n-Octyl α-L-Rhamnopyranosyl-(1→2)-β-D-glucopyranoside derivatives from the glandular trichome exudate of Geranium carolinianum

Article abstract of DOI:10.1248/cpb.59.747

Chemical investigation of the glandular trichome exudate from Geranium carolinianum L. (Geraniaceae) led to the characterization of unique disaccharide derivatives, n-octyl 4-O-isobutyryl-α-L-rhamnopyranosyl-(1→2)-6-O- isobutyryl-β-D-glucopyranoside (1), n-octyl 4-O-isobutyryl-α-L- rhamnopyranosyl-(1→2)-6-O-(2-methylbutyryl)-β-D-glucopyranoside (2) and n-octyl 4-O-(2-methylbutyryl)-α-L-rhamnopyranosyl-(1→2)-6-O- isobutyryl-β-D-glucopyranoside (3), named caroliniasides A - C, respectively. These structures were determined by spectral means. n-Alkyl glycoside derivatives have been isolated from the glandular trichome exudates for the first time. This rare type of secondary metabolites could be applicable to chemotaxonomic perspective because they are found in glandular trichome exudates of plants belonging to the genus Geranium, according to our studies.

Full text of DOI:10.1248/cpb.59.747

June 2011
Note
Chem. Pharm. Bull. 59(6) 747—752 (2011)
747
n-Octyl a-L-Rhamnopyranosyl-(1→2)-b-D-glucopyranoside Derivatives
from the Glandular Trichome Exudate of Geranium carolinianum
Teigo ASAI, Takaomi SAKAI, Kiyoshi OHYAMA, and Yoshinori FUJIMOTO*
Department of Chemistry and Materials Science, Tokyo Institute of Technology; Meguro, Tokyo 152–8551, Japan.
Received November 18, 2010; accepted March 29, 2011; published online April 4, 2011
Chemical investigation of the glandular trichome exudate from Geranium carolinianum L. (Geraniaceae) led
to the characterization of unique disaccharide derivatives, n-octyl 4-O-isobutyryl-a-L-rhamnopyranosyl-(1→2)-
6
-O-isobutyryl-b-D-glucopyranoside (1), n-octyl 4-O-isobutyryl-a-L-rhamnopyranosyl-(1→2)-6-O-(2-methylbu-
tyryl)-b-D-glucopyranoside (2) and n-octyl 4-O-(2-methylbutyryl)-a-L-rhamnopyranosyl-(1→2)-6-O-isobutyryl-
b-D-glucopyranoside (3), named caroliniasides A—C, respectively. These structures were determined by spectral
means. n-Alkyl glycoside derivatives have been isolated from the glandular trichome exudates for the first time.
This rare type of secondary metabolites could be applicable to chemotaxonomic perspective because they are
found in glandular trichome exudates of plants belonging to the genus Geranium, according to our studies.
Key words Geranium carolinianum; caroliniaside; octyl glycoside; Geraniaceae; glandular trichome
Glandular trichomes are micro-organs located on the sur-
face of the leaves, stems, flowers, and fruits of plants that
exude oily substances. The chemical and physiological roles
of the oily substances have been studied in a limited number
of plant species such as those belonging to the Labiatae and
Compositae families. It has been suggested that exudate sub-
stances are related to a plant’s self-protection system, for ex-
ample, to serve as antifeedants, antifungals, antibiotics, or
1,2)
for UV protection.
However, our understanding of the
chemistry and function of glandular trichome exudates re-
main insufficient. Additional systematic chemical and bio-
logical studies of the exudates would aid in further under-
standing of glandular trichomes. From this perspective, we
Fig. 1. Structures of Caroliniasides A—C (1—3)
have been investigating the glandular trichome exudates of now started to investigate glandular trichome exudates from
plants belonging to a variety of families. Our previous stud- plants of the Geraniaceae family. This article describes the
ies led to the characterization of a series of oxygenated fatty isolation and structure elucidation of three unique n-octyl
acyl glycerols, in the glandular trichome exudate on the disaccharides from the glandular trichome exudate of Gera-
leaves, and geranylated flavanones, in the oily secretion on nium carolinianum (Fig. 1).
