Microtropins A-I: 6′-O-(2″S,3″R)-2″-Ethyl-2″, 3″-dihydroxybutyrates of aliphatic alcohol β-d-glucopyranosides from the branches of Microtropis japonica

Article abstract of DOI:10.1016/j.phytochem.2012.11.007

From the branches of Microtropis japonica (Celastraceae), nine aliphatic glucosides, named microtropins A-I, were isolated. The 6-position of glucose was esterified with (2S,3R)-2-ethyl-2,3-dihydroxybutyric acid. Microtropins A-D contained a rare natured product nitrile functional group in their aglycones. The absolute structures of the (2S,3R)-2-ethyl-2,3-dihydroxybutyric acid moiety and aglycone of microtropin A were determined by an X-ray crystallographic method.

Full text of DOI:10.1016/j.phytochem.2012.11.007

Phytochemistry 87 (2013) 140–147
Contents lists available at SciVerse ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Microtropins A–I: 60-O-(200S,300R)-200-Ethyl-200,300-dihydroxybutyrates of aliphatic
alcohol b-D-glucopyranosides from the branches of Microtropis japonica
b
Yuka Uemura a, Sachiko Sugimoto a, Katsuyoshi Matsunami a, Hideaki Otsuka a,b, , Yoshio Takeda ,
⇑
Masatoshi Kawahata c, Kentaro Yamaguchi c
a Department of Pharmacognosy, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan
b Department of Natural Products Chemistry, Faculty of Pharmacy, Yasuda Women’s University, 6-13-1 Yasuhigashi, Asaminami-ku, Hiroshima 731-0153, Japan
c Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Kagawa Campus, 1314-1 Shido, Sanuki-shi, Kagawa 769-2139, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 August 2012
Received in revised form 1 October 2012
Available online 5 December 2012
From the branches of Microtropis japonica (Celastraceae), nine aliphatic glucosides, named microtropins
A–I, were isolated. The 6-position of glucose was esterified with (2S,3R)-2-ethyl-2,3-dihydroxybutyric
acid. Microtropins A–D contained a rare natured product nitrile functional group in their aglycones.
The absolute structures of the (2S,3R)-2-ethyl-2,3-dihydroxybutyric acid moiety and aglycone of micro-
tropin A were determined by an X-ray crystallographic method.
Keywords:
Microtropis japonica
Ó 2012 Elsevier Ltd. All rights reserved.
Celastraceae
Microtropin
(2S,3R)-2-Ethyl-2,3-dihydroxybutyric acid
3-Hydroxy-2-methylbutanenitrile
(E)-4-Hydroxy-2-(hydroxymethyl)but-2-
enenitrile
1. Introduction
2. Results and discussion
Celestraceous plants appeared before the footlight, after a
potent antileukemic ansa-type macrolide, maytansine was isolated
from an Ethiopian shrub, Maytenus serrata (formerly Maytenus
ovatus) (Kupchan et al., 1972; Cassady et al., 2004). In the contin-
uous research on subtropical resource plants, a Celestraceous
plant, Microtropis japonica, collected in Okinawa attracted our
attention and its constituents were investigated. In a previous
paper, the isolation of six ent-labdane-type diterpenoid glucosides,
named microtropiosides A–F were reported, from a MeOH extract
of the leaves of M. japonica (Koyama et al., 2010). M. japonica
Hallier f. is an evergreen tree of about 5 m in height, and has a dis-
tinct distribution in restricted southern parts of Kanto and Kyushu,
Japan, Okinawa Islands and Taiwan (Hatushima, 1975). Isolation
work on the branches of M. japonica afforded nine aliphatic gluco-
sides carrying 2-ethyl-2,3-dihydroxybutyrate at the 6-position of
glucose (1–9). Four of the aliphatic aglycones contained a nitrile
group (1–4), and the structure of 1 including the absolute configu-
rations of the aglycone and acyl moieties was determined by X-ray
crystallographic analysis.
Nine new 2-ethyl-2,3-dihydroxybutyrates of various glucosides,
named microtropins A–I (1–9), were isolated from the 1-BuOH-sol-
uble fraction of a MeOH extract of the branches of M. japonica by a
combination of various separation procedures. Their structures
were elucidated from spectroscopic evidence and the results of
X-ray crystallographic analysis.
Microtropin A (1), [
rods and its elemental composition was determined to be
17H29NO9 by high-resolution (HR)-electrospray ionization (ESI)-
a]
D ꢀ 12.2, was isolated as colorless fine
C
mass spectrometry (MS). The IR spectrum exhibited strong absorp-
tions at 3405 and 1736 cmꢀ1 for hydroxyl and ester carbonyl
groups, respectively, and a characteristic absorption at 2246 cmꢀ1
for a triple bond. In the 1H NMR spectrum, one triplet (dH 0.89)
and three doublet (dH 1.18, 1.34 and 1.36) methyl signals were
observed, together with an anomeric proton resonance at dH 4.39
(J = 7.7 Hz). On acid hydrolysis of 1, D-glucose was detected on
HPLC analysis with an optical rotation detector. The 13C NMR spec-
trum exhibited six signals assignable to 6-acylated glucopyranose,
and ones for four methyls, one methylene, two oxymethines, one
methine, one quaternary carbon with an oxygen functional group,
and an ester carbonyl carbon were also observed. The remaining
one singlet carbon resonance at dC 122.3 must be involved in the
formation of a nitrile functional group, taking into account the
presence of a nitrogen atom and the absorption for a triple bond
⇑
Corresponding author at: Department of Pharmacognosy, Graduate School of
Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima
734-8553, Japan. Tel./fax: +81 82 257 5335.
E-mail address: hotsuka@hiroshima-u.ac.jp (H. Otsuka).
0031-9422/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.phytochem.2012.11.007

Y. Uemura et al. / Phytochemistry 87 (2013) 140–147
141
4
5
3
OH
3"
RO
1
O
2
1"
CN
1
2"
O
4"
HO
O
R:
HO
1'
RO
2
HO
OH
6"
OH
CN
RO
3
1
CN
5
1
2
RO
2
2
RO
1
CN
3
RO
6
5
OH
7
4
OH
4
1
RO
OR
OH
1
2
2
R1
R
OH
OR1
9
8
8a Glc
Fig. 3. ORTEP drawing of 1. The crystal structure has crystallographic numbering.
8c
H
Fig. 1. Structures of isolated compounds.
in the IR spectrum. On 1H–1H correlation spectroscopy (COSY),
three proton spin–spin coupling sequences, i.e., H3-5–H-2–H-3–
H3-4, H-300–H3-400 and H-500–H3-600, were observed (Fig. 2a), and
on heteronulear multiple bond correlation (HMBC) spectroscopy,
assumption (Fig. 2b). Finally, the acyl moiety was found to com-
prise six carbons, i.e., two methyls, one methylene, one oxyme-
thine, one oxygenated secondary and one carbonyl carbon. In the
HMBC spectrum, both of the methyl protons showed correlation
peaks with the oxygenated tertiary carbon and the methylene pro-
tons with the carbonyl carbon. From the above evidence, together
with the results of a 1H–1H COSY experiment, the structure of the
acyl moiety was established to be 2-ethyl-2,3-dihydroxybutyrate.
