Locustoside A - A new purine alkaloid glucoside from seeds of Gleditsia japonica

Article abstract of DOI:10.1016/j.phytol.2010.07.006

A new purine alkaloid glucoside designated locustoside A has been isolated from the seeds of Gleditsia japonica, which has been used in oriental traditional medicine. The structure was determined as 7-β-d-glucopyranosyl- 3-(3-methyl-2-butenyl)-isoguanine by interpretation of spectroscopic data and was confirmed by X-ray crystallographic analysis.

Full text of DOI:10.1016/j.phytol.2010.07.006

Phytochemistry Letters 3 (2010) 198–200
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Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Locustoside A — A new purine alkaloid glucoside from seeds of
Gleditsia japonica
Tadashi Kajimoto, Nobuwa Aoki, Emi Ohta, Shinji Ohta *
Nagahama Institute of Bio-Science and Technology, 1266 Tamura-cho, Nagahama, Shiga 526-0829, Japan
A R T I C L E I N F O
A B S T R A C T
Article history:
A new purine alkaloid glucoside designated locustoside A has been isolated from the seeds of Gleditsia
Received 1 May 2010
japonica, which has been used in oriental traditional medicine. The structure was determined as 7-b-D-
glucopyranosyl-3-(3-methyl-2-butenyl)-isoguanine by interpretation of spectroscopic data and was
confirmed by X-ray crystallographic analysis.
Received in revised form 13 July 2010
Accepted 18 July 2010
Available online 30 July 2010
ß 2010 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Keywords:
Gleditsia japonica
Leguminosae
Fabaceae
Alkaloid
1
. Introduction
2. Results and discussion
Japanese honey locust, Gleditsia japonica Miquel (Leguminosae)
has been used as a diuretic and an expectorant in oriental
traditional medicine (Konoshima et al., 1995). Its seeds have been
reported to contain polyamines (Hamana et al., 1996), phenols,
alkaloids, flavonoids, carbohydrates, saponins, steroids, coumarins,
and amino acids (Wan et al., 2001). During our continuing search
for biologically active compounds from plants (Aoki et al., 2008;
Kajimoto et al., 2010), we isolated a new purine alkaloid glucoside
designated locustoside A (1) from the MeOH extract of the seeds of
G. japonica. This report describes the purification and the structure
The seeds (166 g) of G. japonica were cut into small pieces and
extracted with MeOH. The concentrated MeOH extract was
suspended in water and partitioned successively with hexane
and EtOAc. The aqueous layer was lyophilized to yield a syrup. The
water-soluble fraction (8.4 g) was separated repeatedly on an ODS
column employing MeOH in H
(8.8 mg, 0.0053%) as colorless plates, mp 209–212 8C, [
(c 0.44, H O).
2
O gradient mixtures to afford 1
25
a
]
D
+35.2
2
Locustoside A (1) exhibited a pseudomolecular ion peak at m/z
+
[
(
_
)
T
D
$
F
I
G
]
lucidation of 1.
382 corresponding to [M+H] in the (+)ESIMS. The molecular
formula of 1 was established to be C16
high-resolution ESITOFMS data (m/z 382.1716 [M+H] ,
1.1 mmu). The IR spectrum displayed absorption bands at
23 5 6
H N O on the basis of
+
D
ꢀ
ꢀ
1
3
600–3000 (OH, NH, and CH), 1641, 1616, and 1583 cm (C 55 O,
C 55 N, and C 55 C). The UV (MeOH) absorption maxima at 246 (log
.86) and 290 nm (log 3.67) suggested the presence of a 3,7-
disubstituted isoguanine skeleton (Stewart and Harris, 1977;
e
3
e
1
13
Cafieri et al., 1995), which was supported by the H and C NMR
chemical shifts [ 8.12 (1H, s, H-8), 157.6 (C-2), 153.6 (C-4),
04.5 (C-5), 154.5 (C-6), 144.9 (C-8)]. Inspection of the H and
d
H
d
C
1
13
1
C
NMR spectra (Table 1) together with the DEPT and HMQC
spectral data revealed the presence of two olefinic methyls, two
hetero atom bearing methylenes, five oxygenated methines, an
olefinic methine, and a quaternary olefinic carbon in addition to
the isoguanine unit. The presence of an isopentenyl unit was
suggested by the observation of a fragment ion peak at m/z 314
*
Corresponding author. Tel.: +81 749 64 8101; fax: +81 749 64 8140.
+
E-mail address: s_ohta@nagahama-i-bio.ac.jp (S. Ohta).
[Mꢀ67] in the positive ion MS/MS spectrum, which was
1
874-3900/$ – see front matter ß 2010 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.phytol.2010.07.006

