hz, H-4).
1
0.10), respectively, and their FAB mass, H-NMR and
13C-NMR spectra were the same as those of 10.
3.4.5. 4,6-O-Isopropylidene derivatives of 2±4
3.4.2. Diacetate (11) of 1
Compounds 2±4 (8 mg each) were treated by the same
method as used for 1 to give the 4,6-O-isopropylidene
derivatives (8 mg each, 80% yield). 4,6-O-Isopropylidene
Acetic anhydride (2 ml) was added to a pyridine
solution (3 ml) of 1 (2.0 mg), and left at room tempera-
ture for 3 h. Ice chips were added to the solution, fol-
lowed by concentration to give 11 (2.0 mg). EIMS
(probe) 70 eV, m/z (rel. int.): 252 [M]+ (5), 192 (12), 172
1
derivative of 2. H-NMR (300 MHz, CDCl3): ꢀ 1.469
(3H, s, CH3), 1.474 (3H, s, CH3), 3.20 (1H, dd, J=13.7
and 4.5 Hz, H-5), 3.90 (1H, dd, J=13.7 and 1.0 Hz, H-
5), 4.05 (1H, dd, J=11.1 and 6.0 Hz, H-6), 4.17 (1H,
ddd, J=6.0, 5.3 and 4.5 Hz, H-3), 4.24 (1H, dd, J=11.1
and 5.3 Hz, H-6), 5.33 (1H, td, J=4.5 and 1.0 Hz, H-4).
4,6-O-Isopropylidene derivative of 3. 1H-NMR (300
MHz, CDCl3): ꢀ 1.35 (3H, s, CH3), 1.47 (3H, s, CH3),
3.63 (1H, ddd, J=8.1, 5.1 and 4.6 Hz, H-3), 3.64 (1H,
br. d, J=12.7 Hz, H-5), 4.18 (1H, d, J=12.7 Hz, H-5),
1
(7), 150 (8), 132 (34), 112 (100), 70 (82); H-NMR (500
MHz, CDCl3): ꢀ 2.08 (3H, s), 2.13 (3H, s), 3.64 (1H, dd,
J=13.4 and 4.8 Hz, H-5), 3.75 (1H, dd, J=13.4 and 7.3
Hz, H-5), 4.24 (1H, dd, J=11.7 and 6.8 Hz, H-6), 4.39
(1H, dd, J=11.7 and 6.0 Hz, H-6), 4.65 (1H, dt, J=6.8
and 6.0 Hz, H-3), 6.16 (1H, ddd, J=7.3, 6.0 and 4.8 Hz,
H-4).

N. Hirai et al. / Phytochemistry 55 (2000) 131±140
137
4.19 (1H, dd, J=12.8 and 8.1 Hz, H-6), 4.40 (1H, dd,
J=12.8 and 5.1 Hz, H-6), 5.13 (1H, br. dd, J=4.6 and
2.7 Hz, H-4). 4,6-O-Isopropylidene derivative of 4. H-
NMR (300 MHz, CDCl3): ꢀ 1.428 (3H, s, CH3), 1.433
(3H, s, CH3), 3.42 (1H, ddd, J=8.1, 6.8 and 6.6 Hz, H-
3), 3.89 (1H, dd, J=12.3 and 6.6 Hz, H-5), 3.96 (1H, dd,
J=12.3 and 5.5 Hz, H-5), 4.23 (1H, dd, J=12.0 and 6.8
Hz, H-6), 4.31 (1H, dd, J=12.0 and 8.1 Hz, H-6), 5.00
(1H, td, J=6.6 and 5.5 Hz, H-4).
1H-NMR (500 MHz, CDCl3) of the major isomer: ꢀ
1.42 (3H, d, J=5.0 Hz), 4.04 (1H, dd, J=9.8 and 1.2
Hz), 4.28 (1H, dd, J=9.8 and 5.5 Hz, H-4), 4.53 (1H,
dd, J=12.0 and 0.9 Hz), 4.80 (1H, q, J=5.0 Hz), 5.09
(1H, m), 8.02 (1H, s), 9.61 (1H, s).