the immature fruit surface, from Paulownia tomentosa (Scro-
Geranium carolinianum L. is native to North America and
3,4)
phulariaceae). The glandular trichome exudates of Ibicella was imported into Japan about 100 years ago. The plant is
lutea and Proboscidea louisiana (Martyniaceae) contained widely distributed throughout open roadside in Japan and
5)
11)
glycosylated fatty acids and dammarane triterpenes. Re- comes into flower between April and May. The upper por-
cently we identified a series of cyclic fatty acyl glycosides tion of the aerial part, in particular the calyxes, is rich in cap-
from the glandular trichome exudate of Silene gallica itate glandular trichomes. The aerial part of G. carolinianum
6
)
(
Caryophyllaceae) and 2-acetyl-1-(3-glycosyloxy-fatty was reported to exhibit antimicrobial and anti-hepatitis B
12,13)
acyl)octadecanoyl-sn-glycerol and dammarane triterpenes virus activities.
Previous phytochemical studies of G.
14)
from the glandular trichome-like secretory organs in the carolinianum led to the identification of gallic acid, ethyl
7)
12)
13)
young stipules and leaves of Cerasus yedoensis (Rosaceae).
More recently, we found several plants of the Geraniaceae
family, such as Geranium carolinianum, G. thunbergii, G. Results and Discussion
gallate, ellagic acid, geraniin and flavones.
robertianum and G. maderense, have capitate glandular tri-
chomes in the upper portion of their stems.
In a preliminary study, two exudate samples were obtained
by either gently wiping the surface of calyxes with oil-free
Non-volatile secondary metabolites of the glandular tri- cotton or by briefly rinsing an upper portion of the aerial part
chome exudates of the Geraniaceae plants have been re- in Et O. The two samples showed essentially identical TLC
2
ported only in a few species. Anacardic acids have been iso- spots, with the latter being contaminated with larger amounts
lated from the glandular trichome exudate of Pelargonium x of non-polar materials (exclusively a mixture of hydrocar-
8)
hortorum and their biosynthetic precursors, hexadec-11- bons). To obtain larger samples, an extract was prepared
enoic and octadec-13-enoic acids, have been characterized in using the latter method. This oily material was subjected to
9)
the glandular trichomes of the plant. Flavonoids have been silica gel column chromatography to give the exudate-spe-
identified in the glandular trichome exudates of G. macror- cific fraction, which was a single spot by TLC. HPLC analy-
10)
rhizum and G. lucidum. As a continuation of our work, we sis of this fraction showed essentially two major peaks (see
∗
To whom correspondence should be addressed. e-mail: fujimoto.y.aa@m.titech.ac.jp
© 2011 Pharmaceutical Society of Japan

7
48
Vol. 59, No. 6
Experimental), which were separated to yield compounds 1 A, B and C, respectively. Negligible amounts of these sub-
and 2/3. In contrast, GLC analysis of the fraction as the stances were found in the ether-rinsed residual aerial part,
trimethylsilyl (TMS) ether derivatives exhibited three major thus indicating that compounds 1—3 were distributed in
peaks A, B and C (Fig. 2). Subsequent studies (vide infra) re- glandular trichome exudate.
vealed that compounds 1, 2 and 3 corresponded to the peaks
Compound 1 was obtained as an oil and a major con-
stituent (65%) of the fraction. Its molecular formula
C H O was deduced from high resolution (HR)-FAB-MS
2
8
50 12
data, which exhibited a quasi-molecular ion peak at m/z
ꢀ
6
01.3203 [MꢀNa] (Calcd for C H O Na, 601.3200).
