Absolute configurations of isoflavan-4-ol stereoisomers

Article abstract of DOI:10.1016/j.bmcl.2008.01.116

Isoflavan-4-ol has been synthesized quantitatively from the reduction of isoflavone in the presence of Pd/C and ammonium formate under N₂ atmosphere. Isolation of cis- and trans-isomers was achieved by flash column chromatography and each enant

Full text of DOI:10.1016/j.bmcl.2008.01.116

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Bioorganic & Medicinal Chemistry Letters 18 (2008) 1952–1957
Absolute configurations of isoflavan-4-ol stereoisomers
Dongho Won,a Bok-Kyu Shin,a Suil Kang,b Hor-Gil Hur,b
Mihyang Kimc and Jaehong Hana,*
aMetalloenzyme Research Group and Department of Biotechnology, Chung-Ang University, Gyeonggi, Anseong 456-756,
Republic of Korea
bDepartment of Environmental Science and Engineering, International Environmental Research Center,
Gwangju Institute of Science and Technology, Gwangju 500-712, Republic of Korea
cSchool of Agricultural Biotechnology, Seoul National University, Seoul 151-742, Republic of Korea
Received 30 December 2007; revised 24 January 2008; accepted 30 January 2008
Available online 2 February 2008
Abstract—Isoflavan-4-ol has been synthesized quantitatively from the reduction of isoflavone in the presence of Pd/C and ammo-
nium formate under N2 atmosphere. Isolation of cis- and trans-isomers was achieved by flash column chromatography and each
enantiomer was separated by Sumi-Chiral column chromatography. Absolute configurations of four stereoisomers were determined
by circular dichroism spectroscopy.
Ó 2008 Elsevier Ltd. All rights reserved.
Isoflavonoids form a distinctive subclass of the flavo-
noids1 and are mainly found in Leguminosae species.
They are responsible for the protection against micro-
bial infections in plants2 and for the formation of nod-
ules on Leguminosae species by nitrogen fixing
bacteria.3 Isoflavonoids also exhibit estrogenic, antian-
giogenic, antioxidant, and anticancer activity to human,
probably due to the structural similarity of the metabo-
lites to human estrogens.4–6 These metabolites are found
in blood plasma, urine, and feces as a result of biotrans-
formation by intestinal microorganisms.7–9 For exam-
ple, equol, produced from the sequential reduction
reactions of a natural isoflavonoid daidzein (40,7-dihydr-
oxyisoflavanone), was reported as the most effective
compound for preventing breast cancer10 and stimulat-
ing estrogenic response11 among the isoflavonoid metab-
olites. Interestingly, biological equol production was
found stereoselective with formation of S-equol (3S-
40,7-dihydroxylisoflavan).12 The metabolic pathway for
S-equol production from daidzein has been suggested
based on the isolated biotransformation products
(Fig. 1).13–17 While tetrahydrodaidzein formation from
daidzein seems to be consistent, the next biotransforma-
tion of tetrahydrodaidzein to S-equol is still debatable.
Direct two-electron reduction pathway and dehydra-
tion-reduction pathway of tetrahydrodaidzein seem to
be equally plausible. Besides, the stereochemistry of
dihydrodaidzein and tetrahydrodaidzein has never been
elucidated to the best of our knowledge. For example, it
is not clear yet which enantiomer of dihydrodaidzein or
which stereoisomer of tetrahydrodaidzein is responsible
for S-equol production. Here we report simple prepara-
tion of isoflavan-4-ols from isoflavone and elucidation
of absolute configurations of isoflavan-4-ol stereoiso-
mers, which can provide useful information on the
mechanistic study of isoflavonoid biotransformation.
Due to the structural similarity between isoflavan-4-ol
and tetrahydrodaidzein, elucidation of stereochemistry
of biotransformed tetrahydrodaidzein and biotransfor-
mation of isoflavan-4-ol stereoisomers as tetrahydrod-
aidzein analogs would be possible.
We have previously reported convenient synthetic meth-
od of 20-hydroxydihydrochalcone from flavone, and
isoflavan-4-one from isoflavone by catalytic hydrogen
transfer reaction in the presence of Pd/C.18,19 When we
modified reaction conditions, isoflavan-4-ols were
obtained from isoflavone in quantitative yield. The
reduction of isoflavone was carried out in the presence
of ammonium formate and Pd/C (Aldrich) in an inert
atmosphere glove box. Isoflavone (200 mg, 0.90 mmol),
Pd/C (200 mg), and NH4HCO3 (400 mg, 6.35 mmol)
Keywords: Absolute configuration; Circular dichroism (CD); Flavo-
noids; Isoflavanol; Palladium on activated carbon (Pd/C).
*
Corresponding author. Tel./fax: +82 31 670 4830; e-mail:
jaehongh@cau.ac.kr
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.01.116