1
3.5.3. 2,4-O-Ethylidene-l-erythritol (19)
To a THF solution (50 ml) of LiBH4 (2.3 g) was
added a mixture (7.2 g) of 17 and 18 in THF (60 ml),
and the mixture was stirred at room temperature for 1
h. A THF±H2O (60:40) solution was added to the reac-
tion mixture. The mixture was ®ltered, and the ®ltrate
was concentrated and subjected to silica gel (120 g)
chromatography using a mixture of CHCl3 and MeOH
(7 : 3) as the eluant to give 19 (4.2 g, 69% yield). [ꢁ]D21
+49ꢀ (MeOH; c 0.10); FABMS (matrix: glycerol) m/z
(rel. int.): 149 [MH]+ (65); HR-FABMS (matrix: gly-
cerol): [MH]+ at m/z 149.0801 (C6H13O4 requires
3.5. Synthesis of natural (3R,4R)-1±4
3.5.1. 4,6-O-Ethylidene-ꢁ- and ꢃ-l-glucose (16)
To a ®nely powdered and dried mixture of a- and b-l-
glucose (10.0 g) was added Et2O (5.6 ml), paraldehyde
(5.4 ml) and conc. H2SO4 (0.16 ml). The mixture was
stirred for 1 h, and kept at room temperature for 24 h. To
the mixture was added Et2O until a slurry was formed,
and then K2CO3 (0.28 g). The mixture was kept at room
temperature overnight, and ®ltered. The residue was
extracted with hot Me2CO containing 0.1% NH4OH,
and the extract was ®ltered. The ®ltrate was con-
centrated, and crystallized to give 16 as colorless needles
(3.9 g, mp 149ꢀC). The mother liquor was concentrated
and subjected to silica gel (100 g) chromatography using
a mixture of CHCl3 and MeOH (4:1) as the eluant to give
16 (5.0 g). Total yield of 16, a mixture of a- and b-
anomers (1 : 1), was 78% (8.9 g). [ꢁ]2D3 54ꢀ (MeOH; c
0.10); FABMS (matrix: glycerol) m/z (rel. int.): 207
[MH]+ (16); HR-FABMS (matrix: glycerol): [MH]+ at
1
149.0814) H-NMR (300 MHz, CDCl3): ꢀ 1.34 (3H, d,
J=5.1 Hz), 3.41 (1H, t, J=10.4 Hz), 3.46 (1H, m), 3.78
(1H, m), 3.85 (2H, m), 4.13 (1H, dd, J=10.7 and 5.3
Hz), 4.72 (1H, q, J=5.1 Hz).
3.5.4. 2,4-O-Ethylidene-1,3-O-ditosyl-l-erythritol (20)
To a solution of 19 (4.2 g) in pyridine (50 ml) was
added p-toluenesulfonyl chloride (15.8 g), and the mix-
ture was stirred at room temperature overnight. The
mixture was concentrated and subjected to silica gel
(200 g) chromatography using a mixture of toluene and
Me2CO (19:1) as eluant to give 20 (9.7 g, 76% yield).
MeOH
m/z 207.0866 (C4H9O3S2 requires 207.0869); H-NMR
[ꢁ]2D3 +35ꢀ (MeOH; c 0.10); UV l
nm (log ꢂ): 225
1
max
(300 MHz, CD3OD): ꢀ 1.30 (6H, d, J=5.0 Hz, CH3 of a-
and b-anomers), 3.15±3.55 (8H, m, H-2, -3,-4 and-5 of a-
and b-anomers), 3.78 (2H, m, H-6 of a- and b-anomers),
3.99 (1H, dd, J=10.1 and 4.9 Hz, H-6 of a-anomer), 4.07
(1H, dd, J=10.3 and 4.5 Hz, H-6 of b-anomer), 4.53 (1H,
d, J=7.7 Hz, H-1 of b-anomer), 4.73 (2H, q, J=5.0 Hz,
H-7), 5.09 (1H, d, J=3.8 Hz, H-1of a-anomer).