28 50 12
GLC analysis of the TMS ether derivative of 1 showed a sin-
1
gle peak (peak A in Fig. 2). The H-NMR spectrum of 1 ex-
hibited signals ascribable to hexose and 6-deoxyhexose moi-
eties, two isobutyryl groups and a medium-chain n-alkyloxy
group (Table 1). The hexose moiety was assigned as a b-
linked 6-O-isobutyrylglucopyranosyl (glc) on the basis of the
proton chemical shifts and coupling constants [e.g., d 4.29
(d, Jꢁ7.7 Hz, H-1), 4.33 (dd, Jꢁ12.0, 4.0 Hz, H-6a), 4.34
(
dd, Jꢁ12.0, 4.0 Hz, H-6b)], combined with the data of the
1
1
Fig. 2. GLC Chart of the Fraction Containing Compounds 1—3
H– H correlation spectroscopy (COSY) spectrum. The
1
13
Table 1. H-NMR (500 MHz) and C-NMR (125 MHz) Data of 1, 2 and 3 in CDCl –CD OD (20 : 1)
3
3
a)
a)
1
2
3
Position
dH (mult., J in Hz)
dC
dH (mult., J in Hz)
dC
dH (mult., J in Hz)
dC
Glucopyranosyl
1
2
3
4
5
6
4.29 (d, 7.5)
3.50 (dd, 9.0, 7.7)
3.55 (t, 9.0)
3.30 (t, 9.0)
3.42 (m)
101.69
76.53
77.62
70.44
73.71
63.48
4.29 (d, 7.5)
3.50 (dd, 9.0, 7.5)
3.55 (t, 9.0)
3.30 (t, 9.0)
3.42 (m)
101.69
76.44
77.62
70.41
73.70
63.48
4.29 (d, 7.5)
3.50 (dd, 9.0, 7.5)
3.55 (t, 9.0)
3.30 (t, 9.0)
3.42 (m)
101.69
76.44
77.62
70.41
73.70
63.48
4.33 (dd, 12.0, 4.0)
4.33 (dd, 13.0, 4.0)
4.34 (dd, 13.0, 4.0)
4.33 (dd, 13.0, 4.0)
4.34 (dd, 13.0, 4.0)
4
.34 (dd, 12.0, 4.0)
Rhamnopyranosyl
1
2
3
4
5
6
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
5.28 (s)
3.98 (br s)
3.82 (dd, 9.7, 3.4)
4.83 (t, 9.7)
4.21 (dq, 9.7, 6.3)
1.15 (d, 6.3)
99.74
70.70
69.71
74.65
65.98
17.13
5.28 (s)
3.97 (br s)
3.82 (dd, 9.7, 3.4)
4.82 (t, 9.7)
4.22 (dq, 9.7, 6.3)
1.15 (d, 6.3)
99.71
70.71
69.79
74.62
65.94
17.13
5.28 (s)
3.97 (br s)
3.82 (dd, 9.7, 3.4)
4.83 (t, 9.7)
4.22 (dq, 9.7, 6.3)
1.15 (d, 6.3)
99.71
70.71
69.79
74.62
65.94
17.16
n-Octyl
1
ꢃ
3.85 (dt, 9.8, 6.3)
70.15
3.86 (dt, 9.3, 7.2)
3.46 (dt, 9.3, 7.2)
1.62 (m)
1.22—1.35
1.22—1.35
1.22—1.35
1.22—1.35
1.22—1.35
0.88 (t, 6.9)
70.18
3.86 (dt, 9.3, 7.2)
3.46 (dt, 9.3, 7.2)
1.62 (m)
1.22—1.35
1.22—1.35
1.22—1.35
1.22—1.35
1.22—1.35
0.88 (t, 6.9)
70.18
3
.46 (dt, 9.8, 6.3)
2
3
4
5
6
7
8
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
1.61 (m)
29.61
25.93
29.42
29.19
31.82
22.58
14.02
29.60
25.94
29.43
29.21
31.83
22.59
14.03
29.60
25.94
29.43
29.21
31.83
22.59
14.03
1.22—1.35
1.22—1.35
1.22—1.35
1.22—1.35
1.22—1.35
0.88 (t, 7.0)
Isobutyryl
1
2
3
4
ꢄ
ꢄ
ꢄ
ꢄ
—
—
2.60 (m)
177.87
177.87
33.93
34.18
18.87
18.74
18.96
18.99
—
177.91
34.18
18.74
19.00
—
177.91
33.93
18.87
18.96
2.61 (m)
1.18 (d, 6.3)
1.19 (d, 6.3)
2.61 (m)
1.18 (d, 6.3)
1.19 (d, 6.3)
2
.60 (m)
1.18 (d, 6.3)
.18 (d, 6.3)
1.19 (d, 6.3)
.19 (d, 6.3)
1
1
2
-Methylbutyryl
1
2
3
ꢃꢃ
ꢃꢃ
ꢃꢃ
—
2.43 (m)
1.70 (m)
177.54
41.00
26.75
—
177.54
41.31
26.63
2.42 (m)
1.70 (m)
1.49 (m)
0.94 (t, 7.5)
1.18 (d, 6.3)
1.49 (m)
4
5
ꢃꢃ
ꢃꢃ
0.92 (t, 7.5)
1.18 (d, 6.3)
16.50
11.58
16.61
11.48
a) Recorded as a 1 : 2.5 mixture of 2 and 3.