This was confirmed by hydrolysis of microtropin A (1) under mild
alkaline conditions in MeOH, which gave 3-hydroxy-2-methylbu-
the anomeric proton showed
a correlation peak with C-3
(dC 77.5) and H3-5 (dH 1.36) with dC 122.3 (s) (Fig. 2b), indicating
that the structure of the aglycone was 2-methyl-3-hydroxybutane-
nitrile. The 13C and 1H NMR chemical shifts of the 60-position (C-60
and H2-60) of glucose were shifted downfield (dC 65.1, and dH 4.22
and 4.63), when compared with those of glucose without a substi-
tuent at that position (dC 62.8, and dH 3.69 and 3.85) (Shitamoto
et al., 2010). Thus, this position was assumed to be attached to
some acyl group through an ester bond, which was indicated by
the presence of the carbonyl carbon signal (dC 176.1) in the 13C
NMR spectrum and the IR absorption at 1736 cmꢀ1. The HMBC
correlations from H2-60 to the carbonyl carbon also supported the
tanenitrile b-D-glucopyranoside (1a) and methyl 2-ethyl-2,
3-dihydroxybutyrate (1b). Ribesuvanin A, isolated from Ribes
uva-crispa (Bjarnholt et al., 2008) must be the same compound as
1a, however, only 1H NMR spectroscopic data for ribesuvanin A
are available and 1b is also known as a synthetic material without
characterization data (Yoshimitsu and Ogasawara, 1994). Micro-
tropin A (1) was found to have four asymmetric centers at difficult
positions (C-2, C-3, C-200 and C-300) to be determined, and thus, crys-
talline 1 was subjected to X-ray analysis for unambiguous corrob-
oration of the stereochemistry using glucose as a chiral probe.
Fig. 3 shows an ORTEP drawing of 1, and the absolute stereochem-
istries of the four chiral centers were determined to be 2R, 3S, 200S
and 300R, respectively. Finally, the structure of 1 was elucidated to
OH
O
4
3"
O
4"
OH
3
5
5"
O
HO
O
2
6"
HO
OH
be (2R,3S)-3-hydroxy-2-methylbutanenitrile b-D-glucopyranoside
CN
60-O-(200S,300R)-200-ethyl-200,300-dihydroxybutyrate, as shown in
Fig. 1. A related compound, supinanitriloside D, was isolated from
Euphorbia supina (Cai et al., 2009). The structure of aglycone of 1
was the same as that of supinanitriloside D and supinanitriloside
D which possessed gallate as an acyl moiety, instead of (200S,300R)-
200-ethyl-200,300-dihydroxybutyrate in 1. The absolute configuration
of the aglycone moiety of supinanitriloside D was determined to
be the same as that of 1, since the 13C NMR spectroscopic data of
aglycone and sugar moieties of these two compounds were essen-
tially the same.
a
OH
O
O
OH
O
HO
HO
O
OH
CN
Microtropins B and C (2 and 3), [
a
]
D ꢀ 8.1 and [ D ꢀ 4.3,
a
]
b
H
C
respectively, were isolated as amorphous powders and they exhib-
ited the same elemental composition as that of 1. The IR spectra
exhibited characteristic absorptions for nitrile (2245 and 2248,
respectively) and ester (both 1736 cmꢀ1) functional groups. Their
HMBC
Fig. 2. (a) 1H–1H COSY correlations. (b) Diagnostic HMBC correlations. Dual arrow
curves denote HMBC correlations were observed in both directions.

142
Y. Uemura et al. / Phytochemistry 87 (2013) 140–147
Table 1
13C NMR spectroscopic data for microtropins A–I (1–9) and reference compounds (CD3OD, 100 MHz).
13C
1
1a
1b
2
3
4
5
6
7
7a
7bb
8
8a
22.0
8c
9c
23.6
68.7
35.3
1
2
3
4
5
6
122.3
33.2
77.5
20.0
14.1
122.4
33.0
77.2
19.8
13.9
122.4
34.0
77.0
16.9
14.9
122.3
33.1
77.5
18.9
14.7
116.7
118.2
144.6
68.2
23.9
73.0
22.2
74.8
72.79
27.2
10.3
18.5a
83.5
72.2
18.7a
18.7
72.7
72.7
18.7
18.6
72.6
72.6
18.6
22.2 (ꢀ1.4)d
78.0 (+9.3)d
33.7 (ꢀ0.4)d
33.2
73.8
31.1
23.6
68.7
34.1
30.8
74.0
31.1
10.4
77.9
33.6
33.2
74.0
31.2
10.4
104.0
75.4
77.6
71.8
78.3
63.0
30.6 (ꢀ0.2)e
82.4 (+8.4)e
28.7 (ꢀ2.4)e
10.2
63.2
7
10.3
10
104.9
75.2
77.8
71.3
75.2
65.1
176.1
82.9
72.8
16.8
29.2
8.4
104.8
75.3
78.0
71.6
78.2
62.8
103.6
74.9
77.7
71.3
75.3
65.0
176.1
82.9
72.8
16.8
29.3
8.5
104.2
75.1
77.8
71.4
75.3
65.1
176.1
82.9
72.9
16.9
29.3
8.4
104.2
74.9
77.7
71.4
75.5
65.0
176.2
82.9
72.8
16.9
29.3
8.4
102.8
75.1
77.9
71.6
75.1
65.3
176.1
82.9
72.8
16.8
29.2
8.4
104.6
75.1
77.7
71.5
75.2
65.2
176.1
82.9
72.81
16.9
29.2
8.4
105.7
75.2
77.7
71.5
75.5
65.2
176.1
82.9
72.8
16.8
29.2
8.4
104.2
75.1
77.9
71.6
75.3
65.3
176.1
82.9
72.8
16.8
29.2
8.4
103.9
75.2
78.0
71.6
75.4
65.4
176.2
82.9
72.9
16.9
20
30
40
50
60
100
200
300
400
500
600
–OCH3
176.9
82.9
72.8
16.9
29.3
8.3
29.3
8.5
52.7
a
b
c
Maybe exchangeable.
Although 7b is a diastereomeric mixture, only two signals were observed.
At 150 MHz.
d
e
D
D
d8–8c
d9–8c
.
.
constants. The anti-isomer must have a smaller coupling constant
than that of the syn isomer judging from the results of the X-ray
crystallographic analysis of microtropin A (1). The b-D-glucopyr-
Table 2a
1H and 13C NMR spectroscopic data for (2R⁄,3R⁄)-3-hydroxy-2-methylbutanenitrile
(anti) (11a) and (2R⁄,3S⁄)-3-hydroxy-2-methylbutanenitrile (syn) (11b).
anosylation induced shift-trend rule (Kasai et al., 1977) was ap-
plied to 2 and 3 to establish the absolute configuration at their
3-positions. Table 2b indicates that the b-positions (C-2 and C-4)
11a
11b
H
C
H
C
1
2
3
4
5
–
122.3
34.8
68.6
21.3
14.8
–
122.5
34.8
68.8
19.8
14.3
of 2 were shifted upfield on b-D-glucopyranosylation by 0.8 and
2.74 (qd, J = 7.1, 4.4 Hz)
3.79 (qd, J = 6.2, 4.4 Hz)
1.28 (d, J = 6.2 Hz)
1.32 (d, J = 7.1 Hz)
2.80 (qd, J = 7.1, 5.5 Hz)
3.79 (overlapped)
1.25 (d, J = 5.7 Hz)
1.28 (d, J = 7.1 Hz)
2.9 ppm, respectively, in the 13C NMR spectrum. Thus, C-2 is in
so-called pro-R and C-4 in the pro-S position, respectively. There-
fore, the structure of microtropin B (2) was elucidated to be
(2R,3R)-3-hydroxy-2-methylbutanenitrile
b-D-glucopyranoside
60-O-(200S,300R)-200-ethyl-200,300-dihydroxybutyrate, as shown in
Fig. 1. Similarly, that of 3 was elucidated to be (2S,3S)-3-hydro-
Table 2b
xy-2-methylbutanenitrile b-
ethyl-200,300-dihydroxybutyrate.