(4.38); EIMS (probe) 70 eV, m/z (rel. int.): 456 [M]+
(15), 441 (10), 369 (15), 285 (20), 227 (28), 155 (100);
HR-EIMS: [M]+ at m/z 456.1000 (C20H24O8S2 requires
1
456.0912); H-NMR (300 MHz, CDCl3): ꢀ 1.19 (3H, d,
J=5.0 Hz, CH3), 2.44 (3H, s, CH3), 2.48 (3H, s, CH3),
3.48 (1H, t, J=10.5 Hz, H-4), 3.67 (1H, ddd, J=9.4, 5.6
and 2.0 Hz, H-2), 3.80 (1H, dd, J=11.2 and 5.6 Hz, H-
1), 4.01 (1H, dd, J=11.2 and 2.0 Hz, H-1), 4.13 (1H, dd,
J=10.5 and 5.4 Hz, H-4), 4.23 (1H, ddd, J=10.5, 9.4
and 5.4 Hz, H-3), 4.55 (1H, q, J=5.0 Hz), 7.33 (2H, d,
J=8.3 Hz), 7.39 (2H, d, J=8.3 Hz), 7.74 (2H, d, J=8.3
Hz), 7.78 (2H, d, J=8.3 Hz).
3.5.2. 2,4-O-Ethylidene-l-erythrose (17) and its 3-O-
formate (18)
To a solution of 16 (8.0 g) in dry MeOH (350 ml) was
added NaIO4 (23 g), and the mixture was stirred at
room temperature for 24 h. The mixture was ®ltered,
and the ®ltrate was concentrated and subjected to silica
gel (220 g) chromatography using a mixture of CHCl3
and MeOH (9:1) as the eluant to give 17 and 18 (total
7.7 g, 99% yield). Compound 17; EIMS (probe) 70 eV,
m/z (rel. int.): 146 [M]+ (2), 145 (20), 131 (9), 117 (100);
HR-EIMS: [M]+ at m/z 146.0584 (C6H10O4 requires
3.5.5. (2R,3R)-2,4-O-Ethylidene-1,3-dithiocyanato-1,3-
dithiothreitol (21)
To a solution of 20 (9.4 g) in DMSO (50 ml) was
added KSCN (30 g) and stirred at 120ꢀC for 4 days. The
mixture was diluted with H2O (300 ml), and partitioned
four times with 200 ml of EtOAc. The organic layer was
dried (Na2SO4), ®ltered, concentrated and subjected to
silica gel (200 g) chromatography using a mixture of n-
hexane and EtOAc (1:1) to give 21 (2.3 g, 49% yield).
[ꢁ]2D1 +6ꢀ (MeOH; c 0.10); EIMS (probe) 70 eV, m/z
(rel. int.): 230 [M]+ (10), 229(10), 186 (100), 178 (29),
144 (14), 128 (23); HR-EIMS: [M]+ at m/z 230.0169
1
146.0579); H-NMR (500 MHz, CDCl3): ꢀ 1.40 (3H, d,
J=5.0 Hz), 3.43 (1H, m), 4.16 (2H, m), 4.67 (1H, q,
J=5.0 Hz), 4.98 (1H, d, J=4.9 Hz), 9.77 (1H, s). Com-
pound 18; EIMS (probe) 70 eV, m/z (rel. int.): 173 [M-
H]+ (4), 145 [M-CHO]+ (85), 101 (100); HR-EIMS:
[M-H]+ at m/z 173.0471 (C7H9O5 requires 173.0450);

