T. Asai, Y. Fujimoto / Phytochemistry Letters 4 (2011) 38–42
41
and leaves of the Cerasus species suggests that they may play
3.4. 2-Acetyl-1-{3-[3,4-di-O-acetyl-
b
-
D
-glucopyranosyl-(1 ! 3)-2-
related physiological roles, although their roles remain to be
clarified. We often observe ants being attracted by sugars secreted
from a pair of extrafloral nectarines locating at the boundary
between the petiole and leaf of C. yedoensis like other Cerasus
species (Stephenson, 1982; Heil and Mckey, 2003). However, the
exudates from the trichome-like organs do not appear to have such
attracting effect on ants.
O-acetyl- -rhamnopyranosyloxy]octadecanoyl}-sn-glycerol (1)
a-L
Colorless oil; negative HRFABMS m/z: 849.4509 [MꢀH]ꢀ (calcd
24
for C41H69O18, 849.4484); [
a
]
D
+3.0 (c = 1.3, MeOH); for 1H and
13C NMR (CDCl3) spectroscopic data, see Table 1.
A mixture of 1 (5 mg) and TBDPSCl (8 L) in pyridine (50
m
mL)
was stirred at room temperature for 12 h. Et2O (0.3 mL) was added,
and Et2O-soluble portion was washed with saturated aqueous
NH4Cl, and then brine, dried (Na2SO4) and concentrated. The
resulting di-TBDPS ether (12 mg) was reduced with DIBAL in the
same manner as reported previously (Asai et al., 2009) to yield 3-
O-TBDPS-sn-glycerol (1.5 mg, 77% from 1). The TBDPS ether
3. Experimental
3.1. General experimental procedures
1H and 13C NMR spectra were recorded on a Bruker DRX500
(500 MHz for 1H and 125 MHz for 13C) spectrometer in CDCl3 or
(0.5 mg) was reacted with (R)-MTPACl (1.0 mL) in the same
manner as reported previously (Asai et al., 2009) and a crude
product was purified by PTLC (hexane-AcOEt, 7:1) to give 1,2-di-O-
(S)-MTPA-3-O-TBDPS-sn-glycerol (90% yield). The 1H NMR (CDCl3)
CD3OD solution. Tetramethylsilane (
signals were used as an internal standard for 1H shifts, and CDCl3 (
77.00) and CD3OD (
49.00) signals were used as a reference for 13
d 0.00) and CD2HOD (d 3.30)
d
C
d
spectroscopic data [d: 7.58–7.27 (20H, m), 5.36 (1H, m), 4.75 (1H,
shifts. EIMS, HREIMS (70 eV) and FABMS spectra were obtained on
a JEOL JMS-700 spectrometer. IR spectra were recorded on a JASCO-
FT/IR-5300 spectrometer. Optical rotations were measured on a
JASCO P-2200 polarimeter. Silica gel 60 N (spherical neutral, 40–
dd, J = 12.4, 3.2 Hz), 4.50 (1H, dd, J = 12.4, 6.4 Hz), 3.72 (2H, m), 3.44
(3H, s, MeO), 3.38 (3H, s, MeO), 1.01 (9H, s, tBu)] were identical to
those of authentic sample, but different from those of 2,3-di-O-(S)-
MTPA-1-O-TBDPS-sn-glycerol (Asai et al., 2009).
100
m
m, Kanto Chemical, Japan). TLC analysis was performed using
To a solution of LiOH (10 mg) in water (200
mL) was added a
Merck precoated Si gel 60 F254 glass plates (0.25 mm thickness)
and the spots were detected by treating the plates with a 5%
ethanolic solution of phosphomolybdic acid followed by heating at
120 8C. PTLC was carried out using the same Si gel plates. GLC was
carried out on a Shimadzu GC-14B apparatus equipped with a J&W
solution of 1 (15 mg) in DME (800 L) and the mixture was stirred
m
at room temperature for 48 h. Saturated aqueous NH4Cl was added
and the mixture was partitioned between CHCl3 and H2O. The
CHCl3 layer was concentrated to give a glycosylated fatty acid
(6.5 mg, 90%). Colorless oil; [a]
24–23.1 (c = 8.7, MeOH); negative
D
Scientific DB-5 capillary column (15 m ꢁ 0.25 mm, 0.25
m
m film
HRFABMS m/z 607.3664 [MꢀH]ꢀ (calcd for C30H55O12, 607.3694);
thickness) under the following conditions for the analysis of fatty
acid derivatives: injection temperature 270 8C, column tempera-
ture 208 8C, detection temperature 270 8C, He carrier gas flow rate
of P1, 50 kPa and P2, 125 kPa, H2 flow rate, 50 kPa, air flow rate,
50 kPa, and split (40:1) injection. GLC analysis of trimethylsilylated
sugars was similarly performed under the conditions of injection
temperature 260 8C, column temperature 175 8C and detection
temperature 280 8C.
