Identification of (3S, 9R)- and (3S, 9S)-megastigma-6,7-dien-3,5,9-triol 9-O-β-D-glucopyranosides as damascenone progenitors in the flowers of Rosa damascena Mill.

Article abstract of DOI:10.1271/bbb.66.2692

The progenitors of damascenone (1), the most intensive C ₁₃-norisoprenoid volatile aroma constituent of rose essential oil, were surveyed in the flowers of Rosa damascena Mill. Besides 9-O-β-D-glucopyranosyl-3-hydroxy-7,8-didehydro-β-ionol (4b), a stable progenitor already isolated from the residual water after steam distillation of flowers of R. damascena Mill., two labile progenitors were identified to be (3S, 9R)- and (3S, 9S)-megastigma-6,7-dien-3,5,9-triol 9-O-β-D-glucopyranosides (2b) based on their synthesis and HPLC-MS analytical data. Compound 2b gave damascenone (1), 3-hydroxy-β-damascone (3) and 4b upon heating under acidic conditions.

Full text of DOI:10.1271/bbb.66.2692

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Identification of (3S, 9R)- and (3S, 9S)-
Megastigma-6,7-dien-3,5,9-triol 9-O-β-D-
glucopyranosides as Damascenone Progenitors in
the…
Masayuki SUZUKIa, Shigetaka MATSUMOTOa, Masaya MIZOGUCHIa, Satoshi HIRATAa,
Kazuteru TAKAGIa, Ikue HASHIMOTOa, Yumiko YAMANOb, Masayoshi ITOb, Peter
FLEISCHMANNc, Peter WINTERHALTERc, Tetuichiro MORITAd & Naoharu WATANABEa
a Shizuoka University 836 Ohya, Shizuoka 422-8529, Japan
b Kobe Pharmaceutical University 4-19-1 Motoyamakita, Higashinada-ku, Kobe, Hyogo
658-8558, Japan
c Institut für Lebensmittel Chemie TU-Braunschweig, Schleinitzstr. 20, Braunschweig
D-38106, Germany
d Jeol Co., Ltd., Applied Research Center 3-1-2 Musashino, Akishima, Tokyo 196-8558,
Japan
Published online: 22 May 2014.
To cite this article: Masayuki SUZUKI, Shigetaka MATSUMOTO, Masaya MIZOGUCHI, Satoshi HIRATA, Kazuteru TAKAGI, Ikue
HASHIMOTO, Yumiko YAMANO, Masayoshi ITO, Peter FLEISCHMANN, Peter WINTERHALTER, Tetuichiro MORITA & Naoharu
WATANABE (2002) Identification of (3S, 9R)- and (3S, 9S)-Megastigma-6,7-dien-3,5,9-triol 9-O-β-D-glucopyranosides
as Damascenone Progenitors in the…, Bioscience, Biotechnology, and Biochemistry, 66:12, 2692-2697, DOI: 10.1271/
bbb.66.2692
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Biosci. Biotechnol. Biochem., 66 (12), 2692–2697, 2002
Note
Identiˆcation of (3
S, 9R)- and (3S, 9S)-Megastigma-6,7-dien-3,5,9-triol
9- -glucopyranosides as Damascenone Progenitors in the Flowers
O
-
b
-
D
of Rosa damascena Mill.