138
N. Hirai et al. / Phytochemistry 55 (2000) 131±140
(C8H10N2O2S2 requires 230.0184); 1H-NMR (300 MHz,
CDCl3): ꢀ 1.38 (3H, d, J=5.1 Hz, CH3), 3.13 (1H, dd,
J=13.9 and 5.6 Hz, H-1), 3.29 (1H, dd, J=13.9 and 7.8
Hz, H-1), 3.58 (1H, ddd, J=1.9, 1.8 and 1.7 Hz, H-3),
4.21 (1H, dd, J=12.6 and 1.9 Hz, H-4), 4.28 (1H, ddd,
J=7.8, 5.6 and 1.8 Hz, H-2), 4.40 (1H, dd, J=12.6 and
1.7 Hz, H-4), 4.83 (1H, q, J=5.1 Hz).
ture of toluene, Me2CO and MeOH (40 : 2 :1) as eluant to
give (1R,3R,4R)-2 (16 mg) and (2S,3R,4R)-4 (9 mg).
Fraction 112 was puri®ed with an ODS HPLC column
(YMC SH-342-5, 20 i.d. Â150 mm) with 30% MeOH in
1
0.1% aqueous HOAc at 3.0 ml min with detection at
254 nm to give (1S,3R,4R)-1 (tR 11.4 min, 7 mg). Fraction
115 was puri®ed with an ODS HPLC column according
to the same method as used for 1 to give (2R,3R,4R)-3
(tR 11.5 min, 3 mg). (1S,3R,4R)- (+) -4-Hydroxy-3-
hydroxymethyl-1,2-dithiolane-1-oxide [(1S,3R,4R)-(+)-
1]. [ꢁ]2D5 +497ꢀ (MeOH; c 0.04); HR-FABMS (matrix:
glycerol): [MH]+ at m/z 169.0003 (C4 H9O3S2 requires
168.9993). (1R,3R,4R)-( )-4-Hydroxy-3-hydroxymethyl-
3.5.6. (3R,4R)-4,6-O-Ethylidene-4-hydroxy-3-
hydroxymethyl-1,2-dithiolane (22)
Compound 21 (2.3 g) was dissolved in 60 ml of a
mixture of EtOH and 1 N KOH (3:1), and heated under
re¯ux for 5 h with stirring. The solution was diluted
with 60 ml of H2O and partitioned with 40 ml of EtOAc
four times. The organic layer was dried (Na2SO4), ®l-
tered, concentrated and subjected to silica gel (60 g)
chromatography using a mixture of toluene and Me2CO
(9:1) as the eluant to give 22 (1.2 g, 69% yield). [ꢁ]D25
80ꢀ (CHCl3; c 0.10); EIMS (probe) 70 eV, m/z (rel.
int.): 178 [M]+ (100), 134 (55), 104 (32); HR-EIMS:
[M]+ at m/z 178.0118 (C6H10O2S2 requires 178.0122);
1H-NMR (300 MHz, CDCl3): ꢀ 1.39 (3H, d, J=5.0 Hz,
CH3), 3.27 (1H, dd, J=12.2 and 1.6 Hz, H-5), 3.37 (1H,
dd, J=12.2 and 5.0 Hz, H-5), 3.44 (1H, m, H-3), 4.14
(2H, m, H-4), 4.27 (2H, br. s, H-6), 4.79 (1H, q, J=5.1
Hz).
1,2-dithiolane-1-oxide [(1R,3R,4R)-( )-2]. [ꢁ]D25
302ꢀ
(MeOH; c 0.10); HR-FABMS (matrix: glycerol): [MH]+
at m/z 169.0012 (C4H9O3S2 requires 168.9993).