June 2011
749
deoxy hexose moiety was similarly determined to be an a- FAB-MS that corresponded with the molecular formula
linked 4ꢂ-O-isobutyrylrhamnopyranosyl (rha) [e.g., d 5.28 (s, C H O . GLC analysis of the sample as a TMS ether deriv-
2
9
52 12
H-1ꢂ), 4.83 (t, Jꢁ9.7 Hz, H -4ꢂ), 1.15 (d, Jꢁ6.3 Hz, H -6ꢂ)]. ative showed two peaks (peaks B and C in Fig. 2) in a 1 : 2.5
3
3
1
An a-anomeric configuration of rha was assigned using the ratio. The H-NMR spectrum (recorded in CDCl –CD OD
3
3
nuclear Overhauser effect (NOE) experiments, in which irra- 20 : 1) was essentially identical to that of 1 except that signals
diation of H-3ꢂ caused an NOE enhancement toward H-5ꢂ, of only one isobutyryl group were observed instead of the
1
3
but not toward H-1ꢂ. The C-NMR spectrum of 1 exhibited two discerned in 1, and a doublet methyl and a triplet methyl
8 signals, among which 12 signals were assigned to the car- signals appeared, which are assignable to a branched acyl
2
1
bon atoms of the two sugars and eight signals were assigned group. The H-NMR data suggested that one of the isobu-
to the carbon atoms of the two isobutyryl groups. The n- tyryl groups in 1 was replaced by a new, branched acyl
1
3
alkyloxy group was now defined as an n-octyloxy group on group. The C-NMR spectrum (Table 1) agreed with this hy-
the basis of the number of the remaining carbon signals, pothesis and the new acyl group was assigned as a 2-methyl-
which was in agreement with the calculation from the mole- butyryl group on the basis of the newly appeared signals at d
cular formula of 1. Heteronuclear multiple bond correlations 177.54 (C-1ꢃꢃ), 41.31 (C-2ꢃꢃ), 26.63 (C-3ꢃꢃ), 16.61 (C-4ꢃꢃ)
16,17)
(HMBC) illustrated in Fig. 3, allowed us to connect the and 11.48 (C-5ꢃꢃ).
These signals were accompanied by
above-mentioned partial structures. Thus, a cross peak be- weak (ca. 2.5 : 1 ratio) signals at 41.00 (C-2ꢃꢃ), 26.75 (C-3ꢃꢃ),
1
3
tween the anomeric proton of glc and the oxymethylene car- 16.50 (C-4ꢃꢃ) and 11.58 (C-5ꢃꢃ). The C signals for the
bon (d 70.15) of the n-octyloxy group indicated an n-octyl isobutyryl group were also accompanied by weak signals
1
3
glycosidic linkage. A cross peak between the anomeric pro- (Table 1). The C-NMR data recorded in pyridine-d (Table
5
ton of rha and C-2 of glc (d 76.53) indicated a rha-(1→2)-glc 2) also supported that the sample is a mixture of two com-
linkage. The two isobutyryl groups were also connected to pounds. These NMR data, combined with the GLC behavior
the 6-O of glc and the 4ꢂ-O of rha on the basis of HMBC of the TMS derivative, suggested that the sample could be a
correlations. GC-MS analysis of the TMS ether derivative of 2.5 : 1 mixture of positional isomers with respect to the
1
exhibited ions at m/z 851 [MꢅMe], 737 [MꢅC H O], isobutyryl and 2-methylbutyryl substitution at the 4ꢂ-O of
8 17
488, 361 and 273 (Fig. 4), which supported the presence of rha and 6-O of glc. Alternatively, it can be a mixture of due
the n-octyl moiety and the substitution of an isobutyryl to the co-occurrence of (R)- and (S)-2-methylbutyryl groups.