1
Masayuki SUZUKI,1 Shigetaka MATSUMOTO,1 Masaya MIZOGUCHI,1 Satoshi HIRATA
,
2
Kazuteru TAKAGI,1 Ikue HASHIMOTO,1 Yumiko YAMANO,2 Masayoshi ITO
,
4
Peter FLEISCHMANN,3 Peter WINTERHALTER,3 Tetuichiro MORITA
,
†
1,
and Naoharu WATANABE
1Shizuoka University, 836 Ohya, Shizuoka 422-8529, Japan
2Kobe Pharmaceutical University, 4-19-1 Motoyamakita, Higashinada-ku, Kobe, Hyogo 658-8558, Japan
3Institut fu
ä r Lebensmittel Chemie, TU-Braunschweig, Schleinitzstr. 20, Braunschweig D-38106, Germany
4Jeol Co., Ltd., Applied Research Center, 3-1-2 Musashino, Akishima, Tokyo 196-8558, Japan
Received May 7, 2002; Accepted July 15, 2002
The progenitors of damascenone (1), the most inten-
sive C13-norisoprenoid volatile aroma constituent of
rose essential oil, were surveyed in the ‰owers of Rosa
as glycosidic or polyhydroxylated compounds, by
heat treatment or acidic hydrolysis.6,7) Isoe et al.8) and
OhloŠ et al.9) have originally postulated that the pro-
genitors of 1 were produced by the enzymatic
cleavage of carotenoids, such as neoxanthin, as
shown in Fig. 1. The primary degradation product
was grasshopper ketone, which was expected to yield
the key intermediate, megastigma-6,7-dien-3,5,9-
triol (2a). Compound 2a was transformed to 1 and 3-
damascena Mill. Besides 9-
O
-
b
-
D
-glucopyranosyl-3-
hydroxy-7,8-didehydro- -ionol (4b), a stable progenitor
b
already isolated from the residual water after steam dis-
tillation of ‰owers of R. damascena Mill., two labile
progenitors were identiˆed to be (3
)-megastigma-6,7-dien-3,5,9-triol
S
, 9
9-
R
O
)- and (3
S,
9
S
-
b
-
D
-gluco-
pyranosides (2b) based on their synthesis and HPLC-
MS analytical data. Compound 2b gave damascenone
hydroxy-b-damascone (3) under the acidic condi-
tions.10,11) Several progenitors of 1 have been isolated
(1), 3-hydroxy-
b-damascone (3) and 4b upon heating
from various plant tissues and beverages. Two im-
under acidic conditions.
portant polyols, 2a and 3-hydroxy-7,8-didehydro-b-
ionol (4a), have been identiˆed in wine as progenitors
of 1.10,12) The C-9- and C-3-
-glucopyranosides
(4b, 4c) of the latter polyol, and megastigma-6,7-
Key words: rose ‰ower; damascenone; C13-norisopre-
O
-
b
-
D
noid; progenitor; megastigma-6,7-dien-
3,5,9-triol 9-
O
-
b-
D
-glucopyranoside
dien-3,5,9-triol 9-
O
-
b- -glucopyranoside (2b) have
D
been isolated and characterized from Riesling wine13)
14)
The ‰owers of Rosa damascena Mill. are utilized
for the production of rose essential oil, in which
several volatile C13-norisoprenoids have been identi-
ˆed. Among them, damascenone [1, 1-(2,6,6-
trimethyl-1,3-cyclohexadien-1-yl)-2-buten-1-one] was
the ˆrst constituent to be identiˆed1) in the essential
oil of R. damascena Mill., and showed an extremely
and Lycium halimifolium
,
respectively. Apples
have been reported to contain several disaccharide
glycosides of 4a.15) Quite recently, Kumazawa et al
.
16)
have reported the presence of the glucoside of 4a as a
progenitor of 1 in a black tea extract. Compound 4b
has also been isolated as a progenitor of 1 from the
residual water after steam distillation of the ‰owers
of R. damascena Mill.17) However, 4b would have
been derived from an unstable compound such as 2b
during steam distillaiton of the ‰owers. Therefore,
the genuine progenitor of 1 in rose ‰owers is still
elusive. This paper describes the identiˆcation and
characterization of progenitors of 1 in the ‰owers of
R. damascena Mill.
low-threshold sensory value (2 pg g of water).2) This
W
compound has been also identiˆed in various types of
plant tissue and in plant-derived beverages.3–5) Com-
pound 1 was neither produced nor released from the
‰owers of R. damascena Mill., whereas the essential
oil contained 1. Thus, it has been suggested that 1
was produced from the progenitors during the steam
distillation of the rose ‰owers. Many other C13-
norisoprenoid volatile compounds have been suggest-
ed to also be produced from their progenitors, such
Identiˆcation of damascenone progenitors from
the ‰owers of R. damascena Mill. Frozen ‰owers
†
To whom correspondence should be addressed.