(2R,3R,4R)-( )-4-Hydroxy-3-hydroxymethyl-1,2-dithio-
lane-2-oxide [(2R,3R,4R)-( )-3]. [ꢁ]2D5 213ꢀ (MeOH; c
0.04); HR-FABMS (matrix: glycerol): [MH]+ at m/z
168.9990 (C4H9O3S2 requires 168.9993). (2S,3R,4R)-(+)-
4-Hydroxy-3-hydroxymethyl-1,2-dithiolane-2-oxide [(2S,
3R,4R)-(+)-4]. [ꢁ]D25 +459ꢀ (MeOH; c 0.04); HR-FABMS
(matrix: glycerol): [MH]+ at m/z 168.9993 (C4H9O3S2
1
requires 168.9993). The UV, FAB mass and H-NMR
spectra of the synthesized 1±4 were the same as those of
natural 1±4, respectively, isolated from the weed.
3.5.7. (3R,4R)-4-Hydroxy-3-hydroxymethyl-1,2-
dithiolane (23)
3.6. Synthesis of (3S,4S)-1±4
A solution of 22 (1.10 g) was dissolved in 18 ml of a
mixture of 1 N HCl and MeOH (5:4) and heated under
re¯ux for 8 h with stirring. The solution was neutralized
with 1 N KOH, concentrated and subjected to silica gel
(40 g) chromatography using a mixture of CHCl3 and
MeOH (9:1) as the eluant to give 23 (0.61 g, 65% yield).
[ꢁ]2D3 +18ꢀ (MeOH; c 0.10); EIMS (probe) 70 eV, m/z
(rel. int.): 152 [M]+ (100), 134 (14), 121 (11); HR-EIMS:
[M]+ at m/z 151.9965 (C4H8O2S2 requires 151.9966);
1H-NMR (500 MHz, CD3OD): ꢀ 3.14 (1H, dd, J=11.6
and 3.0 Hz, H-5), 3.33 (1H, dd, J=11.6 and 4.3 Hz, H-
5), 3.65 (1H, ddd, J=8.0, 5.9 and 4.5 Hz, H-3), 3.75
(1H, dd, J=11.3 and 8.0 Hz, H-6), 4.03 (1H, dd, J=11.3
and 5.9 Hz, H-6), 4.78 (1H, ddd, J=4.5, 4.3 and 3.0 Hz,
H-4).
Isomers, (3S,4S)-1±4, were synthesized from 4,6-O-
ethylidene-d-glucose (5.0 g) by the same method as used
for natural (3R,4R)-1-4. (1R,3S,4S)-( )-4-Hydroxy-3-
hydroxymethyl-1,2-dithiolane-1-oxide (10 mg) [(1R,3
S,4S)-( )-1]. [ꢁ]2D6 566ꢀ (MeOH; c 0.10); HR-FABMS
(matrix: glycerol): [MH]+ at m/z 168.9992 (C4H9O3S2
requires 168.9993). (1S,3S,4S)-(+)-4-Hydroxy-3-hydro-
xymethyl-1,2-dithiolane-1-oxide (29 mg) [(1S,3S,4S)-
(+)-2]. [ꢁ]2D6 +292ꢀ (MeOH; c 0.10); HR-FABMS
(matrix: glycerol): [MH]+ at m/z 168.9993 (C4H9O3S2
requires 168.9993). (2S,3S,4S)-(+)-4-Hydroxy-3-hydro-
xymethyl-1,2-dithiolane-2-oxide (18 mg) [(2S,3S,4S)-
(+)-3]. [ꢁ]2D6 +273ꢀ (MeOH; c 0.10); HR-FABMS
(matrix: glycerol): [MH]+ at m/z 168.9996 (C4 H9O3S2
requires 168.9993). (2R,3S,4S)-( )-4-Hydroxy-3-hydro-
xymethyl-1,2-dithiolane-2-oxide (14 mg) [(2R,3S,4S)-
3.5.8. (3R,4R)-4-Hydroxy-3-hydroxymethyl-1,2-
dithiolane-1 and 2-oxides [(3R,4R)-1±4]
( )-4]. [ꢁ]2D6
447ꢀ (MeOH; c 0.11); HR-FABMS
(matrix: glycerol): [MH]+ at m/z 168.9992 (C4H9O3S2
1
To a solution of 23 (0.61 g) in HOAc (5 ml) was
added 30% H2O2 aqueous solution (0.91 ml), and the
mixture was stirred for 1 h at room temperature. The
mixture was concentrated and subjected to silica gel (80 g)
chromatography using a mixture of CHCl3 and MeOH
(39:1). The eluate was collected in fractions of 16 ml.