Microtropin D (4), [
D ꢀ 7.0, was isolated as an amorphous
powder and its elemental composition was determined to be
17H27NO10 by HR-ESI-MS. The IR spectrum also exhibited an
D
-glucopyranoside 60-O-(200S,300R)-200-
Application of b-D-glucopyranosylation-induced shift-trends to microtropins B (2)
and C (3).
a
]
11b
2
3
1
2
3
4
5
122.5
34.8
68.8
19.8
14.3
122.4
122.3
C
34.0 (ꢀ0.8)a
77.0 (+8.2)a
16.9 (ꢀ2.9)a
14.9
33.1 (ꢀ1.7)b
77.5 (+8.7)b
18.9 (ꢀ0.9)b
14.7
absorption band (2224 cmꢀ1) arising from a triple bond. Analysis
of the 1H and 13C NMR spectra established the presence of a
(2S,3R)-2-ethyl-2,3-dihydroxybutyrate moiety, and the six sugar
signals were assignable to 6-O-acylated glucopyranose, whose
absolute configuration was determined to be D on chirality analy-
sis. Five 13C NMR signals, which constituted an aglycone moiety,
were for two oxymethylenes (dC 63.2 and 68.2), a trisubstituted
double bond and an odd quaternary carbon (dC 116.7). The odd
quaternary carbon thus formed a nitrile group, and the double
bond was substituted by the nitrile and two oxymethylene groups.
C-H correlation assignments were performed by means of an HSQC
experiment, and the H2-5 protons [dH 4.16 (2H, ddd, J = 1.4, 1.4,
1.4 Hz)] showed long-range couplings with H-3 and H2-4. A direct
coupling H-3 olefinic proton and H2-4 were observed in the 1H
NMR spectrum, and in the differential NOE experiment, signal
strength enhancement of the H-3 proton on irradiation of H2-5
and vice versa assigned the geometry of the double bond as E.
The attachment site of the sugar moiety was determined to be to
the hydroxyl group at the 4-position by the HMBC spectrum and
the mode of linkage was b judging from the coupling constant of
the anomeric proton (J = 7.9 Hz) in the 1H NMR spectrum.
a
D
D
d2–11b
d3–11b
.
.
b
spectroscopic data indicated that they were similar compounds to
1. The NMR spectroscopic chemical shifts of the acyl and sugar
moieties were essentially superimposable on those of 1, but those
of the aglycones showed slight differences. The coupling constant
between H-2 and H-3 of microtropin A (1) was J = 4.3 Hz, whereas
those for microtropins B and C (2 and 3) were 5.2 and 5.5 Hz.
Although the stereochemistry of 3-hydroxy-2-methylbutanenitrile
was examined by enzyme-catalyzed reduction of 3-keto-2-meth-
ylbutanenitrile, ambiguous data have been still reported (Ito
et al., 1989). 3-Hydroxy-2-methylbutanenitrile (11) was indepen-
dently prepared according to the method reported by Nishimura
et al. (Nishimura et al., 1987), the final products containing anti
(11a) and syn isomers (11b) (Table 2a), which were clearly distin-
guishable from the unequal product ratio and their J2–3 coupling

Y. Uemura et al. / Phytochemistry 87 (2013) 140–147
143
Table 3
OH
OH
Partial 1H NMR spectroscopic data for microtropin F (6) and reference compounds,
(2R)- and (2S)-1,2-propanediol 1-O-b-D-glucopyranosides (6a and 6b, respectively)
(CD3OD, 400 MHz).
O
O
HO
O
H
1
6
6a
6b
HO
HO
O
3.73 dd J = 10.3, 5.9 Hz 3.71 dd J = 10, 7 Hz
3.58 dd J = 10.3, 3.6 Hz 3.56 dd J = 10, 4 Hz
3.86 dd J = 10, 3 Hz
3.35 dd J = 10, 8 Hz
OH
2
3.62–3.68 m
3.94 dqd J = 7, 7, 4 Hz 3.95 dqd J = 8, 7, 3 Hz
Fig. 4. Important HMBC correlations of 8.
NMR data for 6a and 6b are from Kijima et al. (1996).
methyl [dH 0.93 (t, J = 6.4 Hz) on dC 10.3], one doublet methyl [dH
1.22 (d, J = 6.4 Hz) on dC 22.2], three methylenes (dC 33.7, 33.2
and 31.1), and two secondary (dC 78.0 and 73.8) alcohols (Table 1).
In the HMBC spectrum, the anomeric proton (dH 4.34) showed a
correlation peak with C-2 (dC 78.0), and H3-7 (dH 0.93, t,
J = 6.4 Hz) with C-5 (dC 78.0). Therefore, the structure of 8 was ten-
Therefore, the structure of microtropin D (4) was elucidated to be
(E)-4-hydroxy-2-(hydroxymethyl)but-2-enenitrile 4-O-b-D-gluco-
pyranoside 60-O-(200S,300R)-200-ethyl-200,300-dihydroxybutyrate, as
shown in Fig. 1.
Microtropin E (5), [
powder and its elemental composition was determined to be
15H28O9. The spectroscopic data implied 5 was a similar com-
a]
D ꢀ 38.6, was isolated as an amorphous
tatively elucidated to be 2,5-heptanediol 2-O-b-D-glucopyranoside
60-O-(200S,300R)-200-ethyl-200,300-dihydroxybutyrate, as shown in
Fig. 1. Microtropin H (8) was hydrolyzed with CH3ONa in MeOH
to give deacylated compound 8a and methyl (200S,300R)-200-ethyl-
200,300-dihydroxybutyrate (8b = 1b), followed by enzymatic
C
pound to the aforementioned ones with a different aglycone. The
aglycone comprised three carbon atoms, two doublet methyls [dH
1.18 (d, J = 6.2 Hz) on dC 22.2 and 1.21 (d, J = 6.2 Hz) on dC 23.9],
and a secondary alcohol [dH 3.97 (septet, J = 6.2 Hz) on dC 73.0]
(Table 1). Therefore, microtropin E (5) was elucidated to be 2-pro-
hydrolysis of 8a that gave an aglycone (8c). When the b-D-gluco-
pyranosylation-induced shift-trend rule was applied for 8a and
8c (Kasai et al., 1977) (Table 1), the absolute configuration at the
2-position was determined to be R.
panol
b-
D
-glucopyranoside
60-O-(200S,300R)-200-ethyl-200,300-
-gluco-
dihydroxybutyrate, as shown in Fig. 1. According to the b-
D
Microtropin I (9), [
a
]
D ꢀ 16.1 was isolated as an amorphous
pyranosylation-induced shift-trend rule, C-1 is pro-R methyl and
C-3 pro-S.
powder and it had the same elemental composition as that of 8.