(3
S
, 9
R
)- and (3
S
, 9
S
)-Megastigma-6,7-dien-3,5,9-triol 9-
O
-
b
-
D
-glucopyranosides in Rose Flowers
2693
Fig. 2. HPLC Analyses of a Sample (A) Puriˆed from the Flow-
ers of Rosa damascena Mill., and of Authentic (3
and (3 , 9 )-2b (C).
S
, 9
R
)-2b (B)
Fig. 1. Hypothetical Biogenetic Pathway for Damascenone
Progenitors 2a–2c and 4a–4c, and Their Transformation to
S
S
Damascenone (1) and 3-Hydroxy-b-damascone (3).
11)
none progenitor in wine by Sefton et al
.
and
(10 kg fr. wt) at the full bloom stage were extracted
with 70 MeOH, and the resulting extract was sub-
jected to chromatography on an Amberlite XAD-2
column (water, MeOH). The MeOH eluate was
acetylated and puriˆed by chromatography on silica
gel cartridge column with hexane-EtOAc as the
solvent. The progenitor fractions of 1 yielded 3-
Skouroumounis et al.
13) Although at least three addi-
z
tional fractions were obtained as the progenitors be-
sides 4b, we could not isolate any others due to their
instability. This fact strongly suggests the presence of
other labile progenitors such as allenic triol (2a), its
9-O-glucopyranoside (2b), and 3-O-glucopyranoside
(2c) in these fractions. As already suggested by
Juglisi et al.,19) 2a was quite labile to yield 4a, 1, and 3
at room temperature and at pH 3.0 in an aqueous
hydroxy-
b-damascone (3) together with 1 after an
acid treatment according to the method of Skourou-
11,12)
mounis et al
.
Each fraction was further puriˆed,
environment. They have also postulated that the
b- -
D
being guided by detection of the ion peaks for m z
glucosyl moiety at C-3 and or C-9 stabilized 2a,
W
W
190 (M+) and m z 175 (M+-CH3) at tR 29.0 min for
although it may lower the rate of dehydration at
＝
W
1, and m z 208 (M+) at tR
＝
52.2 min for 3 by GC-
these positions.20) The progenitor fractions can thus
W
MS chromatography. Successive ‰ash chromato-
be expected to contain 9-
O
-
b
-D
-glucopyranoside (2b)
graphy on an LP-75 silica gel cartridge (hexane-
t
-
or 3- -glucopyranoside (2c) as a progenitor of 1.
O
-
b
-
D
As 4b had already been isolated from the residual
water after steam distillation of the ‰owers, 4b is
thought to be one of the products transformed from
2b during steam distillation of the ‰owers and or
W
evaporation of aqueous MeOH used as an extraction
solvent. We therefore surveyed 2b in the ‰owers of
R. damascena.
To conˆrm the chemical structures of the progeni-
tors, we ˆrst tried to synthesize 2b. Two diastereom-
ers of 2b were obtained from grasshopper ketone8,21)
in four steps. The absolute stereo-structures were de-
termined by a direct comparison with (3
S, 9R)- and
(3 , 9 )-2b, respectively, which had been enantio-
S
S
selectively synthesized. This enantio-selective synthe-
sis will be reported elsewhere.
The stability of 2b and its 3,2?,3?,4?,6?-penta-O-

2694
M. SUZUKI et al.
reliable due to the low (1 5–1 10th) intensity being
W
W
compared with those for m z 420.3 and m z 433.3.
W
W
Although Kotseridis et al.