group on each sugar moiety. Finally, it was established that HMBC correlations observed for the mixture are depicted in
the sugar fraction obtained by hydrolysis of 1 was composed
of L-rhamnose and D-glucose by the GLC analysis of their
TMS ethers of the thiazolidine derivatives according to the
15)
method of Hara et al. Hence, the structure of compound 1
was determined to be n-octyl 4-O-isobutyryl-a-L-rhamno-
pyranosyl-(1→2)-6-O-isobutyryl-b-D-glucopyranoside as
shown in Fig. 1, and named caroliniaside A.
Compounds 2 and 3 were obtained as an inseparable mix-
ꢀ
ture and showed an [MꢀNa] quasi-molecular ion at m/z
6
15.3386 (Calcd for C H O Na, 615.3356) in the HR-
29 52 12
Fig. 3. 2D-NMR Correlations for Compounds 1 and 3
Fig. 4. Mass Fragmentation for the TMS Ether Derivatives of 1—3 (Cor-
2D-NMR correlations for 3 were extracted from the spectrum recorded for a 1 : 2.5
mixture of 2 and 3 in pyridine-d5.
responding to Peaks A, B and C, Respectively, in Fig. 2)

7
50
Vol. 59, No. 6
1
13
Table 2. H-NMR (500 MHz) and C-NMR (125 MHz) Data of 1, 2 and 3 in Pyridine-d6
a)
a)
1
2
3
Position
dH (mult., J in Hz)
dC
dH (mult., J in Hz)
dC
dH (mult., J in Hz)
dC
Glucopyranosyl
1
2
3
4
5
6
4.77 (d, 7.5)
4.19 (m)
4.19 (m)
3.94 (m)
3.94 (m)
102.71
77.56
79.22
71.61
75.12
64.26
4.77 (d, 7.5)
4.19 (m)
4.19 (m)
3.94 (m)
3.94 (m)
102.69
77.54
79.18
71.66
75.09
64.26
4.77 (d, 7.5)
4.19 (m)
4.19 (m)
3.94 (m)
3.94 (m)
102.69
77.54
79.18
71.60
75.09
64.26
4.87 (dd, 9.9, 3.6)
4.87 (dd, 9.9, 3.6)
4.79 (dd, 9.9, 3.6)
4.87 (dd, 9.9, 3.6)
4.79 (dd, 9.9, 3.6)
4
.79 (dd, 9.9, 3.6)
Rhamnopyranosyl
1
2
3
4
5
6
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
6.35 (s)
4.73 (br s)
4.61 (m)
5.83 (t, 9.9)
4.86 (m)
1.46 (d, 6.0)
101.89
72.46
70.32
75.67
67.02
18.07
6.34 (s)
4.73 (br s)
4.60 (m)
5.83 (t, 9.9)
4.86 (m)
1.46 (d, 6.1)
101.88
72.45
70.30
75.64
67.00
18.05
6.34 (s)
4.73 (br s)
4.60 (m)
5.83 (t, 9.9)
4.86 (m)
1.46 (d, 6.1)
101.88
72.45
70.30
75.52
67.00
18.09
n-Octyl
1
ꢃ
4.11 (dt, 9.3, 7.0)
69.91
4.11 (dt, 9.3, 7.0)
3.68 (dt, 9.3, 7.0)
1.78 (m)
69.90
4.11 (dt, 9.3, 7.0)
3.68 (dt, 9.3, 7.0)
1.78 (m)
69.90
3
.68 (dt, 9.3, 7.0)
2
3
4
5
6
7
8
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
1.78 (m)
1.43 (m)
30.27
26.48
29.75
29.52
32.07
22.88
14.24
30.52
26.47
29.75
29.52
32.07
22.86
14.22
30.52
26.47
29.75
29.52
32.07
22.86
14.22
1.43 (m)
1.43 (m)
1.22—1.35
1.22—1.35
1.22—1.35
1.22—1.35
0.85 (t, 6.9)
1.22—1.35
1.22—1.35
1.22—1.35
1.22—1.35
0.85 (t, 6.9)
1.22—1.35
1.22—1.35
