Revised chirality of the acyl group of 8′-O-(3-hydroxy-3- methylglutaryl)-8′-hydroxyabscisic acid

Article abstract of DOI:10.1016/j.phytochem.2004.07.012

The chirality of the acyl group 8′-O-(3-Hydroxy-3-methylglutaryl)- 8′-hydroxyabscisic acid was revised to S based on an HPLC analysis of the diastereomer derived from mevalonolactone obtained by reduction of the conjugate with lithium borohydride. 8′-O-(3

Full text of DOI:10.1016/j.phytochem.2004.07.012

PHYTOCHEMISTRY
Phytochemistry 65 (2004) 2517–2520
www.elsevier.com/locate/phytochem
Revised chirality of the acyl group of
80-O-(3-hydroxy-3-methylglutaryl)-80-hydroxyabscisic acid
a,
b
c
c
Tsunashi Kamo *, Nobuhiro Hirai , Chiaki Matsumoto , Hajime Ohigashi ,
a
Mitsuru Hirota
a
Department of Bioscience and Biotechnology, Faculty of Agriculture, Shinshu University, 8304 Minami-minowa, Kami-ina, Nagano 399-4598, Japan
b
International Innovation Center, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
c
Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
Received 26 January 2004; received in revised form 7 May 2004
Abstract
80-O-(3-Hydroxy-3-methylglutaryl)-80-hydroxyabscisic acid is a stable conjugate of the first metabolite of abscisic acid, 80-
hydroxyabscisic acid, that is spontaneously isomerized to phaseic acid. The chirality of the 3-hydroxy-3-methylglutaryl group of
the conjugate was revised to S based on an HPLC analysis of the diastereomer derived from mevalonolactone obtained by reduction
of the conjugate with lithium borohydride.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Keywords: Robinia pseudo-acacia; Leguminosae; 3-hydroxy-3-methylglutaryl (HMG); Abscisic acid; Chirality; Lithium borohydride; Diborane;
Mevalonolactone
1. Introduction
1978). One of the authors previously reported the chiral-
ity of 3-hydroxy-3-methylglutaryl (HMG) group of 4 to
be R (Hirai and Koshimizu, 1981). This identification
was based on the chirality of mevalonolactone obtained
by the reduction of 4 with diborane that is regarded to
reduce the carboxyl group and not the ester group
(Yoon et al., 1973). However, Tanaka et al. (1992)
reported that the HMG group of gympolilins, bitter
terpenoids from Gymnopilus spectabilis, had the S-con-
figuration. They used lithium borohydride, which re-
duces the ester group and not the carboxyl group, to
obtain mevalonolactone from the gympolilins. Natu-
rally occurring HMG esters would be formed via the
acylation of alcohols by (S)-HMG-CoA, meaning that
they should possess the (S)-HMG group (Bergot et al.,
1979). Thus, we reinvestigated the chirality of the
The plant hormone, abscisic acid (ABA, 1) is metab-
olized to 80-hydroxy-ABA (2) by ABA 80-hydroxylase in
plants (Fig. 1) (Crozier et al., 2000; Kushiro et al., 2004).
80-Hydroxy-ABA (2) spontaneously isomerizes to pha-
seic acid (3), although the isomerization in vivo seems
to be enzyme catalyzed (Milborrow et al., 1988; Tod-
oroki et al., 1995). The instability of 80-hydroxy-ABA
(2) limits confirmation of its occurrence in plants, but
isolation of a stable conjugate of 80-hydroxy-ABA, 80-
O-(3-hydroxy-3-methylglutaryl)-80-hydroxy-ABA (4),
from Robinia pseudo-acacia has established 80-hydroxy-
ABA (2) as a metabolite of ABA (1) (Hirai et al.,
*
Corresponding author. Tel.: +81 265 77 1602; fax: +81 265 77
HMG group of
borohydride.
4 by reduction using lithium
1629.
E-mail address: kamo274@gipmc.shinshu-u.ac.jp (T. Kamo).
0031-9422/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2004.07.012