22) have reported a quan-
titative determination of free and hydrolytically
liberated 1 in wine by a stable isotope dilution assay,
there is almost no quantitative data on the generation
of 1 from 2b. To conˆrm the transformation of 2b to
1, we surveyed the reaction conditions for hydrolysis.
Compound (3S, 9S)-2b was treated under the condi-
tions shown in the Experimental section. Compound
1 was produced only when 2b was treated in an aque-
ous solution at pH 2.0 at 90
(3 , 9
(4.8 mol
and (3
z
9C. Within half an hr,
S
S
)-2b was transformed or degraded to give 1
) and 3 (8.2 mol ) as volatile compounds,
, 9 )-4b (13.0 mol ), as well as several
z
z
Fig. 3. Selected Ion Traces from the LC-APCI MS Analysis at
W
R
S
m z 387.2, m z 420.3, and m z 433.3 of Authentic (3
S
, 9
R)-2b
W
W
W
unidentiˆed compounds which were found in the
non-volatile fraction. A longer reaction period
(1.0 hr) did not signiˆcantly increase the amount of 1
and (3
S
, 9
S
)-2b (A) and of a Sample (B) Puriˆed from the Flow-
ers of Rosa damascena Mill.
(5.1 mol
nor 3 (data not shown). As a conclusion, labile pro-
genitors (3 , 9 )- and (3 , 9 )-2b for 1 were identi-
z). The reaction at pH 4.0 gave neither 1
acetate was investigated by evaporating in vacuo the
samples with MeOH or MeCN in water. Compound
2b was revealed to be unstable, whereas its acetate
was relatively stable. Thus, the majority of the pro-
genitors found in the MeOH extract already men-
tioned must have been gradually transformed to
other compound(s) during the course of the puriˆca-
tion.
S
R
S
S
ˆed for the ˆrst time in the ‰owers of R. damascena
Mill. The instability of 2b was conˆrmed to explain
the reason for the disappearance of progenitors dur-
ing puriˆcation of the MeOH extract from R.
damascena Mill. A portion of (3
the residual water after steam distillation was strong-
ly suggested to be one of the artifacts from (3 , 9 )-
2b. We are now surveying all the possible progenitors
of 1 in the ‰owers of R. damascena Mill. by LC-MS.
R, 9R)-4b found in
Based on the empirical results just mentioned, the
‰owers (50 g fr. wt) were extracted with an
HCOOH–NH4OH buŠer to give a glycosidic progen-
itor fraction. The extract was lyophilized, and the
materials were acetylated and then puriˆed by succes-
sive chromatography on a silica gel cartridge column
S
R
Experimental
(hexane-
t
-butyl methyl ether) and by preparative
Instruments. Optical rotation data were measured
with a DIP-370 (Jasco) polarimeter at ambient tem-
perature. 1H- and 13C-NMR and two-dimensional
spectra were recorded by either a Jeol JNM-LA500
or a Jeol JNM-EX270 spectrometer. Mass spectra
(MS) were acquired with a Jeol JMS-DX303-HF
spectrometer. GC-MS data were measured with a
Jeol JMS-DX302 instrument with a Jeol JMA-
DA5000 MS data system equipped with GC appara-
tus (Yokogawa Hewlett Packard 5890) was used un-
der the following conditions: column, PEG-20M
TLC (hexane-diethyl ether) guided by the chromato-
graphic behavior of the pentaacetate of 2b. After
treating with MeONa, the sample was further puri-
ˆed by preparative HPLC (ODS-AM, MeCN-water)
to give the desired fraction. This fraction was ana-
lyzed by HPLC apparatus equipped with a pho-
todiode array detector. As shown in Fig. 2, peaks at
tR 24.04 and 26.51 min were detected at the same
retention times for synthetic (3
S
, 9
R
)-2b and (3
S
S
,
,
9
9
S)-2b, respectively. Finally, the presence of (3
R
)-2b and (3
S
, 9
S
)-2b was conˆrmed by an LC-
(0.25 mm
gradient from 60
temp., 150 C; carrier gas, He (1 ml min). To detect
×
50 m); oven temp., a linear temperature
APCI MS analysis. As shown in Fig. 3, the progeni-
9
C to 220 C (3 C min); injector
9
9
W
W
tor fraction gave two prominent peaks with selected
9
W
ion monitoring: SIM traces at m z 387.2 (M„H)„,
1 and 3 after the acid treatment, ions were monitored
W
420.3 (M+O2)„ and 433.3 (M„H+HCOOH)„ at tR
at m z 208 [M]+ for 3, 190 [M]+ for 1 and or
W
W
19.26 and 21.34 min, respectively. The ion traces of
[M„H2O]+ for 3, and 175 [190-CH3]+. The
t
Rs for 1
the authentic compounds, (3
S
, 9
R
)-2b and (3
S
, 9
S
)-
and 3 were 29.0 and 52.2 min, respectively.