The spectroscopic data indicated that 9 was a similar compound
to 8. The NMR spectroscopic chemical shifts of the aglycone
showed slight differences, especially for one doublet methyl
[dH 1.15 (d, J = 6.2 Hz) on dC 23.6], one triplet methyl [dH 0.90
(t, J = 7.6 Hz) on dC 10.2], and two secondary alcohols (dC 68.7
and 82.4) (Table 1). In the HMBC spectrum, the anomeric proton
(dH 4.32) and H3-7 (dH 0.93) showed correlation peaks with C-5
(dC 82.4) (Fig. 4). Therefore, the structure of 9 was elucidated to
Microtropin F (6), [
powder and its elemental composition was determined to be
16H30O10 by HR-ESI-MS. From analysis of the spectroscopic data,
a]D + 3.2, was also isolated as an amorphous
C
6 was also expected to be a congeneric compound with a different
aglycone. The aglycone moiety comprised four carbons, a triplet
methyl [dH 0.95 (t, J = 7.5 Hz) on dC 10.3], a methylene (dC 27.2),
and primary (dC 74.8) and secondary (dC 72.79) alcohols (Table 1).
In the HMBC spectrum, the anomeric proton (dH 4.28) showed a
correlation peak with the primary alcohol carbon (dC 74.8).
Therefore, the structure of 6 was elucidated to be 1,2-butanediol
be 2,5-heptanediol 5-O-b-
ethyl-200,300-dihydroxybutyrate, as shown in Fig. 1. Similarly the
b- -glucopyranosylation-induced shift-trend rule for 9 and 8c
D
-glucopyranoside 60-O-(200S,300R)-200-
1-O-b-D
-glucopyranoside 60-O-(200S,300R)-200-ethyl-200,300-dihydroxybu-
D
showed the absolute configuration at the 5-position to be R
(Table 1).
tyrate, as shown in Fig. 1. The stereochemistry at the 2-position
was determined by comparison of 1H NMR spectroscopic data for
synthetic (2R)- and (2S)-1,2-propanediol 1-O-b-D-glucopyrano-
sides (6a and 6b, respectively) (Kijima et al., 1996) with those for
6. The protons on the primary alcohols of 6 resonated at dH 3.73
and 3.58 showed a closer resemblance to those of the (2R)-isomer
(6a) than those of the (2S)-isomer (6b), and the coupling constants
[dH 3.73 (dd, J = 10.3, 5.9 Hz) and 3.58 (dd, J = 10.3, 3.6 Hz)] of these
protons were closer to those of the (2R)-isomer (6a) as well
(Table 3). From this evidence, the chirality at the 2-postion was
determined to be R.
3. Concluding remarks
A series of glucosides with an unusual (2S,3R)-2-ethyl-2,3-
dihydroxybutyric acid as the acyl moiety at the 6-position of glu-
cose was isolated. Of the compounds isolated, microtropins A–D
(1–4) contained a nitrile functional group in their aglycone moie-
ties, and the aglycones of the other microtropins were relatively
simple alcohols. 2-Ethyl-2,3-dihydroxybutyric acid is known to
be found in cider fermentations (Whiting, 1959), and probably an
intermediate in biosynthesis of isoleucine (Satyanarayana and
Microtropin G (7), [
powder and its elemental composition was determined to be
16H30O10. From analysis of the spectroscopic evidence, 7 was
a
]D ꢀ 0.87, was isolated as an amorphous
C
Radhakrishnan, 1962).
A
related compound, 2-methyl-2,3-
judged to be an analogous compound with a different aglycone.
The aglycone possessed two doublet methyls [dH 1.13
(d, J = 6.4 Hz) and 1.18 (d, J = 6.3 Hz)] and two secondary alcohols
(dC 72.2 and 83.5), indicating its structure of the aglycone to be
2,3-butandiol. Since the two methyl signals of 7, and those of race-
mic 2,3-butandiol (7a) and (2R,3R)-2,3-butanediol (7b) resonated
dihydroxybutyric acid was found in wine (Würdig et al., 1969). Iso-
lation of this acid as a natural compound is the first time at has
been reported.
4. Experimental
at very close frequencies, the b-D-glucopyranosylation-induced
shift-trend rule could not be applied. Thus, the absolute configura-
tion at the 2- and 3-positions remains to be determined.
4.1. General experimental procedures
Microtropin H (8), [
a
]
D ꢀ 17.8, was also isolated as an amor-
Optical rotations were measured on a JASCO P-1030 digital
polarimeter. IR and UV spectra were measured on Horiba FT-710
and JASCO V-520 UV/Vis spectrophotometers, respectively. 1H
phous powder and its elemental composition was determined to
be C19H36O10 by HR-ESI-MS. From spectroscopic data, 8 was also
expected to be a congeneric compound with a different aglycone.
The NMR spectra of the aglycone moiety exhibited one triplet
and 13C NMR spectra were taken on a JEOL JNM
a
-400 or ECA-
600 at 400 or 600 MHz, or 100 or 150 MHz, respectively, with

144
Y. Uemura et al. / Phytochemistry 87 (2013) 140–147
tetramethylsilane as an internal standard. Positive-ion HR-ESI-MS
was performed with an Applied Biosystems QSTAR XL NanoSpray™
System.
(H2O-MeOH, 3:1) to afford 5 (1.7 mg) and 3 (0.2 mg) from the
peaks at 29 and 32 min, respectively.
The residue (2.91 g out of 3.10 g) in fractions 30–38 obtained on
A highly-porous synthetic resin (Diaion HP-20) was purchased
from Mitsubishi Kagaku (Tokyo, Japan). Silica gel column chroma-
tography (CC) was performed on silica gel 60 (70–230 mesh, E.
Merck, Darmstadt, Germany), and ODS open CC on Cosmosil
75C18-OPN (Nacalai Tesque, Kyoto). Analytical and preparative
TLC were carried out on precoated silica gel plates (CHCl3–
MeOH–H2O: 15:6:1) [Kiesel Gel 60 F254 (E. Merck), 0.25 mm thick-
ness] and spots were detected under UV light (254 nm) and/or
visualized by spraying 10% H2SO4 and heating at 120 °C. The drop-
let counter-current chromatograph (Tokyo Rikakikai, Tokyo, Japan)
silica gel CC was separated by ODS CC (U = 25 mm, L = 25 cm) with a
linear gradient solvent system [H2O–MeOH (1:9, 2 L) ? (9:1 2 L)],
10 g-fractions being collected. The residue (88.4 mg) in fractions
73–80 was separated by DCCC and then the residue (21.3 mg) in
fractions 57–64 was finally purified by HPLC (H2O–MeOH, 17:3) to
yield 4 (6.0 mg) from the peak at 31 min. The residue (306 mg) in
fractions 81–93 was applied to DCCC and the residue (64.8 mg) in
fractions 42–47 was separated by HPLC (H2O–MeOH, 3:1) to give
two fractions. The former (3.9 mg from the peak at 21 min) was fi-
nally purified by HPLC again (Inertsil; Ph-3, GL Science, Tokyo, Japan;
was equipped with 500 glass columns (
U
= 2 mm, L = 40 cm), the
U = 6 mm, L = 250 mm, 1.6 mL/min) (H2O–MeOH, 1:4) to give 6
lower and upper layers of a solvent mixture of CHCl3–MeOH–
H2O–1-PrOH (9:12:8:2) being used as the stationary and mobile
phases, respectively. Five-gram fractions were collected and num-
bered according to their order of elution with the mobile phase.
HPLC was performed on an ODS column (Inertsil; ODS-3, GL Sci-
(1.0 mg) from the peak at 13 min. The latter (10.9 mg from the peak
at 22 min) was similarly purified by HPLC to give 7 (5.2 mg) from the
peak at 13 min. The residue (134 mg) in fractions 151–168 was sub-
jected to DCCC and the residue (30.3 mg) in fractions 64–85 was
purified by HPLC (H2O–MeOH, 3:2) to give 8 (5.4 mg) and 9
(2.2 mg) from the peaks at 21 and 22 min, respectively.
ence, Tokyo, Japan;
U = 6 mm, L = 250 mm, 1.6 mL/min), and the
eluate was monitored with a UV detector at 254 nm, and a refrac-
tive index monitor.
