Stereochemical determination of O-desmethylangolensin produced from daidzein

Article abstract of DOI:10.1016/j.foodchem.2014.08.122

We had isolated an O-desmethylangolensin (O-DMA)-producing bacterium, Clostridium rRNA cluster XIVa strain SY8519. According to chiral separation using HPLC, the SY8519-produced O-DMA exhibited high optical purity. To determine the absolute stereochemistry of O-DMA, we prepared 2-(4-hydroxyphenyl)propionic acid (2-HPPA) from the O-DMA using the Baeyer-Villiger reaction. From chiral analysis of the product, the major peak had the same stereochemistry to that of 2-HPPA produced from genistein by the same bacteria. As we have determined the stereochemistry of SY8519-produced 2-HPPA to have an R configuration, by the chemical synthesis of (S)-2-HPPA, the SY8519-produced O-DMA must also possess R stereochemistry at the 2-position. To study the stereoselective metabolism, we applied racemic dihydrodaidzein to SY8519. The O-DMA was isolated from the culture media and starting material was also recovered. The O-DMA produced was optically active in a similar manner to that produced from daidzein. However, the remaining dihydrodaidzein exhibited no difference between the enantiomers. These results suggested that SY8519 produces (R)-O-DMA from both enantiomers of dihydrodaidzein.

Full text of DOI:10.1016/j.foodchem.2014.08.122

Food Chemistry 171 (2015) 153–156
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Stereochemical determination of O-desmethylangolensin produced
from daidzein
Toshio Niwa a, , Shin-ichiro Yokoyama , Natsuki Matsugasaki , Eri Inomata , Asako Taira ,
⇑
b
a
a
a
c
Toshihiko Osawa
a
Department of Human Health and Nutrition, Shokei Gakuin University, 4-10-1, Yurigaoka, Natori, Miyagi 981-1295, Japan
Department of Food Technology, Industrial Technology Center, Gifu Prefectural Government, 47 Kitaoyobi, Kasamatsu-cho, Hashima-gun, Gifu 501-6064, Japan
Department of Health and Nutrition, Faculty of Psychological & Physical Science, Aichi Gakuin University, 12, Araike, Iwasaki-cho, Nisshin, Aichi 470-0195, Japan
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 March 2014
Received in revised form 25 July 2014
Accepted 27 August 2014
Available online 10 September 2014
We had isolated an O-desmethylangolensin (O-DMA)-producing bacterium, Clostridium rRNA cluster XIVa
strain SY8519. According to chiral separation using HPLC, the SY8519-produced O-DMA exhibited high
optical purity. To determine the absolute stereochemistry of O-DMA, we prepared 2-(4-hydroxy-
phenyl)propionic acid (2-HPPA) from the O-DMA using the Baeyer–Villiger reaction. From chiral analysis
of the product, the major peak had the same stereochemistry to that of 2-HPPA produced from genistein
by the same bacteria. As we have determined the stereochemistry of SY8519-produced 2-HPPA to have
an R configuration, by the chemical synthesis of (S)-2-HPPA, the SY8519-produced O-DMA must also pos-
sess R stereochemistry at the 2-position. To study the stereoselective metabolism, we applied racemic
dihydrodaidzein to SY8519. The O-DMA was isolated from the culture media and starting material was
also recovered. The O-DMA produced was optically active in a similar manner to that produced from
daidzein. However, the remaining dihydrodaidzein exhibited no difference between the enantiomers.
These results suggested that SY8519 produces (R)-O-DMA from both enantiomers of dihydrodaidzein.
Ó 2014 Published by Elsevier Ltd.
Keywords:
O-Desmethylangolensin
Human
Metabolism
Soy isoflavonoids
1
. Introduction
Considerable attention has been focused on certain foods which
et al., 2005) exhibits potent activity compared to that of the unnat-
ural form (Muthyala et al., 2004). Therefore, the stereochemistry of
the metabolites is important for determining the in vivo activity
after soy intake.
In 2004, Wang et al. reported that an anaerobic bacteria enanti-
oselectively produced 2-HPPA and O-DMA from genistein and
daidzein, respectively. The results suggested that the metabolites
produced in the human intestine would be produced enantioselec-
tively. However, the stereochemistry of 2-HPPA and O-DMA were
not determined in the previous study. Therefore, we are interested
in the determination of the absolute stereochemistry of the
metabolites.
can potentially prevent diseases or ageing. Soy isoflavones are
amongst the most interesting food ingredients due to the variety
of uses. However, previous studies have demonstrated that soy iso-
flavones, such as daidzein and genistein, are metabolized by intes-
tinal bacteria in certain populations. The study of this metabolism
is ongoing, but three metabolites have been identified in animal
urine (Coldham et al., 1999; Yasuda, Ueda, & Ohsawa, 2001),
human urine (Kelly, Nelson, Waring, Joannou, & Reeder, 1993)
and bacterial fermentations (Schoefer, Mohan, Braune, Birringer,
&
Blaut, 2002; Wang, Kim, Lee, Hur, & Kim, 2004). Daidzein is con-
We have isolated a Clostridium rRNA cluster XIVa strain SY8519
as an O-DMA-producing bacterium (Yokoyama, Niwa, Osawa, &
Suzuki, 2010). SY8519 also decomposes genistein to 2-HPPA. We
determined the absolute stereochemistry of the biosynthetic
2-HPPA by preparing (S)-2-HPPA from commercially available
2-(S)-phenylpropionic acid (Niwa, Yokoyama, & Osawa, 2013).
We believe that this analytical method would be applicable for
the determination of the stereochemistry of O-DMA if 2-HPPA
was obtained from O-DMA. We then examined the preparation of
2-HPPA from O-DMA, which was produced by SY8519.
verted to equol and O-desmethylangolensin (O-DMA) and geni-
stein produces 2-(4-hydroxyphenyl)propionic acid (2-HPPA).
Because all three metabolites have an asymmetric carbon, they
have enantiomers. Enantiomers often show significantly different
biological activity in vivo. Indeed, natural (S)-equol (Setchell
⇑
Corresponding author. Tel.: +81 22 381 3351; fax: +81 22 381 3325.
E-mail address: nbononaka@hotmail.co.jp (T. Niwa).
http://dx.doi.org/10.1016/j.foodchem.2014.08.122
308-8146/Ó 2014 Published by Elsevier Ltd.
0