2b, showed the peaks at the same retention times as
those for the desired fraction, indicating the presence
Isolation and characterization of damascenone
progenitors from the ‰owers of R. damascena Mill.
The ‰owers of Rosa damascena Mill. were harvested
at the full-bloom stage in Fukuroi (Shizuoka, Japan)
in 1998, then stored in a freezer („30
cultivar was grown at the University Farm of
of (3
S
, 9
R
)-2b and (3
S, 9S)-2b. The ratio of (3S,
9
R
)-2b and (3
S
, 9S)-2b could be roughly calculated
to be ca. 10–4 to 1 based on the ion intensities at m z
W
420.3 and 433.3, although the ratio of the ion inten-
sity at m z 387.2 was 1 to 1. This latter ratio is not
9C). The same
W

(3
S
, 9
R
)- and (3
S
, 9
S
)-Megastigma-6,7-dien-3,5,9-triol 9-
O
-
b
-
D
-glucopyranosides in Rose Flowers
2695
Shizuoka University (Japan) in 2000. The frozen
‰owers (10 kg) were extracted by 70 MeOH under
ice-cooling, and the extract was loaded into a column
of Amberlite XAD-2 (120 660 mm, Vt 7500 ml)
that has been equilibrated with water and developed
with MeOH to give a progenitor fraction. After con-
centration, the MeOH extract was acetylated
(pyridine-acetic anhydride) and puriˆed by ‰ash
chromatography on an LP-75 silica gel cartridge
partially puriˆed by preparative silica gel TLC
＝
z
(CH2Cl2:hexane:EtOAc 6:2.5:1.5) to give the (3
S,
9
S
)- and (3 , 9 )-diastereomers. These were treated
S
R
×
＝
・
with LiOH H2O in the usual manner and further
puriˆed by HPLC (MeCN-water, YMC pack ODS-
×
AQ, 20 250 mm) to give (3
0.75 yield) and (3 , 9 )-2b (3.2 mg, 2.9z
S
, 9
S
)-2b (0.8 mg,
yield).
z
S
R
Spectral data:
3-O-Acetyl-megastigma-6,7-diene-3,5,9-triol (3-O-
acetyl-2a). 1H-NMR (270 MHz, CDCl3)
: 1.08, 1.10
6.6 Hz, H-10),
1.36 (3H, s, H-13), 1.37 (1H, overlapped with H-13,
H-2ax), 1.38, 1.39 (each 3 2H, s, H-12), 1.47 (1H,
(75
×
300 mm, Vt
＝
1325 ml; Wako, Japan; hexane-
＝
EtOAc 8:2–3:7 v v) to yield eight fractions.
d
W
＝
(each 3 2H, s, H-11), 1.30 (3H, d, J
To detect the progenitors, a portion (10–20 g fr.