1H NMR (CD3OD) : 4.81 (d, J = 1.5 Hz, H-10), 4.50 (d, J = 7.7 Hz, H-
d
100), 4.08 (m, H-3), 4.04 (dd, J = 3.0, 1.8 Hz, H-20), 3.80 (dd, J = 11.9,
2.4 Hz, Ha-600), 3.72 (m, Hb-600), 3.72 (m, H-50), 3.70 (dd, J = 9.5,
2.4 Hz, H-30), 3.53 (t, J = 9.5 Hz, H-40), 3.37 (m, H-300), 3.36 (m, H-400),
3.28 (m, H-200), 3.28 (m, H-500), 2.49 (d, J = 6.4 Hz, H2-2), 1.61–1.25
(CH2), 1.22 (d, J = 7.4 Hz, H3-60), 0.89 (t, J = 6.7 Hz, CH3). 13C NMR
(CDCl3) d
: 175.2 (C-1), 105.8 (C-100), 99.9 (C-10), 83.2 (C-30), 77.7 (C-
300), 77.7 (C-500), 75.5 (C-3), 75.4 (C-200), 72.6 (C-40), 71.9 (C-20), 70.9
(C-400), 69.9 (C-50), 62.1 (C-600), 41.2 (C-2), 34.4 (C-4), 33.1 (C-16),
30.8–30.5 (CH2), 25.9 (C-5), 23.7 (C-17), 18.0 (C-60), 14.4 (C-18).
A mixture of the glycosylated fatty acid (5 mg) and 1.5 N HCl
3.2. Plant material
Stipules and young leaves of the Prunus yedoensis (Rosaceae)
were collected in May in 2008 on the campus of Tokyo Institute of
Technology. The plant was identified by Prof. S. Kohshima,
Department of Biological Sciences, Graduate School of Bioscience
and Biotechnology, Tokyo Institute of Technology. A voucher
specimen (CMS20-04) was deposited in the Department of
Chemistry and Materials Science, Tokyo Institute of Technology.
(200 mL) was heated at 80 8C for 6 h and cooled down to room
temperature. The solution was partitioned between ether and
water, and the ether layer was concentrated to dryness in vacuo.
The residue was treated with excess ethereal diazomethane, and
the product was separated by PTLC (hexane-AcOEt, 6:1) to give
methyl 3-hydroxyoctadecanoate (2.2 mg) as a white powder, (+)-
FABMS m/z: 315 [M+H]+; 1H and 13C NMR spectroscopic data was
identical with those reported in our previous paper (Asai et al.,
2009). The water layer was concentrated and analyzed by TLC (Rf
values 0.55 and 0.85, developed with CH3CN–H2O 6:1, three times)
in comparison with authentic glucose and rhamnose. A part
3.3. Extraction and isolation
Fresh young stipules (fresh wt. 7.2 g, ca. 1.0 cm wide and 2.5 cm
long, 800 stipules) were briefly (ca. 2 s) rinsed twice in a beaker
containing Et2O (total volume 100 mL), the Et2O solution being
concentrated to dryness (230 mg) under reduced pressure. The
extract was treated with hexane (10 mL ꢁ 2) and the hexane
soluble part (40 mg) was subjected to silica gel (10 g) CC. Elution
with a gradient of hexane–AcOEt (10:1 ! 1:1, total volume 50 mL)
to yield 2 (18 mg, eluted with hexane–AcOEt 6:1) and 3 (12 mg,
eluted with hexane–AcOEt 4:1). The hexane insoluble part
(190 mg) was applied to silica gel (20 g) CC. Elution with a
gradient of CHCl3–MeOH (30:1 ! 10:1, total volume 60 mL) gave 1
(160 mg, eluted with CHCl3–MeOH 20:1).
(100
TMSCl in anhydrous pyridine) (50
to room temperature. Hexane (200
m
g) of the residue was treated with TMS-HT (HMDS and
L) at 70 8C for 1.5 h and cooled
L) and H2O (100 L) were
m
m
m
added and a portion of the hexane layer was analyzed by GLC. The
chromatogram showed two intense peaks at 3.1 and 9.1 min,
corresponding to rhamnose tetra-TMS ether (
glucose penta-TMS ether ( -anomer) in ca. 1:1 ratio.
Methyl 3-hydroxyoctadecanoate (0.5 mg) was treated with (R)-
MTPACl (1.2 L) in pyridine (30 L) at room temperature. After
30 min, MeOH (100 L) was added to the reaction mixture and the
a-anomer) and
a
m
m
m
solution was purified by PTLC (hexane/AcOEt, 6:1) to give the (S)-
MTPA ester (0.7 mg, 93% yield). Colorless oil; FABMS m/z 531
[M+H]+; 1H NMR (CDCl3)
d: 7.58–7.54 (m, 5H), 5.47 (m, H-3), 3.59
(s, CO2Me), 3.47 (d, J = 1.0 Hz, OMe), 2.65 (dd, J = 16.1, 8.0 Hz, Ha-2),
2.57 (dd, J = 16.1, 5.1 Hz, Hb-2), 1.25 (CH2), 0.88 (t, J = 6.7 Hz, H3-
Fresh young leaves (fresh wt. 700 g, ca. 3.0 cm wide and 5.0 cm
long, 400 leaves) were similarly processed to give the Et2O extract
(30 mg), from which compounds 1 (20 mg), 2 (1.5 mg) and 3
(0.8 mg) were isolated.