D. Won et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1952–1957
1953
HO
O
HO
O
O
O
OH
OH
Dihydrodaidzein (4',7-dihydroxyisoflavanone)
Daidzein (4',7-dihydroxyisoflavone)
8
HO
O
HO
O
2
7
2'
3
5
4
4'
OH
5'
S-Equol (3S-4',7-dihydroxyisoflavan)
OH
OH
Tetrahydrodaidzein (4',7-dihydroxyisoflavanol)
Figure 1. Suggested metabolic pathway for biological S-equol production.
were dissolved in anhydrous EtOH (25 ml). The reaction
mixture was stirred at room temperature and the reac-
tion was monitored by silica gel TLC. Isoflavone
(Rf = 0.53) almost disappeared in an hour and isofla-
van-4-one (Rf = 0.65) was observed as a major product
on TLC (100% CHCl3). In 6 h, cis-isoflavan-4-ol
(Rf = 0.37) and trans-isoflavan-4-ol (Rf = 0.29) were
observed on TLC, and the reaction was completed in
12 h. The reaction was stopped by filtering Pd/C, and
the product was isolated by evaporating reaction
solvent. The product was only isoflavan-4-ol stereoiso-
mers and no other byproduct was observed. Although
many hydrogenation reactions of isoflavanone were
reported,20,21 this is the first report that produced exclu-
sively isoflavan-4-ol products, to our knowledge.
of trans-isoflavan-4-ol have suggested equilibrium of
diaxial and diequatorial conformers in MeOH solution,
preferring thrice more diequatorial conformers.22
Accordingly, it was found that the diequatorial confor-
mation mainly exists from our experiment. The observed
JH3,4 = 8 Hz has confirmed diequatorial and diaxial con-
formers of trans-isoflavan-4-ol co-exist22 (Fig. 3). Be-
cause the detection time scale of NMR is relatively
long, the observed JH3,4 coupling constant reflects the
statistic mean value of the coupling constants of both
conformation (JH3,4 of diequatorial conformer = 9.95
and JH3,4 of diaxial conformer = 1.66).
The 1H NMR spectrum of cis-isoflavan-4-ol is first
reported in this work. It was characterized by broad sin-
glet of H-4 at 4.852 ppm, and H-3, H-2a, and H-2b
were observed at 3.374 ppm (dt), 4.377 ppm (ddd)
and 4.652 ppm (dd), respectively. The conformation of
cis-isoflavan-4-ol can be determined based on 1H
UV–vis spectra of the isoflavan-4-ol stereoisomers in
MeOH were taken on Scinco S-3100 spectrophotome-
ter, and showed kmax at 283 nm (e = 1820 Mꢀ1 cmꢀ1),
275 nm (e = 2100 Mꢀ1 cmꢀ1
)
and 220 nm (e =
NMR measurement as above. The observed JH3,4 =
3950 Mꢀ1 cmꢀ1) (Fig. 2). H NMR spectra of cis- and
trans-isoflavan-4-ol in CDCl3 were obtained on a
400 MHz NMR spectrometer. In case of trans-isofla-
van-4-ol, half-chair conformation of C-ring can form
two conformers of diaxial and diequatorial in solution,
due to the Ph-3 and OH-4 position (Fig. 3). Extensive
1H NMR data assignment and conformation analysis
3.4 Hz corresponds to H–C3–C4–H dihedral angle of
ca. 50°.23 Because both conformers have similar dihedral
angles close to 50° based on molecular mechanics calcu-
lations, actual conformation of cis-isoflavan-4-ol could
not be elucidated based on the NMR data (Fig. 3).
But diequatorial conformation is favored because of
steric hindrance between phenyl and hydroxyl groups
of diaxial conformers.24
1
1H NMR of trans-isoflavan-4-ol (400 MHz, CDCl3, ppm): 5.017
(dd, JH-2a = 0.3 Hz (unr), JH-2b = 5.2 Hz, JH-3 = 8 Hz, H-4), 4.400
(dd, JH-2a = 11.2 Hz, JH-3 = 3.6 Hz, JH-4 = 5.2 Hz (unr), H-2b), 4.292
(dd, JH-2b = 11.2 Hz, JH-3 = 8.4 Hz, JH-4 = 0.3 Hz (unr), H-2a), 3.211
(dt, JH-2a = 8.4 Hz, JH-2b = 3.6 Hz, JH-4 = 8 Hz, H-3), 1H NMR of
cis-isoflavan-4-ol (400 MHz, CDCl3, ppm): 4.852 (br s, JH-2a = 12 Hz
(unr), JH-2b = 0.2 Hz (unr), JH-3 = 3.4 Hz (unr), H-4), 4.652 (dd,
Because hydrogenation of C-ring would generate four
stereoisomers with C-3 and C-4 chiral centers, the dia-
stereomeric mixture of isoflavan-4-ol was first separated
by flash column chromatography and each enantiomer
was purified further by a preparative Sumi-Chiral col-
umn. The enantiomers of trans-isoflavan-4-ol were sepa-
rated isocratically with 35:65 ratio of MeCN:potassium
phosphate (20 mM, pH 3.0) eluent system and the enan-
tiomers of cis-isoflavan-4-ol were separated isocratically
JH-2a = 12 Hz, JH-3 = 11.8 Hz, JH-4 = 0.2 Hz (unr), H-2b), 4.377 (ddd,
JH-2b = 12 Hz, JH-3 = 3.6 Hz, JH-4 = 1.2 Hz, H-2a), 3.374 (dt,
JH-2a = 3.6 Hz, JH-2b = 11.8 Hz, JH-4 = 3.4 Hz, H-3).