Fractions 65±71 were combined and concentrated to give
a mixture (85 mg) of (3R,4R)-2 and-4. The mixture was
subjected to silica gel (15 g) chromatography using a mix-
requires 168.9993). The UV, FAB mass and H-NMR
spectra of the synthesized 1±4 were the same as those of
natural 1±4, respectively, isolated from the weed.
3.7. Isomerization of 1±4, and incubation of 3 in an
H218O solution
A D2O solution (0.5 ml) of 1 (2.5 mg) was added to an
NMR tube (5 mm i.d.), and heated at 50ꢀC for 10 days.

N. Hirai et al. / Phytochemistry 55 (2000) 131±140
139
Isomerization was monitored by measurement of its 1H-
3.9. Contents of 1±7 in S. zeylanica
NMR spectra every 24 h. Ratios of 1±4 in the solution
were calculated from signal integrals of each isomer.
Isomerization of 2±4 was examined by the same method
as used for 1.
Compound 3 (2.8 mg) was dissolved in 0.1 ml of H218ꢀO
(95 atom% 18O, Aldrich), heated in a sealed tube at 50 C
for 24 h, and its FAB mass spectrum was measured.
The MeOH extracts of the fresh and dried S. zeyla-
nica were analyzed with an ODS HPLC column (YMC
AQ-311, 6 i.d.Â100 mm) by eluting with 100% H2O for
1±4, and with 30% MeOH in 0.1% HOAc aqueous
solution for 5±7 at 1.0 ml min 1, and the eluate was
monitored at 254 nm. Compounds 1 and 3, 2 and 4, 5, 6
and 7 were eluted at tR 5.7 min, 6.7 min, 12.5 min, 9.6
min and 6.7 min, respectively. The amounts of 1 and 3,
2 and 4, 5, 6, and 7 were calculated from the calibration
curves between peak area and amount of authentic
samples. The contents in the dried plant were corrected
by H2O content (80%) of the fresh plant.
3.8. Bioassays
For rice seedling assay, seeds of rice (Oryza sativa L.
cv. Nihonbare) were soaked in EtOH for 5 min, ster-
ilized with 1% antiformin (NaClO4) for 1 h, and washed
with running tap water for 3 h. The seeds were allowed
to germinate in H2O for 2 days at 30ꢀC. The resulting
seedlings were placed in a glass tube containing 2 ml of
test solution, and grown with the tube sealed with a
sheet of polyethylene ®lm under continuous illumina-
tion at 30ꢀC. The weight of the root was measured after
7 days, and the inhibition ratio was calculated. The
inhibition ratio was de®ned as [(A B)/A]Â100%, where
A=the mean weight of the root when H2O was used,
and B=the mean weight of the root when test com-
pound was used.
Acknowledgements
This study was supported by a Grant-in-Aid (Bio-
cosmos program) from the Ministry of Agriculture,
Forestry and Fisheries of Japan (BCP-00-I-A-1). We
thank Professor Naohito Takeda at Faculty of Phar-
macy, Meijo University for measurement of a high
resolution FAB mass spectrum of compound 5.
For lettuce seed germination assay, 50 seeds of lettuce
(Lectuca sativa L. cv. Cisco) were placed on two sheets
of No. 2 ®lter paper (5.5 cm in diameter, Toyo Roshi
Ltd.) soaked in 3 ml of a test solution, and allowed to
germinate under illumination at 25ꢀC. After 48 h, the
inhibition ratio was calculated. The inhibition ratio was
de®ned as [(A B)/A]Â100%, where A=the number of
seeds that germinated when H2O was used, and B=the
number of seeds that germinated when test compound
was used.