wt. eq.) of each fraction was deacetylated by treating
W
with MeONa and then heated at 909C for 2 h at pH 2
W
in a sealed tube.6,7) The volatile compounds formed
were extracted by an azeotropic mixture of pentane-
CH2Cl2 (2:1) under neutral conditions and analyzed
by GC-MS. Two desired fractions were separately
further puriˆed by silica gel ‰ash chromatography on
dd,
J 12.9, 8.8 Hz, H-4ax), 1.96 (1H, ddd, J 2.3,
＝ ＝
4.2, 12.2 Hz, H-2eq), 2.03 (3H, s, acetate), 2.25 (1H,
＝
2.3, 4.2, 12.9 Hz, H-4eq), 4.33 (1H, m,
ddd,
H-9), 5.33 (1H, m, H-3), 5.41 (1H, d,
H-8); 13C-NMR (67.5 MHz, CDCl3)
: 23.3 (C10),
J
＝
J
5.7 Hz,
d
an LP-40 cartridge (40
×
＝
150 mm, Vt
＝
188 ml; hex-
29.2 (C12), 31.2 (C13), 32.2 (C11), 35.1 (C1), 45.1,
(C4), 45.3 (C2), 66.3 (C3), 67.9 (C9), 72.9 (C5), 100.1
(C8), 117.5 (C6), 197.6 (C7), 21.4 and 170.5
(acetate).
ane-
t-butyl methyl ether 9:1–3:7 v v) and then by
W
×
HPLC (column: ODS-AM 20 250 mm, YMC,
Japan; solvent: MeCN-water 50:50–75:25 v v) to
W
give four progenitor fractions. Compound 4b
(1.2 mg) was isolated from one of the less-polar frac-
tions as its pentaacetate.
(3S, 9S)-megastigma-6,7-diene-3,5,9-triol 9-O-
glucopyranoside ((3S, 9S)-2b). 1H-NMR (500 MHz,
D2O) : 1.07 (3H, s, H-11), 1.26 (3H, s, H-12), 1.32
(1H, dd, 11.9, 8.9 Hz, H-2ax), 1.36 (3H, d,
b
-
D
-
d
3,2
7,8-didehydro-
(3,2 ,3 ,4 ,6
-penta-O-acetyl-(3R, 9R)-4b). 13C-NMR
(125 MHz, CDCl3) : 22.3 (CH3-C5), 23.1 (CH3-C9),
28.5 (CH3-C1), 30.1 (CH3-C1), 35.9 (C1), 37.3 (C4),
42.1 (C2), 62.0 (C6 ), 67.5 (C9), 67.8 (C3), 68.4
(C4 ), 71.8 (C2 ), 71.9 (C5 ), 73.0 (C3 ), 84.0 (C7),
91.9 (C8), 98.9 (C1 ), 123.2 (C6), 137.9 (C5),
20.6–21.4 (CH3 5 of acetyl group), 169.3–170.7 (C
?
,3
?
,4
?
,6
?
-penta-O-acetyl-(3R, 9R)-3-hydroxy
J
＝
D
J
＝
6.4 Hz, H-10), 1.41 (1H, dd,
J
＝
12.0, 8.8 Hz, H-
b
-ionol 9-O- -glucopyranoside
b
-
?
?
?
?
4ax), 1.41 (3H, s, H-13), 1.89 (1H, ddd,
11.9 Hz, H-2eq), 2.16 (1H, ddd,
J
＝
2.0, 4.5,
＝
d
J
2.0, 4.5,
＝
8.5 Hz, H-2?), 3.33
12.0 Hz, H-4eq), 3.27 (1H, t,
(1H, ddd, 1.2, 5.8, 8.8 Hz, H-5
8.5, 8.8 Hz, H-4 ), 3.45 (1H, dd,
H-3 ), 3.70 (1H, dd, 5.8, 12.5 Hz, H-6
(1H, dd, 1.5, 12.5 Hz, H-6 b), 4.21 (1H, m, H-3),
4.54 (1H, m, H-9), 4.64 (1H, d,
5.29 (1H, d,
8.0 Hz, H-8); 13C-NMR (125 MHz,
D2O) : 23.7 (C10), 30.9 (C12), 33.1 (C13), 34.6
(C11), 37.3 (C1), 50.1 (C4), 50.4 (C2), 63.6 (C6 ),
66.7 (C3), 71.3 (C4 ), 74.7 (C5), 75.8 (C2 ), 76.8
(C9), 78.7 (C3 ), 78.9 (C5 ), 98.5 (C8), 101.9 (C1 ),
J
?