1.22—1.35
1.22—1.35
0.85 (t, 6.9)
Isobutyryl
1
2
3
4
ꢄ
ꢄ
ꢄ
ꢄ
—
—
2.56 (m)
176.77
177.73
34.18
34.58
19.10
19.12
19.03
19.44
—
176.72
34.57
19.09
19.42
—
176.77
34.16
19.08
19.00
2.69 (m)
2.56 (m)
2
.69 (m)
1.084 (d, 7.0)
.252 (d, 7.0)
1.101 (d, 6.3)
.211 (d, 6.5)
1.252 (d, 7.3)
1.211 (d, 6.3)
1.084 (d, 7.3)
1.101 (d, 6.3)
1
1
2
-Methylbutyryl
1
2
3
ꢃꢃ
ꢃꢃ
ꢃꢃ
—
2.54 (m)
1.83 (m)
176.39
41.22
27.56
—
176.35
41.67
27.11
2.54 (m)
1.83 (m)
1.52 (m)
0.96 (t, 7.4)
1.25 (d, 7.0)
1.52 (m)
4
5
ꢃꢃ
ꢃꢃ
0.97 (t, 6.8)
1.25 (d, 7.0)
16.68
11.72
17.12
11.80
a) Recorded as a 1 : 2.5 mixture of 2 and 3.
Fig. 3, which indicated that 2-methylbutyryl group was lo- rhamnopyranosyl-(1→2)-6-O-(2-methylbutyryl)-b-D-glu-
cated at the 4ꢂ-O of rha and isobutyryl group at the 6-O of copyranoside, while compound 3 was defined as n-octyl 4-O-
glc in the major constituent 3 (the major and minor con- (2-methylbutyryl)-a-L-rhamnopyranosyl-(1→2)-6-O-isobu-
stituents were designated as 3 and 2, respectively).
tyryl-b-D-glucopyranoside. Compounds 2 and 3 were named
This was further supported by the GC-MS analysis of the caroliniasides B and C, respectively. The chirality of the 2-
TMS derivative. Fragment ions in the electron impact mass methylbutyryl group remains unknown and will be investi-
spectra (EI-MS) of peak B (corresponding to 2-TMS ether) gated in the future.
and peak C (corresponding to 3-TMS ether) are shown in
The present study demonstrated the occurrence of a rare
Fig. 4, which exhibited diagnostic fragment ions at m/z 502 class of secondary metabolites, n-alkyl disaccharide deriva-
and 361 for peak B and 488 and 375 for peak C, in addition tives, in the glandular trichome exudate of G. carolinianum.
to common ions at m/z 865 [MꢅMe] and 751 [MꢅC H ]. This is the first report of the isolation of n-alkyl glycoside
8
17
These MS data clearly indicated that the isobutyryl and 2- derivatives from glandular trichome exudates. n-Octyl gluco-
methylbutyryl groups were linked to the 6-O of glc and the sides have been isolated previously from Rhodiola species
1
8,19)
20)
4
ꢂ-O of rha, respectively, in compound 3, and were attached (Crassulaceae),
Circaea lutetiana (Onagraceae) and
21)
to the 4ꢂ-O of rha and the 6-O of glc, respectively, in com- Eucalyptus globules (Myrtaceae). n-Hexyl glucosides have
pound 2. The presence of only two sets of C-NMR signals been isolated previously from Rhodiola species (Crassu-
1
3
22,23)
23,24)
in the sample could rule out the possibility of the occurrence laceae).