2,3-Butanediol, (2R,3R)-butanediol and 3-chloro-2-butanone
were purchased from Wako Pure Chemical Industries, Ltd. (Osaka,
Japan). b-Glucosidase from almond was also a product of Wako
Pure Chemical Industries, Ltd.
4.3.1. Microtropin A (1)
23
Colorless rods; mp 153–154 °C (2-PrOH); [
a
]
D
ꢀ 12.2 (c 1.27,
MeOH); IR mmax (film): 3405, 2980, 2246, 1736, 1456, 1382, 1171,
1079, 1053 cmꢀ1 1H NMR (CD3OD, 400 MHz) d: 4.63 (1H, dd,
;
J = 11.7, 2.2 Hz, H-60a), 4.39 (1H, d, J = 7.7 Hz, H-10), 4.22 (1H, dd,
J = 11.7, 5.2 Hz, H-60b), 3.93 (1H, q, J = 6.5 Hz, H-300), 3.88 (1H, qd,
J = 6.4, 4.3 Hz, H-3), 3.49 (1H, ddd, J = 9.9, 5.2, 2.2 Hz, H-50), 3.35
(2H, m, H-30 and 40), 3.20 (1H, dd, J = 9.2, 7.7 Hz, H-20), 2.94 (1H,
qd, J = 6.8, 4.3 Hz, H-2), 1.74 (1H, dq, J = 13.9, 7.5 Hz, H-500a), 1.55
(1H, dq, J = 13.9, 7.5 Hz, H-500b), 1.36 (3H, d, J = 6.8 Hz, H3-5), 1.34
(3H, d, J = 6.4 Hz, H3-4), 1.18 (3H, d, J = 6.5 Hz, H3-400), 0.89 (3H, t,
J = 7.5, 7.5 Hz, H3-600); 13C NMR (CD3OD, 100 MHz): Table 1; HR-
ESI-MS (positive-ion mode) m/z: 414.1728 [M+Na]+ (calcd for C17-
H29NO9Na, 414.1734).
4.2. Plant material
Branches of M. japonica Hallier f. (Celastraceae) were collected
in Kunigami Village, Kunigami County, Okinawa, Japan, in July
1997, and a voucher specimen was deposited in the Herbarium
of Pharmaceutical Sciences, Graduate School of Biomedical Sci-
ences, Hiroshima University (97-MJ-Okinawa-0716).
4.3. Extraction and isolation
Branches of M. japonica (13.0 kg) were extracted three times
with MeOH (30 L ꢁ 3) at room temperature for 1 week and then
concentrated to 3 L in vacuo. The concentrated extract was washed
with n-hexane (3 L, 50.8 g), and then the MeOH layer was concen-
trated to a gummy mass. The latter was suspended in H2O (3 L) and
then extracted with EtOAc (3 L) to give an EtOAc-soluble fraction
(103 g). The aqueous layer was extracted with 1-BuOH (3 L) to give
a 1-BuOH-soluble fraction (40.9 g), and the remaining H2O-solu-
ble-layer was concentrated to furnish a H2O-soluble fraction
(107 g). The 1-BuOH-soluble fraction (39.9 g) was subjected to Dia-
4.3.2. Microtropin B (2)
24
Amorphous powder; [
a
]
ꢀ 8.1 (c 0.25, MeOH); IR
mmax (film):
D
3393, 2980, 2245, 1736, 1456, 1391, 1238, 1170, 1089, 1052 cmꢀ1
;
1H NMR (CD3OD, 400 MHz) d: 4.62 (1H, dd, J = 11.7, 2.1 Hz, H-60a),
4.37 (1H, d, J = 7.7 Hz, H-10), 4.21 (1H, dd, J = 11.7, 5.3 Hz, H-60b),
3.91 (1H, q, J = 6.4 Hz, H-300), 3.83 (1H, qd, J = 6.3, 5.2 Hz, H-3),
3.47 (1H, ddd, J = 9.7, 5.3, 2.1 Hz, H-50), 3.35 (2H, m, H-30 and 40),
3.17 (1H, dd, J = 9.3, 7.7 Hz, H-20), 3.13 (1H, qd, J = 7.3, 5.2 Hz, H-
2), 1.73 (1H, dq, J = 13.7, 7.4 Hz, H-500a), 1.54 (1H, dq, J = 13.7,
7.4 Hz, H-500b), 1.31 (3H, d, J = 6.3 Hz, H3-4), 1.25 (3H, d,
J = 7.3 Hz, H3-5), 1.17 (3H, d, J = 6.4 Hz, H3-400), 0.88 (3H, dd,
J = 7.4, 7.4 Hz, H3-600); 13C NMR (CD3OD, 100 MHz): Table 1; HR-
ESI-MS (positive-ion mode) m/z: 414.1719 [M+Na]+ (calcd for C17-
H29NO9Na, 414.1734).
ion HP-20 CC (
U = 50 mm, L = 40 cm), using H2O–MeOH (4:1, 2 L),
(3:2, 2 L), (2:3, 2 L), and (1:4, 2 L), and MeOH (2 L), 500 mL-frac-
tions being collected. The residue (7.53 g out of 8.73 g) in fractions
7–10 was subjected to silica gel (250 g) CC with increasing
amounts of MeOH in CHCl3 [CHCl3 (3 L), and CHCl3–MeOH (49:1,
1.5 L), (24:1, 1.5 L), (23:2, 1.5 L), (9:1, 1.5 L), (17:3, 1.5 L), (4:1,
1.5 L), (3:1, 1.5 L), and (7:3, 1.5 L)], CHCl3–MeOH–H2O (35:15:2,
1.5 L), and MeOH (1.5 L), 500 mL-fractions being collected. The res-
idue (962 mg) in fractions 23–29 was separated by ODS CC
4.3.3. Microtropin C (3)
25
Amorphous powder; [
a
]
D
ꢀ 4.3 (c 0.012, MeOH); IR mmax
(film): 3391, 2981, 2248, 1736, 1456, 1391, 1238, 1168, 1080,
(U
= 50 mm, L = 25 cm) with a linear gradient solvent system
1044 cmꢀ1 1H NMR (CD3OD, 400 MHz) d: 4.61 (1H, dd, J = 11.9,
;
[H2O–MeOH (1:9, 2 L) ? (9:1 2 L)], 10 g-fractions being collected.