T. Kajimoto et al. / Phytochemistry Letters 3 (2010) 198–200
199
Table 1
the structure of locustoside A was elucidated as 7-
anosyl-3-(3-methyl-2-butenyl)-isoguanine (1).
b
-
D
-glucopyr-
a
NMR spectral data of 1 in D
2
O–CD
3
OD (9:1).
6
No.
d
C
d
H
multiplicity (J in Hz)
HMBC
Most of naturally occurring prenylated purine alkaloids are N -
6
prenylated adenine derivatives, e.g. N -isopentenyladenine
2
4
5
6
8
157.6 s
153.6 s
104.5 s
154.5 s
144.9 d
42.6 t
(Robins et al., 1967), which are known as plant cytokinins. So
far, only a few 3-prenylated purine alkaloids have been reported:
triacanthine (Belikov et al., 1954; Leonard and Deyrup, 1962),
saikachinoside A (Kajimoto et al., 2010), and dioicine (Fitch et al.,
2009) have been isolated from the leaves of Gleditsia triacanthos,
the seeds of G. japonica, and the leaves and seeds of the Kentucky
coffeetree, Gymnocladus dioicus, respectively.
0
0
0
0
8.12 s
C-4, C-5, C-1
0
0
0
1
4.52 dd (15.8, 7.0)
C-2, C-4, C-2 , C-3
4
.56 dd (15.8, 7.0)
C-2, C-4, C-2 , C-3
0
0
0
2
118.5 d
138.9 s
25.8 q
18.4 q
87.9 d
73.3 d
76.5 d
69.0 d
79.9 d
60.0 t
5.12 br t (7.0)
C-4 , C-5
0
3
0
0
0
0
0
0
0
4
1.65 br s (3H)
1.75 br s (3H)
5.57 d (8.4)
3.70 m
C-2 , C-3 , C-5
0
5
C-2 , C-3 , C-4
0
0
0
0
0
0
0
0
0
0
0
0
00
00
3. Experimental
1
2
3
4
5
6
C-5, C-8, C-2 , C-3
00
00
00
00
C-1 , C-3
00
3.68 m
C-2 , C-4
3.1. General
00
3.76 t (9.5)
3.78 m
C-3 , C-5
00
C-4
The optical rotation was measured using a Horiba SEPA-300
polarimeter. UV and IR spectra were recorded on a JASCO V-630
00
00
00
3.94 dd (12.5, 2.2)
C-4 , C-5
00
3
.90 dd (12.5, 1.8)
C-4 , C-5
1
13
and a Horiba FT720 spectrometer, respectively. H and C NMR
spectra were recorded on a JEOL AL400 spectrometer (400 MHz for
a
1
13
The H and C NMR were measured at 400 and 100 MHz, respectively.
3 H C
Chemical shifts were referenced to CD OD (d 3.30 and d 49.0).
1
13
H, 100 MHz for C) at 25 8C. High-resolution ESITOFMS was
measured on a Shimadzu LCMS-IT-TOF mass spectrometer. GC–MS
were measured on a Shimadzu GCMS-QP2010 Plus mass spec-
trometer. Single-crystal X-ray analysis was carried out on a Rigaku
RAXIS RAPID imaging plate area detector with graphite mono-
˚
chromated Mo Ka radiation (l = 0.71075 A). Column chromatog-
raphy was carried out using ODS (Wacogel LP40C18). TLC was
carried out on precoated Kieselgel 60 F254 (Merck, Darmstadt,
Germany) and RP18 F254 plates (Merck).
0 0
2
established on the basis of the HMBC correlations (H -1 /C-2 and
0
0
0
0
0
0
0
0
0
C-3 ; H-2 /C-4 and C-5 ; H
3
-4 /C-2 ,
C-3 , and C-5 ; H
3
-5 /C-
0
0
0
0
2
4
, C-3 , and C-4 ). The HMBC correlations from H-1 to C-2 and C-
indicated the attachment of the isopentenyl unit at N-3 of the
1
1
1
isoguanine unit. The interpretation of H NMR and H– H COSY