For antimicrobial assay against Bacillus subtilis,
Escherichia coli, Pseudomonas aeruginosa, Staphylo-
coccus aureus, Candida albicans, Saccharomyces cerevi-
siae, Aspergillus niger, Fusarium oxysporum, Pyricularia
oryzae, a pulp disk (5 mm in diameter) soaked in MeOH
solution (1000 ppm) of test compound was dried, put on
potato±dextrose±agar medium containing the micro-
organism, and incubated at 37ꢀC for the bacteria and at
25ꢀC for the fungi in the dark. The diameter of the zone
showing growth inhibition was measured after 3 days
for the bacteria, and after 7 days for the fungi. For
antimicrobial assay against Pythium debaryanum,
Phytophthora infestans, Pyricularia oryzae, Botrytis
cinerea, Rhizoctonia solani, and Septoria tritici, the fungi
on an agar disk (5 mm in diameter) were incubated on
References
Allen Jr., P., Brook, J.W., 1962. Preparation of alkyl thiolsul®nates,
thiolsulfonates, and a-disulfones. Journal of Organic Chemistry 27,
1019±1020.
Boros, C.A., Stermitz, F.R., 1991. Iridoids. An updated review, part
II. Journal of Natural Products 54, 1173±1246.
Buck, K. W., Foster, A. B., Pardoe, W.D., Qadir, M.H., Webber,
J.M., 1966. Observations on the n.m.r. spectra of some derivatives
of 1,4-oxathian S-oxide. Anisotropy of the S!O bond. Chemical
Communications, 759±761.
Calis, I., Sticher, O., 1984. Secoiridoid glucosides from Lonicera peri-
clymenum. Phytochemistry 23, 2539±2540.
Cavallito, C.J., Bailey, J.H., 1944. Allicin, the antimicrobial principle
of Allium sativum. I. Isolation, physical properties and antibacterial
action. Journal of American Chemical Society 66, 1950±1951.
Corey, E.J., Mitra, R.B., 1962. l(+)-2,3-Butanedithiol: synthesis and
application to the resolution of racemic carbonyl compounds.
Journal of American Chemical Society 84, 2938±2941.
Dale, J.A., Mosher, H.S., 1973. Nuclear magnetic resonance enantio-
mer reagents. Con®gurational correlations via nuclear magnetic
resonance chemical shifts of diastereomeric mandelate, O-methyl-
mandelate, and a-methoxy-a-tri¯uoromethylphenylacetate (MTPA)
esters. Journal of American Chemical Society 95, 512±519.
Damtoft, S., Franzyk, H., Jensen, S.R., 1994. Fontanesioside and 5-
hydroxysecologanol from Fontanesia phillyreoides. Phytochemistry
35, 705±711.
Foster, A.B., Duxbury, J.M., Inch, T.D., Webber, J.M., 1967. Sulph-
oxides of methyl 2,6-anhydro-2-thio-a-d-altropyranoside. Com-
ments on the assignment of sulphoxide con®guration by the nuclear
magnetic resonance method. Chemical Communications 881±883.
Hall, D.M., Stamm, O.A., 1970. 4,6-O-Alkylidene derivatives of d-
glucose and its methyl pyranosides. An improved acetalation pro-
cedure. Carbohydrate Research 12, 421±428.
potato±dextrose±agar
medium
containing
test
compound at 100 ppm at 25ꢀC in the dark. The dia-
meters of fungal colonies were measured after 5 days,
and compared with those of the respective organisms
grown on potato±dextrose±agar medium without the
compound.

140
N. Hirai et al. / Phytochemistry 55 (2000) 131±140
Harborne, J.B., Baxter, H., Moss, G.P., 1999. Phytochemical Dic-
tionary: A Handbook of Bioactive Compounds from Plants. 2nd
Edition. Taylor & Francis, London (pp. 621±622).