The residue (147 mg) in fractions 88–98 was separated by DCCC
and the residue (36.8 mg) in fractions 73–84 was purified by HPLC
(H2O–MeOH, 7:3) to give three fractions, 6.9, 19.0 and 3.3 mg from
the peaks at 15, 17 and 19 min, respectively. The residue in the first
peak was further purified by HPLC (H2O-MeOH, 3:1) to give 2
(3.7 mg) from the peak at 24 min. The residue in the second peak
was recrystallized from 2-PrOH to give 1 (10.4 mg) as colorless
rods. The residue in the third peak was repeatedly purified by HPLC
2.2 Hz, H-60a), 4.40 (1H, d, J = 8.0 Hz, H-10), 4.22 (1H, dd, J = 11.9,
5.2 Hz, H-60b), 3.91 (1H, q, J = 6.4 Hz, H-300), 3.84 (1H, qd, J = 6.4,
5.5 Hz, H-3), 3.48 (1H, m, H-50), 3.35 (2H, m, H-30 and 40), 3.17
(1H, dd, J = 9.2, 8.0 Hz, H-20), 3.02 (1H, qd, J = 7.1, 5.5 Hz, H-2),
1.73 (1H, dq, J = 13.6, 7.4 Hz, H-500a), 1.54 (1H, dq, J = 13.6, 7.4 Hz,
H-500b), 1.34 (3H, d, J = 6.4 Hz, H3-4), 1.30 (3H, d, J = 7.1 Hz, H3-5),
1.17 (3H, d, J = 6.4 Hz, H3-400), 0.88 (3H, dd, J = 7.4, 7.4 Hz, H3-600);
13C NMR (CD3OD, 100 MHz): Table 1; HR-ESI-MS (positive-ion
mode) m/z: 414.1733 [M+Na]+ (calcd for C17H29NO9Na, 414.1734).

Y. Uemura et al. / Phytochemistry 87 (2013) 140–147
145
4.3.4. Microtropin D (4)
J = 9.2, 7.9 Hz, H-20), 1.75 (1H, m, H-500a), 1.71 (1H, m, H-4a), 1.51
(1H, m, H-6a), 1.50 (2H, m, H-4b and 500b), 1.48 (2H, m, H2-3),
1.45 (1H, m, H-6b), 1.22 (3H, d, J = 6.4 Hz, H3-1), 1.17 (3H, d,
J = 6.4 Hz, H3-400), 0.93 (3H, t, J = 6.4 Hz, H3-7), 0.88 (3H, t,
J = 7.4 Hz, H3-600); 13C NMR (CD3OD, 100 MHz); Table 1; HR-ESI-
MS (positive-ion mode) m/z: 447.2198 [M+Na]+ (calcd for C19H36-
O10Na, 447.2201).
24
Amorphous powder; [
a
]
ꢀ 7.0 (c 0.40, MeOH); IR
mmax (film):
D
3366, 2977, 2224, 1735, 1649, 1455, 1241, 1170, 1079, 1012 cmꢀ1
;
UV kmax (MeOH): 213 (3.75) nm (log e
); 1H NMR (CD3OD, 400 MHz)
d: 6.65 (1H, ddt, J = 6.7, 6.1, 1.4 Hz, H-3), 4.64 (1H, dd, J = 11.8,
2.2 Hz, H-60a), 4.53 (1H, ddt, J = 12.6, 6.1, 1.4 Hz, H-4a), 4.50 (1H,
ddt, J = 12.6, 6.7, 1.4 Hz, H-4b), 4.34 (1H, d, J = 7.9 Hz, H-10), 4.25
(1H, dd, J = 11.8, 5.3 Hz, H-60b), 4.16 (2H, ddd, J = 1.4, 1.4, 1.4 Hz,
H2-5), 3.94 (1H, q, J = 6.4 Hz, H-300), 3.50 (1H, m, H-50), 3.36 (2H,
m, H-30 and 40), 3.20 (1H, m, H-20), 1.75 (1H, dq, J = 13.9, 7.4 Hz,
H-500a), 1.54 (1H, dq, J = 13.9, 7.4 Hz, H-500b), 1.18 (3H, d,
J = 6.4 Hz, H3-400), 0.89 (3H, dd, J = 7.4, 7.4 Hz, H3-600); 13C NMR
(CD3OD, MeOH): Table 1; HR-ESI-MS (positive-ion mode) m/z:
428.1530 [M+Na]+ (calcd for C17H27NO10(Na, 428.1527).
4.3.9. Microtropin I (9)
26
Amorphous powder; [
a]
ꢀ 16.1 (c 0.15, MeOH); IR mmax
D
(film): 3365, 2967, 1735, 1456, 1377, 1241, 1169, 1077,
1042 cmꢀ1 1H NMR (CD3OD, 600 MHz) d: 4.61 (1H, dd, J = 11.7,
;
2.2 Hz, H-60a), 4.32 (1H, d, J = 7.8 Hz, H-10), 4.19 (1H, dd, J = 11.7,
5.3 Hz, H-60b), 3.92 (1H, q, J = 6.5 Hz, H-300), 3.74 (1H, m, H-2),
3.60 (1H, m, H-5), 3.44 (1H, ddd, J = 9.3, 5.3, 2.2 Hz, H-50), 3.36
(2H, m, H-30 and 40), 3.17 (1H, dd, J = 9.3, 7.8 Hz, H-20), 1.73 (1H,
dq, J = 13.8, 7.4 Hz, H-500a), 1.67 (1H, m, H-4a), 1.60 (2H, m, H2-6),
1.56 (1H, m, H-500b), 1.55 (1H, m, H-4b), 1.54 (2H, m, H2-3), 1.18
(3H, d, J = 6.5 Hz, H3–400), 1.15 (3H, d, J = 6.2 Hz, H3-1), 0.90 (3H, t,
J = 7.6 Hz, H3-7), 0.88 (3H, t, J = 7.4 Hz, H3-600); 13C NMR (CD3OD,
150 MHz); Table 1; HR-ESI-MS (positive-ion mode) m/z:
447.2200 [M+Na]+ (calcd for C19H36O10Na, 447.2201).
4.3.5. Microtropin E (5)
23
Amorphous powder; [
a
]
D
ꢀ 38.6 (c 0.11, MeOH); IR mmax
(film): 3398, 2975, 1733, 1446, 1377, 1241, 1171, 1083,
1038 cmꢀ1 1H NMR (CD3OD, 400 MHz) d: 4.59 (1H, dd, J = 11.7,
;
2.2 Hz, H-60a), 4.34 (1H, d, J = 7.8 Hz, H-100), 4.22 (1H, dd, J = 11.7,
5.5 Hz, H-60b), 3.97 (1H, septet, J = 6.2 Hz, H-2), 3.92 (1H, q,
J = 6.4 Hz, H-300), 3.47 (1H, ddd, J = 9.5, 5.5, 2.2 Hz, H-50), 3.35 (2H,
m, H-30 and 40), 3.14 (1H, dd, J = 9.2, 7.8 Hz, H-20), 1.73 (1H, dq,
J = 13.9, 7.4 Hz, H-500a), 1.54 (1H, dq, J = 13.9, 7.4 Hz, H-500b), 1.21
(3H, d, J = 6.2 Hz; H3-1), 1.18 (3H, d, J = 6.2 Hz, H3-3), 1.17 (3H, d,
J = 6.4 Hz, H3-400), 0.88 (3H, dd, J = 7.4, 7.4 Hz, H3-600); 13C NMR
(CD3OD, 100 MHz): Table 1; HR-ESI-MS (positive-ion mode) m/z:
375.1622 [M+Na]+ (calcd for C15H28O9Na, 375.1625).
4.4. Sugar analysis
Microtropins A–I (1–9) (ca. 500 lg each) were hydrolyzed with
1 M HCl (0.2 mL) at 90 °C for 2 h. Each reaction mixture was parti-
tioned with an equal amount of EtOAc (0.2 mL), and then the aque-
ous layers were individually analyzed with a chiral detector (JASCO
OR-2090plus) on an amino column [Asahipak NH2P-50 4E, CH3CN-
H2O (3:1), 1 mL/min]. All hydrolyzates gave a peak for D-glucose at
8.8 min with a positive optical rotation sign. The peaks were iden-
tified by co-chromatography with authentic samples.