1954
D. Won et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1952–1957
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
200
220
240
260
280
300
λ (nm)
Figure 2. UV spectrum of isoflavan-4-ol.
O
2
3
4
OH
H-2β
Ph
H-2α
H
H-4
H
O
O
HO
Ph
HO
H
H-3
H
Diequatorial
Diaxial
H-4
Ph
H-4
Ph
C2
Φ
Φ
H-3
C2
HO
H-3
HO
Figure 3. Half-chair conformations of trans-isoflavan-4-ol.
with 30:70 ratio of eluent system (Fig. 4). Each fraction
was extracted with ethyl acetate and the solvent was
removed under reduced pressure. CD spectra of four
stereoisomers in MeOH were obtained on J-715 CD
spectrometer and are shown at Figure 5. The trans-isofl-
avan-4-ol enantiomer isolated from the peak 1 showed
negative Cotton effect between 250 and 300 nm, which
corresponds to 1Lb band p ! p* transition, and positive
Cotton effect between 220 and 240 nm, which corre-
CD spectrum symmetric to peak 1. The conformation
of isoflavan-4-ol can be visualized as half-chair confor-
mation, due to the rigid chroman-4-ol structure
(Fig. 6), and the peak 1 showing negative Cotton effect
at the range between 250 and 300 nm was assigned as
3R, 4S-stereoisomer, and the peak 2 showing positive
Cotton effect at the same region as 3S, 4R-stereoisomer,
according to Snatzke’s modified octant rule.26–28 Like-
wise, the cis-isoflavan-4-ol enantiomer isolated from
peak 1, showing positive Cotton effect between 245
and 300 nm, can be assigned as 3S, 4S-isoflavan-4-ol,
1
sponds to La.25 On the contrary, the other trans-isofla-
van-4-ol enantiomer isolated from the peak 2 showed

D. Won et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1952–1957
1955
Figure 4. HPLC elution profiles for chiral separation of cis-isoflavan-4-ol and trans-isoflavan-4-ol.
and the other enantiomer from peak 2 with negative
Cotton effect at the same region can be assigned as
3R, 4R-isoflavan-4-ol (Fig. 6). Interestingly, all 3S-enan-
tiomers of (3S, 4R)-trans- and (3S, 4S)-cis-isoflavanol,
as well as 3S-equol,17 and 3S-isoflavanone,19 showed
short retention time on the Sumi-Chiral column, com-
pared to the other relevant enantiomers.
was twice smaller than that of cis-isoflavanol
stereoisomers.
In summary, we have successfully prepared isoflavan-4-ol
from catalytic hydrogenation of isoflavone without
other byproducts. The four isoflavan-4-ol stereoisomers
were efficiently isolated by Sumi-Chiral column chroma-
tography and their absolute configurations were
assigned by CD spectroscopy. This work could provide
valuable information to the mechanistic study of the
isoflavonoid biotransformation.
We have observed co-existence of diequatorial and
diaxial conformers of trans-isoflavan-4-ol from the
NMR data. In case of (3R, 4S)-trans-isoflavan-4-ol,
diequatorial conformer with P-helicity and diaxial
conformer with M-helicity exist in about 3:1 ratio.
Accordingly, the difference between absorbance of
left circularly polarized and right circularly polarized
light would be reduced, due to the circularly polar-
ized lights mixing. In fact, it was observed that the
molar ellipticity of trans-isoflavanol stereoisomers
Acknowledgments
This work was supported by the Korea Science and
Engineering Foundation (KOSEF) grant funded by

1956
D. Won et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1952–1957
O
3S
4R
OH
O
3R
4S
OH
220
230
240
250
260
270
280
290
300
λ(nm)
O
3S
4S
OH
O
3R
4R
OH
220
230
240
250
260
270
280
290
300
λ(nm)
Figure 5. CD spectra of isoflavan-4-ol stereoisomers.
O
O
O
O
3S
3R
3R
3S
4R
4S
4R
4S
OH
OH
OH
OH
H
H
H
H
H
OH
Ph
H
H
O
Ph
Ph
OH
H
O
O
O
OH
H
Ph
H
H
H
OH
H
H
H
H
P-helicity: negative 1Lb band
M-helicity: positive 1Lb band
M-helicity: positive 1Lb band
P-helicity: negative 1Lb band
(3R, 4S)-trans-isoflavan-4-ol
(3S, 4S)-cis-isoflavan-4-ol
(3S, 4R)-trans-isoflavan-4-ol
(3R, 4R)-cis-isoflavan-4-ol
Figure 6. Half-chair conformations of isoflavan-4-ol stereoisomers.
the Korea government (MOST) (No. R01-2007-000-
21067-0). This work was also supported by the Korea
Research Foundation Grant funded by the Korean
Government (MOEHRD, Basic Research Promotion
Fund) (KRF-2006-311-F00053).
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