of both antipodes of the 2-methylbutyryl group. Thus, com- and creoside IV, have been isolated from Rhodiola species.
pound 2 was determined to be n-octyl 4-O-isobutyryl-a-L- Our preliminary study indicated that glandular trichome
n-Alkyl di-saccharides, rhodiooctanoside
23)

June 2011
751
exudates of G. thunbergii, G. robertianum and G. maderense HPLC (solvent, MeOH–H O 6 : 1, flow rate 1 ml/min) to give compound 1
2
(
30 mg, eluted at 8.82 min) and compound 2/3 (10 mg, eluted at 10.76 min).
also contained n-octyl rhamnopyranosyl-(1→2)-glucopyra-
nosides esterified with various shorter-chain acyl groups.
These findings suggested that n-octyl disaccharide deriva-
ꢀ
Caroliniaside A (1): Colorless oil; HR-FAB-MS m/z: 601.3203 [MꢀNa]
2
5
(
(
Calcd for C H O Na, 601.3200); [a] ꢅ82.0 (cꢁ0.41, CHCl ); IR
28 50 12 D 3
ꢅ1
CHCl ) cm : 3450, 2920, 2880, 2860, 1720, 1480, 1390, 1190, 1160,
3
1
13
tives occur in the Geranium genus as components of glandu- 1140, 1090; H- and C-NMR data, see Tables 1 and 2.
lar trichome exudates and this type of compounds can be a
taxonomical marker of this genus. To this end, it is interest-
ing to explore glandular trichome exudates of the other major
genera of the Geraniaceae family. Sugar esters, in particular
Caroliniaside B (2) and Caroliniaside C (3): Colorless oil; HR-FAB-MS
ꢀ
25
m/z: 615.3386 [MꢀNa] (Calcd for C H O Na, 615.3356); [a] ꢅ72.9
29 52 12
D
ꢅ1
(
1
cꢁ0.31, CHCl ); IR (CHCl ) cm : 3450, 2980, 2920, 2880, 2860, 1720,
3
3
1
13
480, 1390, 1190, 1160, 1140, 1090; H- and C-NMR data (recoded in
CDCl –CD OD 20 : 1 and pyridine-d ), see Tables 1 and 2.
3
3
5
25)
esters with shorter-chain acyl groups, glucose esters, su-
GLC and GC-MS Analysis A mixture of hexamethyldisilazane and
26,27)
28)
crose esters
and inositol esters, have been character- TMSCl in pyridine (TMS-HT) (30 ml) and compounds 1 (0.4 mg) was
heated at 75 °C for 1.5 h, and then the mixture was cooled to room tempera-
ized as glandular trichome exudates from a limited genera of
different families. Our present and previous results indicated
that secondary metabolites contained in glandular trichome
ture. Hexane (50 ml) and H O (50 ml) were added to the reaction mixture and
2
a part of the hexane layer was analyzed by GLC and GC-MS. The fraction
containing compounds 1—3 and a mixture of compounds 2 and 3 were simi-
exudates would be much more structurally diverse than ever larly converted to the TMS ether and analyzed. EI-MS of 1 (peak A in Fig.
2
) m/z: 851 [MꢅMe] (tr), 737 (tr), 720 (tr), 695 (tr), 607 (1), 578 (1), 488
before expected.
(1), 361 (3), 273 (17), 204 (43), 143 (17), 73 (100). EI-MS of 2 (peak B in
Experimental
Fig. 2) m/z: 865 [MꢅMe] (tr), 751 (tr), 735 (tr), 709 (tr), 622 (tr), 578 (tr),
502 (tr), 475 (tr), 449 (tr), 361 (3), 273 (17), 231 (3), 204 (40), 182 (6), 143
(14), 103 (3), 73 (100). EI-MS of 3 (peak C in Fig. 2) m/z: 865 [MꢅMe]
(tr), 751 (tr), 720 (tr), 695 (tr), 621 (tr), 607 (tr), 592 (tr), 519 (tr), 488 (tr),
375 (3), 361 (3), 273 (26), 231 (6), 204 (66), 182 (11), 143 (25), 103 (9), 73
(100).