J
＝
?), 3.37 (1H, dd,
＝
＝
?
?
?
?
J
?
J
8.9, 8.5 Hz,
a), 3.89
?
?
J
＝
?
×
＝
J
?
＝ ×
O
＝
8.3 Hz, H-1
5 of acetyl group). All the signals were as-
J
?),
signed based on the 1H–1H-COSY, HMQC, and
HMBC spectra.12,17)
J
＝
d
?
Synthesis of (3S, 9S)- and (3S, 9R)-megastigma-
?
?
6,7-diene-3,5,9-triol 9-O-
b
-
D
-glucopyranosides ((3S,
-acetyl-
?
?
?
9S)- and (3S, 9R)-2b). To a solution of 3-
O
117.9 (C6), 203.2 (C7); FABMS (negative ion,
grasshopper ketone, which had been prepared
according to the literature method,18) (96 mg,
0.36 mmole) in 3 ml of MeOH was added NaBH4
(38 mg, 1.0 mmole), and the mixture stirred for
glycerol) m z 387 (M„H)„; [
a
]2D7„29.1
9
(
c
1.00,
W
MeOH).
(3S, 9R)-megastigma-6,7-diene-3,5,9-triol 9-O-
-glucopyranoside ((3S, 9R)-2b). 1H-NMR (500
MHz, D2O) : 1.10 (3H, s, H-11), 1.24 (3H, s,
H-12), 1.29 (1H, dd,
b-
30 min to give 3-
3,5,9-triol (3- -acetyl-2a; 81 mg, 0.30 mmole, 83
-Acetyl-2a (76 mg, 0.28 mmole) was
with 2,3,4,6-tetra- -pivaloyl- -gluco-
O
-acetyl-megastigma-6,7-diene-
D
O
z
d
＝
yield). 3-
stirred
O
J
11.5, 9.2 Hz, H-2ax), 1.32
＝
J
O
D
(3H, d,
(1H, dd,
2.3, 4.2, 11.5 Hz, H-2eq), 2.15 (1H, dd, J
＝
6.1 Hz, H-10), 1.36 (3H, s, H-13), 1.42
＝
pyranosyl bromide (489 mg, 0.84 mmole) and
tetramethylurea (252 mg, 2.2 mmole) in dry CH2Cl2
J
11.9, 8.8 Hz, H-4ax), 1.89 (1H, dd,
2.3,
8.2 Hz, H-2 ),
1.2, 4.6, 7.9 Hz, H-5 ), 3.37 (1H,
7.5, 7.9 Hz, H-4 ), 3.39 (1H, t,
J
＝
＝
at 0
9C for 1 h in the presence of molecular sieves
4.2, 13.2 Hz, H-4eq), 3.23 (1H, t,
J
?
＝
4A (2 g). To this solution, silver tri‰uoroacetate
(292 mg, 1.0 mmole) was added, and the solution
3.33 (1H, ddd,
J
?
＝
＝
dd,
H-3
(1H, dd,
J
?
J
7.5 Hz,
＝
4.6, 12.2 Hz, H-6?a), 3.88
further stirred for 5.5 hours at 0
9
C. The diastereo-
?
), 3.71 (1H, dd,
J
meric mixture of tetra- -pivaloyl-2b obtained was
O
J
＝
1.2, 12.2 Hz, H-6?b), 4.20 (1H, m, H-3),

2696
M. SUZUKI et al.
＝
4.48 (1H, m, H-9), 4.56 (1H, d,
J
7.9 Hz, H-1
?