spectra revealed the presence of a glucopyranosyl unit which was
supported by the observation of a fragment ion peak at m/z 152
+
[
Mꢀ67ꢀ162] in the positive ion MS/MS spectrum. The HMBC
3.2. Plant material
0
0
correlations (Fig. 1) from H-1 to C-5 and C-8 and from H-8 to C-
0
0
1
1
as well as the H vicinal coupling constant of the anomeric
proton indicated the attachment of the glucopyranosyl unit at N-
of the isoguanine unit via an N- -glycosidic linkage. The
structure of 1 was confirmed by X-ray crystallographic analysis
Fig. 2).
Absolute configuration of glucose obtained after acid hydrolysis
The seeds of G. japonica were collected in Shiga Prefecture,
Japan in March 2009. Voucher specimens (No: SS090301) are kept
at Nagahama Institute of Bio-Science and Technology.
7
b
(
3.3. Extraction, isolation and characterization
of 1 was determined as D by GC–MS analysis of the trimethylsi-
The seeds (166 g) of G. japonica were cut into small pieces
lylated thiazolidine derivative (Hara et al., 1987). Consequently,
[(Fig._1)TD$FIG]
and extracted with MeOH. The concentrated MeOH extract
(
10 g) was suspended in water and partitioned successively
with hexane and EtOAc. The aqueous layer was lyophilized to
yield syrup. The water-soluble fraction (8.4 g) was separated on
an ODS column (50 g, 2.5 cm ꢁ 30 cm) employing MeOH in
2
H O gradient mixtures [20:80, 40:60, 60:40, 80:20, 100:0
(
6
100 mL of each solvent mixture)] yielding 10 fractions. Fraction
(38 mg, eluted with MeOH–H O, 60:40) was applied to an ODS
O [40:60, 60:40,
0:20 (10 mL of each solvent mixture)] to afford 1 (8.8 mg,
.0053%).
2
column (4 g, 0.8 cm ꢁ 20 cm) with MeOH–H
2
8
0
Fig. 1. Key HMBC correlations observed for 1.
3.4. Locustoside A (1)
[(Fig._2)TD$FIG]
2
5
Colorless needles; mp 209–212 8C; [
a
]
D
+35.28 (c 0.44, H
2
O);
UV (MeOH) 246 (log
e
3.86), 290 nm (log
e
3.67); IR max (KBr):
n
ꢀ1
1
13
3600–3000, 1641, 1616 and 1583 cm ; for H and C NMR
+
spectroscopic data see Table 1; HRESITOFMS m/z 382.1716 [M+H]
calc. for C16 , 382.1727).
(
24 5 6
H N O
3.5. X-ray crystallographic study of 1
Formula
C
16
H
23
N
5
O
6
,
M = 381.39, 0.40 mm ꢁ 0.20 mm ꢁ
0
.10 mm, orthorhombic, space group P2
a = 8.298 (3) A, b = 12.837 (4) A, c = 18.160 (8) A,
V = 1934.4 (12) A , F(0 0 0) = 808,
Dc = 1.309 g cm , T = 100 K. 18 662 reflections measured, 2507
1
2
1
2
˚
1
(#19), Z = 4,
˚
˚
a
=
b
=
g
= 908,
3
ꢀ1
˚
m(Mo
K
a) = 1.015 cm
,
ꢀ
3
Fig. 2. Perspective view of the crystal structure of 1.

2
00
T. Kajimoto et al. / Phytochemistry Letters 3 (2010) 198–200
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He; injection temperature 300 8C, column temperature 150 8C,
m
m
1
1
R
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