2518
T. Kamo et al. / Phytochemistry 65 (2004) 2517–2520
9'
8'
HO
[O]
5
3
4
6'
3'
5'
4'
1'
2'
2
OH
OH
OH
CO2H
CO2H
O
CO2H
1
O
O
O
7'
2
1
3
CO2H
O
HO
S
O
O
OH
CO2H
4
Fig. 1. Early metabolic pathway of ABA (1) in plants
2. Results and discussion
the absolute configuration of the HMG group of 4 was
assigned as R (Hirai and Koshimizu, 1981). This incor-
rect assignment was derived from the preconception for
the reducing characteristic of diborane. Other research-
ers also determined the absolute configuration of the
HMG groups in natural products using diborane.
Macrophorin D (Sassa and Nukita, 1984) and viscum-
neoside IV (Kong et al., 1990) were suggested to pos-
sess the (R)-HMG group, the former of which has
been revised as S by the reduction using lithium trieth-
ylborohydride (Fujimoto et al., 2001), while the HMG
groups of tubeimoside I (Kasai et al., 1986) and tangs-
henosides I and IV (Mizutani et al., 1988; Yuda et al.,
1990) were revealed to have the S-configuration.
Although the reason remains to be elucidated, the re-
sults of these experiments using diborane are not con-
sistent. This situation would be attributed to revealing
the nature of the reducing characteristic of diborane,
not to the co-occurrence of the (R)- and (S)-HMG
groups in natural products. We should keep in mind
that diborane sometimes seems to reduce the ester
group rather than the carboxyl group. On the other
hand, reduction using lithium borohydride always give
an identical and reasonable result, the S-configuration,
for the HMG groups (Tanaka et al., 1992, 1993; Mim-
aki et al., 1993; Kamo et al., 2003). All of these results
strongly recommend the use of lithium borohydride or
lithium triethylborohydride to confirm the absolute
configuration of the HMG groups.
Compound 4 was reduced by lithium borohydride to
give mevalonolactone (5) from the HMG group, and the
reduced derivatives from 80-hydroxy-ABA (2) (Scheme
1). The 1H NMR spectrum suggested that the latter con-
sisted of several components structurally related to 80-
hydroxy-ABA (2) but their structures could not be
determined due to the small amounts of the individual
compounds. Compound 5 was converted to a diastereo-
mer, 5-O-acetyl-1-[(S)-phenylethyl]mevalonamide (6),
by treatment with (S)-(ꢀ)-1-phenylethylamine followed
by acetylation. The chirality at C-3 of 6 was deter-
mined by comparison of its retention time during HPLC
with those of authentic diastereomers, (3R)-5-O-acetyl-
1-[(S)-phenylethyl]mevalonamide and its (3S)-diastereo-
mer (Hirai and Koshimizu, 1981). The HPLC analysis
showed that 6 was identical to the (3R)-diastereomer,
indicating that the chirality of 5 is R. Thus, the absolute
configuration of 4 should be revised to 80-O-[(S)-3-hyd-
roxy-3-methylglutaryl]-80-hydroxy-ABA. This configu-
ration corresponds well to the presumed in vivo
formation of 4 via the acylation of 80-hydroxy-ABA
(2) by (S)-HMG-CoA.
Compound 6, previously obtained by reduction of 4
with diborane, also had the 3R-configuration, by which
OH
OH
3
c) d)
CH3
S
4
5
2
1
R
CONH
O
O
AcO
H
C6H5
3. Experimental
a) b)
5
6
4
3.1. General
The 1H NMR spectra were recorded by a Bruker
DRX-500 FT-NMR spectrometer operating at
500.1 MHz for protons with TMS as the internal
standard.
a mixture of the reduced derivatives of 2
Scheme 1. (a) LiBH4/THF, (b) H+ (10%), (c) (S)-1-(ꢀ)-phenylethyl-
amine, (d) Ac2O/pyridine (ca. 100%).