1
13
General Experimental Procedures H- and C-NMR spectra were
recorded on a Bruker DRX-500 (500 MHz for H and 125 MHz for C)
1
13
spectrometer in CDCl –CD OD or pyridine-d solution. Tetramethylsilane (d
3
3
5
1
0
.00) was used as an internal standard for H-NMR shifts, and CDCl (d
3
13
77.00) was used as a reference for C-NMR shifts. For the spectra taken in
pyridine-d , the residual nondeuterated solvent signal at d 7.19 and the sol-
Sugar Analysis A solution of LiOH (0.65 mg) in H O (100 ml) was
5
2
1
13
vent signal at d 123.50 were referenced for H and C shifts, respectively.
Positive mode FAB-MS and HR-FAB-MS spectra, using 3-nitrobenzyl alco-
hol as the matrix, were obtained on a JEOL JMS-700 spectrometer. IR spec-
tra were recorded on a JASCO-FT/IR-5300 spectrometer. Optical rotations
were measured on a JASCO P-2200 polarimeter. TLC analysis was per-
formed using Merck precoated Si gel 60 F254 glass plates and the spots
were detected by treating the plates with a 5% ethanolic solution of phos-
phomolybdic acid followed by heating at 120 °C. Silica gel 60 N (spherical
neutral, 40—100 mm, Kanto Chemical, Japan) was used for column chro-
matography. HPLC was carried out on a Shimadzu LC-6A apparatus
equipped with a UV detector (monitored at 215 nm) using a reversed-phase
column (Shimadzu Shim-Pack CLC-ODS, 15 cmꢆ4.6 mm i.d.) under iso-
cratic solvent conditions. GLC for the trimethylsilyl derivatives of com-
pounds 1, 2 and 3 were carried out on a Shimadzu GC-14B apparatus (FID
detector) equipped with a DB-5 capillary column (15 mꢆ0.25 mm, 0.25 mm
film thickness, J & W Scientific, U.S.A.) under the following conditions: in-
jection temperature of 275 °C, column temperature of 275 °C, detection tem-
perature of 280 °C, He carrier gas flow rate of 50 kPa (P1) and 120 kPa (P2),
added to a solution of 1 (2 mg) in dimethoxyethane (400 ml) and the mixture
was stirred at room temperature for 20 h. The mixture was neutralized by the
addition of sat. aq. NH Cl and partitioned between n-BuOH and H O. Con-
4
2
centration of the n-BuOH layer under reduced pressure gave an oily residue.
This was mixed with 2 M HCl (200 ml), and heated at 80 °C for 48 h. The
mixture was partitioned between Et O and H O. The aqueous layer was con-
2
2
centrated and the residual material was identified as glucose and rhamnose
by TLC [Rf values, 0.24 (glucose) and 0.55 (rhamnose), developed with
CH CN–H O 85 : 15] by comparison with authentic sugars. The sugar frac-
3
2
tion was converted to the TMS ether of the thiazolidine derivative, as re-
15)
ported by Hara et al., and analyzed by GLC, which showed two peaks at
11.0 and 18.4 min, identical to those of the TMS ethers derived from L-
rhamnose and D-glucose.
Acknowledgement The authors thank Prof. Shiro Kohshima, Depart-
ment of Biological Sciences, Graduate School of Bioscience and Biotech-
nology, Tokyo Institute of Technology for botanical identification.
H flow rate of 50 kPa, air flow rate of 50 kPa and split (40 : 1) injection.
References
2
GLC of the TMS derivatives of the sugars was carried out under similar con-
ditions except for injection temperature of 270 °C, column temperature of
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95 °C, detection temperature of 270 °C. GC-MS of the trimethylsilylated
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2
2
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3
ture of hydrocarbons), Fr. 2 (15 mg, CHCl –MeOH 20 : 1), Fr. 3 (35 mg,
3
CHCl –MeOH 10 : 1), Fr. 4 (120 mg, CHCl –MeOH 8 : 1) and Fr. 5 (5 mg,
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3
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