Sphenoclea zeylanica showing di€erential response to 2,4-D. Weed
Research 30, 245±250.
Noda, K., Teerawatsakul, M., Prakongvongs, C., Chaiwiratnukul, L.,
1994. Major weeds in Thailand. National weed science research
institute project, Bangkok, p. 109.
Holm, L.G., Plucknett, D.L., Pancho, J.V., Herberger, J.P., 1977. The
World's Worst Weeds. The University Press of Hawaii, Honolulu,
pp. 446±449.
Pons, T.L., 1982. Factors a€ecting weed seed germination and seedling
growth in lowland rice in Indonesia. Weed Research 22, 155±161.
Premasthira, C., Zungsontiporn, S., 1996a. Allelopathic e€ect of
extract substances from gooseweed (Sphenoclea zeylanica) on rice
seedlings. Weed Research, Japan 41, 79±83.
Ishii, A., Nakamura, S., Yamada, T., Nakayama, J., 1997. Stereo-
chemistry of dithiirane 1-oxides: optical resolution, absolute
con®guration, and racemization and isomerization of dithiirane 1-
oxides. Tetrahedron 53, 12203±12214.
Kato, A., Numata, M., 1972. Brugierol and isobrugierol, trans- and
cis-1,2-dithiolane-1-oxide, from Brugiera conjugata. Tetrahedron
Letters 203±206.
Premasthira, C., Zungsontiporn, S., 1996b. Allelopathic potential of
gooseweed (Sphenoclea zeylanica Gaertn.) in submerged soil. Weed
Research, Japan 41, 103±106.
Kawai, H., Kuroyanagi, M., Ueno, A., 1988. Iridoid glucosides from
Lonicera japonica Thunb. Chemical & Pharmaceutical Bulletin 36,
3664±3666.
Rice, E.L., 1984. Allelopathy. 2nd Edition, Academic Press, New
York.
Singh, P.K., Field, L., Sweetman, B.J., 1988. Organic disul®des and
related substances. 49. Preparation of cyclic thiosul®nates and
reaction with thiols. Journal of Organic Chemistry 53, 2608±
2612.
Kiso, M., Nakamura, A., Tomita, Y., Hasegawa, A., 1986. A novel
route to d-erythro-shingosine and related compounds from mono-
O-isopropylidene d-xylose or-d-galactose. Carbohydrate Research
158, 101±111.
Soerjani, M., Kostermans, A.J.G.H., Tjitrosoepomo, G., 1987. Weeds
of rice in Indonesia. Balai Pustaka, Jakarta, pp. 552±553.
Tomassini, L., Cometa, M.F., Sera®ni, M., Nicoletti, M., 1995. Isola-
tion of secoiridoid artifacts from Lonicera japonica. Journal of
Natural Products 58, 1756±1758.
Konno, K., Hirayama, C., Yasui, H., Nakamura, M., 1999. Enzy-
matic activation of oleuropein: a protein crosslinker used as a che-
mical defense in the privet tree. Proceedings of the National
Academy of Sciences of the United States of America 96, 9159±
9164.
Wudl, F., Gruber, R., Padwa, A., 1969. Absence of pyramidal
inversion in a cyclic thiolsul®nate. Tetrahedron Letters, 2133±2136
Yanagawa, H., Kato, T., Kitahara, Y., 1973. Asparagusic acid-S-
oxides, new plant growth regulators in etiolated young asparagus
shoots. Tetrahedron Letters, 1073±1075.
Lindberg, B., Bergson, G., 1965. Oxidation of 1,2-dithiolanes. Arkiv
for Kemi 23, 319±333.
Mercado, B.L., De Datta, S.K., Migo, T.R., Baltazar, A.M., 1990.
Growth behavior and leaf morphology of Philippine strains of