T. Kamo et al. / Phytochemistry 65 (2004) 2517–2520
2519
3.2. Reduction of 80-O-(3-hydroxy-3-methylglutaryl)-80-
hydroxy-ABA (4) and derivatization of mevalonolactone
5.13 (1H, quintet, J = 6.9 Hz, H-10), 4.22 (2H, m, H-
5), 2.40, 2.28 (each 1H, d, J = 14.8 Hz, H-2), 2.03 (3H,
s, 5-OCOCH3), 1.85 (2H, m, H-4), 1.49 (3H, d, J = 6.9
Hz, H-20), 1.23 (3H, s, 3-CH3). Hydrolysis of this dia-
Compound 4 (30 mg) was isolated from fresh imma-
ture fruits (6.4 kg) of Robinia pseudo-acacia L. (Hirai et
al., 1978). Compound 4 (15 mg) in lithium borohydride
solution (0.5 mL, 2.0 M in THF; Aldrich) was stirred at
room temperature for 12 h. The reaction was quenched
with H2O and then 1 N HCl. The solution was extracted
three times with EtOAc at pH 3, and the organic layers
were combined and concentrated to give a mixture of
the reduced derivatives of 80-hydroxy-ABA (10 mg).
The aqueous layer was acidified to pH 1 with 1 N HCl,
stirred for 3 days at room temperature, and then ex-
tracted three times with EtOAc. The organic layers were
combined, and purified by HPLC with an ODS column
(YMC RS-323, 250 · 10 mm) eluting with MeOH–H2O
(1:1) at a flow rate of 3.0 mL/min with detection at 200
stereomer (16.5 mg) gave (R)-(ꢀ)-mevalonolactone (1.8
21
mg), ½aꢁ ꢀ14ꢁ (c 0.18, EtOH). These data were identical
D
with those already reported (Hirai and Koshimizu, 1981;
Fujimoto et al., 2001).
3.3.2. (3S)-5-O-acetyl-1-[(S)-phenylethyl]mevalonamide
25
½aꢁ ꢀ53ꢁ (c 2.39, EtOH). 1H NMR (CDCl3): d
D
7.25–7.36 (5H, m, 10-C6H5), 6.34 (1H, d, J = 7.4 Hz,
NH), 5.13 (1H, quintet, J = 6.9 Hz, H-10), 4.20 (2H,
m, H-5), 2.40, 2.28 (each 1H, d, J = 14.8 Hz, H-2),
2.00 (3H, s, 5-OCOCH3), 1.82 (2H, m, H-4), 1.49
(3H, d, J = 6.9 Hz, H-20), 1.24 (3H, s, 3-CH3). Hydrol-
ysis of this diastereomer (17.4 mg) gave (S)-(+)-meval-
21
D
onolactone (1.8 mg), ½aꢁ +13ꢁ (c 0.18 EtOH). These
1
nm to give 5 (0.5 mg). The H NMR spectrum of 5 was
data were identical to those already reported (Hirai
and Koshimizu, 1981).
identical to that of authentic mevalonolactone. (S)-1-
(ꢀ)-Phenylethylamine (2 lL) was added to 5 in CHCl3,
then stored overnight at room temperature after remov-
ing the solvent in vacuo. The resulting material was acet-
ylated with Ac2O in pyridine, and purified by HPLC
using an ODS column (YMC RS-323) eluting with
MeOH–H2O (4:1) at a flow rate of 3.0 mL/min with
detection at 254 nm to give 6 (1.0 mg). Its 1H NMR spec-
trum was consistent with that of the authentic (3R)-5-O-
acetyl-1-[(S)-phenylethyl] mevalonamide.
3.4. HPLC analysis of 6 and the authentic diastereomers
Compound 6 was subjected to HPLC with a silica
gel column (YMC A-004, 300 · 4.6 mm) eluting with
n-hexane–CH2Cl2-i-PrOH (20:20:1) at a flow rate of
2.0 mL/min with detection at 254 nm. The tRs of 6
and authentic (3R)-5-O-acetyl-1-[(S)-phenylethyl]meva-
lonamide, and authentic (3S)-5-O-acetyl-1-[(S)-phenyl-
ethyl]mevalonamide were 14.0 and 14.9 min,
respectively.
3.3. Preparation and identification of authentic (3R)-5-
O-acetyl-1-[(S)-phenylethyl]mevalonamide and (3S)-5-
O-acetyl-1-[(S)-phenylethyl]mevalonamide
Acknowledgements
These diastereomers were synthesized by the reported
procedure except for reacting without solvent (Hirai and
Koshimizu, 1981). The diastereomers (60 mg) were sub-
jected to preparative HPLC using a silica gel column
(YMC SH-043-5, 250 · 20 mm) eluting with CH2Cl2-i-
PrOH (20:1) at a flow rate of 8.0 mL/min with detection
at 254 nm. Materials eluted at tRs 18.3 and 19.3 min
were separately collected, and concentrated to give
(3R)-5-O-acetyl-1-[(S)-phenylethyl]mevalonamide (20.9
mg) and (3S)-5-O-acetyl-1-[(S)-phenylethyl]mevalona-
mide (23.9 mg), respectively. These diastereomers were
We thank Ms. M. Miyazawa of Shinshu University
for providing the NMR spectra.
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