),
9
S
)-2b was dissolved in 100
conditions for the acid treatment were as follows: pH
was set at 2.0 or 4.0; temperature was set to 40 C,
60 C or 90 C; incubation period was 0.5, 1.0 or
4.0 hr. After cooling to below „20 C, the reaction
mixture was then neutralized with 0.1 NaOH. Af-
ter adding 1 g of ethyl decanoate as an internal stan-
ml of acidic water. The
6.7 Hz, H-8); 13C-NMR (125 MHz,
＝
5.45 (1H, d,
D2O)
(C11), 37.3 (C1), 50.1 (C4), 50.5 (C2), 63.5 (C6
66.8 (C3), 72.3 (C4 ), 74.9 (C5), 76.0 (C2 ), 78.6
(C9), 78.8 (C3 ), 79.0 (C5 ), 99.6 (C8), 103.7 (C1 ),
J
d
: 22.5 (C10), 31.0 (C12), 33.0 (C13), 34.3
),
9
?
9
9
?
?
9
?
?
?
M
118.2 (C6), 202.4 (C7); FABMS (negative ion,
m
glycerol) m z 387 (M„H)„; [
a
]2D3 0
9
(
c
0.77, MeOH).
dard, each solution was extracted three times with
1 ml each of a mixture composed of pentane:CH2Cl2
W
Identiˆcation of allenic triol 9-O-
b
-D
-glucopyrano-
＝
2:1. The organic layers were combined and dried
over MgSO4, and then concentrated to 10–20 l. One
l of the concentrate was injected for the GC-MS
analysis as already mentioned. To analyze the non-
volatile compounds, 10 l of the aqueous layer was
side (2b) in ‰owers of R. damascena Mill. The frozen
‰owers (50 g fr. wt., harvested in 2000) at the full-
bloom stage were homogenized with 100 ml of a
m
m
10 m
M
HCOOH–NH4OH buŠer (pH 7.0) under ice-
m
cooling. The homogenate was extracted by stirring in
400 ml of the same buŠer at room temperature. After
analyzed by HPLC without further pre-treatment.
The HPLC conditions were the same as those de-
scribed for the synthesis and identiˆcation of the al-
lenic triol (2a) and its 9-
der these conditions, (3
were detected at 26.3 and 29.3 mins, respectively.
the centrifugation (3800 g, 09C, 15 min), the super-
natant was lyophilized. The resulting powdery
material (3.2 g) was acetylated (pyridine-acetic anhy-
dride) and puriˆed by silica gel column chro-
O
S
-glucopyranoside (2b). Un-
, 9 )-2b and (3 , 9 )-4b
S
R
S
matography (hexane-t-butyl methyl ether, diethyl
ether, stepwise) to yield ten fractions. The diethyl
ether fraction was further puriˆed by preparative
Acknowledgments
TLC (Merck 1.13792) in hexane-
1:6 as the developing solvent. The silica gel in the
Rf region, corresponding to those for both the di-
astereomers of 3,2 ,3 ,4 ,6 -penta- -acetyl-2b, was
t-butyl methyl ether
The authors thank Dr. Skouroumounis of the Aus-
tralian Wine Research Institute for his kind sugges-
tions on the synthesis of glycosidic C13-noriopre-
noids. We appreciate the work of Mr. Akihito Yagi
of Shizuoka University in recording NMR and GC-
MS data. This work was partly supported by grant in
aid A(2) from the Japanese Ministry of Education,
Science and Sports (to NW), and B1 from Japan So-
ciety for the Promotion of Science (to NW).
＝
?
?
?
?
O
scrapped oŠ and extracted with dichloromethane.
The resulting extract was deacetylated by treating
with NaOMe, and then further puriˆed by HPLC un-
der the following conditions: column, YMC-ODS-
×
AM5, 4.6 250 mm (YMC Co., Kyoto, Japan); sol-
vent, 5 MeCN (0–10 min), the concentration of
MeCN then being increased to 30 in 30 min at a
‰ow rate of 1.0 ml min at 40 C; detector, pho-
z
z
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