4.3.6. Microtropin F (6)
Amorphous powder; [
a
]
23 + 3.2 (c 0.06, MeOH); IR
mmax (film):
D
3384, 2970, 2933, 1736, 1457, 1378, 1241, 1171, 1079, 1014 cmꢀ1
;
1H NMR (CD3OD, 400 MHz) d: 4.60 (1H, dd, J = 11.9, 2.2 Hz, H-60a),
4.28 (1H, d, J = 7.9 Hz, H-10), 4.24 (1H, dd, J = 11.9, 5.7 Hz, H-60b),
3.93 (1H, q, J = 6.5 Hz, H-300), 3.73 (1H, dd, J = 10.3, 5.9 Hz, H-1a),
3.65 (1H, m, H-2), 3.58 (1H, dd, J = 10.3, 3.6 Hz, H-1b), 3.49 (1H,
m H-50), 3.36 (2H, m, H-30 and 40), 3.21 (1H, dd, J = 9.3, 7.9 Hz, H-
20), 1.74 (1H, dq, J = 13.9, 7.4 Hz, H-500a), 1.55 (2H, m, H-3a and
500b), 1.44 (1H, dqd, J = 13.9, 7.5, 7.4 Hz, H-3b), 1.18 (3H, d,
J = 6.5 Hz, H3-400), 0.95 (3H, dd, J = 7.5, 7.5 Hz, H3-4), 0.88 (3H, dd,
J = 7.4, 7.4 Hz, H3-600); 13C NMR (CD3OD, 100 MHz): Table 1; HR-
ESI-MS (positive-ion mode) m/z: 405.1751 [M+Na]+ (calcd for C16-
H30O10Na, 405.1731).
4.5. Mild alkaline hydrolysis of 1
Microtropin A (1) (6.9 mg) in MeOH (450
l
L) was hydrolyzed
with 1 M CH3ONa (50
l
L) at 25 °C for 40 h. The reaction mixture
was neutralized with Amberlite IR-120B (H+) and filtered. The fil-
trate was evaporated, and the residue was purified by HPLC
(MeOH–H2O, 3:17) to give 1a (2.0 mg) from the peak at 8 min
and 1b (0.1 mg) from the peak at 28 min. 3-Hydroxy-2-methylbun-
tanenitrile b-D-glucopyranoside (1a) Amorphous powder,
[a]
D
ꢀ 26.9 (c 0.13, MeOH); IR mmax (film) cmꢀ1: 3396, 2924,
22
4.3.7. Microtropin G (7)
2886, 2245, 1455, 1383, 1353, 1164, 1076, 1045; 1H NMR (CD3OD,
400 MHz) d: 4.36 (1H, d, J = 7.9 Hz, H-10), 3.95 (1H, qd, J = 6.4,
4.2 Hz, H-3), 3.86 (1H, dd, J = 11.8, 1.9 Hz, H-60a), 3.67 (1H, dd,
J = 11.8, 5.4 Hz, H-60b), 3.36–3.27 (3H, m, H-30, 40 and 50), 3.20
(1H, dd, J = 9.0, 7.9 Hz, H-20), 2.96 (1H, qd, J = 7.1, 4.2 Hz, H-2),
1.37 (3H, d, J = 6.4 Hz, H3-4), 1.36 (3H, d, J = 7.1 Hz, H3-5); 13C
NMR (CD3OD, 100 MHz): Table 1; HR-ESI-MS (positive-ion mode)
m/z: 284.1102 [M+Na]+ (calcd for C11H19NO6Na: 284.1104). Methyl
22
Amorphous powder; [
a
]
D
ꢀ 0.87 (c 0.34, MeOH); IR mmax
(film): 3395, 2977, 2935, 1736, 1455, 1389, 1241, 1172, 1077,
1054 cmꢀ1 1H NMR (CD3OD, 400 MHz) d: 4.61 (1H, dd, J = 11.7,
;
2.2 Hz, H-60a), 4.41 (1H, d, J = 7.8 Hz, H-10), 4.20 (1H, dd, J = 11.7,
5.3 Hz, H-60b), 3.92 (1H, q, J = 6.2 Hz, H-300), 3.62 (1H, dq, J = 6.4,
6.4 Hz, H-3), 3.52 (1H, dq, J = 6.4, 6.3 Hz, H-2), 3.47 (1H, ddd,
J = 7.8, 5.3, 2.2 Hz, H-50), 3.36 (1H, dd, J = 9.0, 8.8 Hz, H-30), 3.34
(1H, dd, J = 8.8, 7.8 Hz, H-40), 3.20 (1H, dd, J = 9.0, 7.8 Hz, H-20),
1.73 (1H dq, J = 13.8, 7.4 Hz, H-500a), 1.54 (1H, dq, J = 13.8, 7.4 Hz,
H500b), 1.18 (3H, d, J = 6.3 Hz, H3-1), 1.17 (3H, q, J = 6.2 Hz, H3-400),
1.13 (3H, d, J = 6.4 Hz, H3-4), 0.88 (3H, dd, J = 7.4, 7.4 Hz, H3-600);
13C NMR (CD3OD, 100 MHz); Table 1; HR-ESI-MS (positive-ion
mode) m/z: 405.1738 [M+Na]+ (calcd for C16H30O10Na, 405.1731).
(200S,300R)-200-ethyl-200,300-dihydroxybutyrate (1b) Colorless liquid;
[a
]
D
ꢀ 3.2 (c 0.09, MeOH); IR mmax (film) cmꢀ1: 3411, 2978,
22
1735, 1511, 1457, 1381, 1172, 1078; 1H NMR (CD3OD, 400 MHz)
d: 3.88 (1H, q, J = 6.4 Hz, H-300), 3.75 (3H, s, –OCH3), 1.70 (1H, dq,
J = 13.8, 7.4 Hz, H-500a), 1.53 (1H, dd, J = 13.8, 7.4 Hz, H-500b), 1.16
(3H, d, J = 6.4 Hz, H3-400), 0.84 (3H, dd, J = 7.4, 7.4 Hz, H3-600); 13C
NMR (CD3OD, 100 MHz): Table 1; HR-ESI-MS (positive-ion mode)
m/z: 185.0786 (calcd for C7H14ONa: 185.0784).
4.3.8. Microtropin H (8)
25
Amorphous powder; [
a
]
D
ꢀ 17.8 (c 0.36, MeOH); IR mmax
(film): 3373, 2968, 1736, 1457, 1378, 1241, 1170, 1077,
4.6. X-ray crystallographic analysis of 1
1041 cmꢀ1 1H NMR (CD3OD, 400 MHz) d: 4.71 (1H, dd, J = 11.8,
;
2.2 Hz, H-60a), 4.34 (1H, d, J = 7.9 Hz, H-10), 4.21 (1H, dd, J = 11.8,
5.4 Hz, H-60b), 3.92 (1H, q, J = 6.4 Hz, H-300), 3.79 (1H, m, H-2),
3.47 (2H, m, H-5 and 50), 3.34 (2H, m, H-30 and 40), 3.15 (1H, dd,
A suitable crystal (0.28 ꢁ 0.05 ꢁ 0.01 mm) was used for analy-
sis. The data were measured using a Rigaku AFC-5 four circle dif-
fractometer, using MoKa graphite-monochromated radiation

146
Y. Uemura et al. / Phytochemistry 87 (2013) 140–147
(k = 0.71073 Å). The structure was solved by a direct method using
the program SHELXTL-97 (Sheldrick, 2008). The refinement and all
further calculations were carried out using SHELXTL-97 (Sheldrick,
2008). The absorption correction was carried out utilizing the SAD-
ABS routine (Sheldrick, 1996). The H atoms were included at calcu-
lated positions and treated as riding atoms using the SHELXTL
default parameters. The non-H atoms were refined anisotropically,
using weighted full-matrix least-squares on F2. Fig. 4 was drawn
2.1 Hz, H-60), 3.85 (1H, m, H-2), 3.66 (1H, dd, J = 11.9, 5.3 Hz,
H-60), 3.47 (1H, m, H-5), 3.25–3.37 (3H, m, H-30, 40 and 50), 3.15
(1H, dd, J = 9.2, 7.8 Hz, H-20), 1.74 (1H, m, H-3), 1.38–1.63 (5H, m,
H-3, H2-4 and H2-6), 1.24 (3H, d, J = 6.4 Hz, H3-1), 0.94 (3H, t,
J = 7.5 Hz, H3-7); 13C NMR (CD3OD, 100 MHz): Table 1; HR-ESI-
MS (positive-ion mode) m/z: 317.1575 [M+Na]+ (calcd for C13H26-
O7Na: 317.1571). Methyl (200S,300R)-200-ethyl-200,300-dihydroxybuty-
rate (8b = 1b): Colorless liquid; HR-ESI-MS (positive-ion mode)
m/z: 185.0788 (calcd for C7H14ONa: 185.0784).
with ORTEP32 (Farrugia, 1997). Crystal data:
C17H29NO9,
M = 391.41 g molꢀ1, monoclinic, P21, a = 12.631(5) Å, b = 6.611(4) Å,
c = 13.0534(5) Å, b = 114.160(3)°, V = 994.5(6) Å3, T = 173 K, Z = 2,
Dc = 1.307 Mg/m3,
l(MoKa , F(000) = 420, 2064
) = 0.881 mmꢀ1
4.10. Enzymatic hydrolysis of 8a
reflections were measured in the range of 2h 6 146.54°, 1977 being
unique (Rint = 0.0588), and used in all calculations. The final good-
ness-of-fit on F2 was 0.982, and the final R indices were R1 = 0.0427
Compound 8a (1.8 mg) in H2O (1 mL) was hydrolyzed with
b-glucosidase (5 mg) at 37 °C for 2 h. The reaction mixture was
evaporated, and the residue was purified by preparative TLC
[8 cm width and developed for 9 cm with CHCl3-MeOH, 9:1 and
for visualization one side (about 5 mm) of the TLC was cut off] to
give 8c (0.6 mg) and 8d from the spots at Rf = 0.35 and Rf = 0.01,
respectively. Compound 8d was repeatedly purified by preparative
TLC [8 cm width and developed for 9 cm with CHCl3–MeOH–H2O,
15:6:1 and for visualization, one side (about 5 mm) of the TLC
was cut off] to give pure 8d (0.3 mg) from the spot at Rf = 0.11.
2,5-Dihydroxyheptane (8c): 1H NMR (CD3OD, 400 MHz) d: 3.72
(1H, m, H-2), 3.45 (1H, m, H-5), 1.39–1.61 (6H, m, H2-3, H2-4,
H2-6), 1.16 (3H, d, J = 6.4 Hz, H3-1), 0.94 (3H, t, J = 7.4 Hz, H3-7);
13C NMR (CD3OD, 100 MHz): Table 1; HR-ESI-MS (positive-ion
mode) m/z: 155.1041 [M+Na]+ (calcd for C7H16O2Na: 155.1043).
and wR2 = 0.1077 based on I > 2r(I), and R1 = 0.1006 and
wR1 = 0.1302 with all data. The largest differences of the peak
and the hole were 0.241 and ꢀ0.269 eÅꢀ3, respectively. The CCDC
deposit contains supplementary crystallographic data (CCDC No.
892883). These data can be obtained free of charge via http://
www.ccdc.cam.ac.uk/conts/retrieving.html, or from the Cambridge
Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ,
UK; fax: (+44) 1223 336 033; or e-mail: deposit@ccdc.cam.ac.uk.
4.7. Preparation of 3-chloro-2-hydroxybutane (10)
3-Chloro-2-butanone (4.03 g) in MeOH (40 mL) was reduced
with NaBH4 (1.44 g) at 25 °C for 18 h. The reaction mixture was
evaporated and the resultant residue was extracted with CH2Cl2
(50 mL). The organic layer was washed with H2O (30 mL ꢁ 6) and
dried over Na2SO4. Removal of the solvent gave a liquid material
(10). 3-Chloro-2-hydroxybutane (10): volatile oil, anti-isomer
(10a), 1H NMR (CD3OD, 400 MHz) d: 3.95 (1H, qd, J = 6.8, 4.2 Hz,
H-3), 3.79 (1H, qd, J = 6.3, 4.2 Hz, H-2), 1.46 (3H, d, J = 6.8 Hz, H3-
4), 1.21 (3H, d, J = 6.3 Hz, H3-1); 13C NMR (CD3OD, 100 MHz) d:
71.9 (C-2), 63.6 (C-3), 20.9 (C-1), 19.3 (C-4). Syn isomer (10b), 1H
NMR (CD3OD, 400 MHz) d: 3.94 (1H, qd, J = 6.8, 5.1 Hz, H-3), 3.76
(1H, qd, J = 6.3, 5.1 Hz, H-2), 1.45 (3H, d, J = 6.8 Hz, H3-4), 1.22
(3H, d, J = 6.3 Hz, H3-1); 13C NMR (CD3OD, 100 MHz) d: 72.4
(C-2), 63.7 (C-3), 20.8 (C-1), 19.1 (C-4).
D-Glucose (8d): D-Glucose was confirmed by HPLC analysis with
a chiral detector as described in Section 4.4.
Acknowledgements
The authors are grateful for access to the superconducting NMR
instrument (JEOL JNM a-400) at the Analytical Center of Molecular
Medicine of the Hiroshima University Faculty of Medicine and an
Applied Biosystem QSTAR XL system ESI (Nano Spray)-MS at the
Analysis Center of Life Science of the Graduate School of Biomedi-
cal Sciences, Hiroshima University. This work was supported in
part by Grants-in-Aid from the Ministry of Education, Culture,
Sports, Science and Technology of Japan (Nos. 22590006 and
23590130), the Japan Society for the Promotion of Science, and
the Ministry of Health, Labour and Welfare. Thanks are also due
to the Research Foundation for Pharmaceutical Sciences and the
Takeda Science Foundation for the financial support.
4.8. Preparation of (2R⁄,3R⁄)-3-hydroxy-2-methylbutanenitrile (11a)
and (2R⁄,3S⁄)-3-hydroxy-2-methylbutanenitrile (11b)
A solution of 3-chloro-2-hydroxybutane (10) (2.07 g) in H2O
(40 mL) was heated until reflux began with NaCN (1.00 g) and
tetraethylammonium bromide (1.00 g) for 2 h. After cooling, the
aqueous layer was extracted with CH2Cl2 (30 mL ꢁ 5) and the
organic layer was washed with H2O (100 mL ꢁ 4). The organic
layer was dried over Na2SO4 and evaporated to leave a liquid mate-
rial (1.41 g) (11). 3-Hydroxy-2-methylbutanenitrile (11): volatile
oil, anti-isomer (2R⁄,3R⁄) (11a), NMR(CD3OD): Table 2a. Syn isomer
(2R⁄,3S⁄) (11b), NMR (CD3OD): Table 2a.
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