1
54
T. Niwa et al. / Food Chemistry 171 (2015) 153–156
2
. Materials and methods
2.6. Isolation of O-DMA from human urine
2
.1. Materials
Five female volunteers (21 and 22 years old) from our labora-
tory participated in our study with informed consent and approval
from the Research ethical committee of our university (#13–007).
In the first screening, they drank soy milk (200 ml; Kikkoman,
Chiba, Japan) purchased from a local market prior to sleeping. A
small amount of urine from each student was collected the next
morning. The samples were treated with enzymes to liberate the
polyphenols (Hall, O’Brien, & McCormack, 2007). Briefly, each
9 ml sample was mixed with 1 ml of a 0.5 M acetate buffer (pH
2 3
Racemic 2-HPPA, m-chloroperbenzoic acid (mCPBA) and K CO
were obtained from Tokyo Kasei Kogyo (Tokyo, Japan). Chemically
synthesized O-DMA and biosynthesized (R)-2-HPPA were prepared
according to previously published protocols (Niwa, Yokoyama, &
Osawa, 2009; Niwa et al., 2013). Dihydrodaidzein was obtained
from Toronto Research Chemicals Inc. (Ontario, Canada) or synthe-
sized in our laboratory (Niwa et al., 2009). b-Glucuronidase was
purchased from Sigma (St. Louis, MO). Sulfatase and NaHCO
obtained from Wako Pure Chemical Industries (Osaka, Japan).
3
were
5.5). Then, the samples were treated with 250
(1000 U/ml) and 250 l of b-glucuronidase (2500 U/ml) at 37 °C
for 2 h. The enzyme-treated samples were extracted with EtOAc
3 Â 3 ml), and the extracts were dried under reduced pressure.
The residual samples were re-dissolved by the addition of 300
of MeOH and then 200 l of H O. The samples were subjected to
ll of sulfatase
l
(
2.2. Preparation of O-DMA from daidzein by a SY8519 strain
ll
l
2
The fermentation and isolation of O-DMA from the culture med-
HPLC with an ODS column used to identify the producer of O-DMA.
ium was performed according to a previously reported protocol
Yokoyama et al., 2010). For this study, O-DMA was purified by
(
semi-preparative HPLC on a 250 Â 8.0 mm i.d. Develosil ODS-5-
2.7. Reversed-phase HPLC analysis
HG column (Nomura chemical, Aichi, Japan). A solvent mixture
containing H
2
O/MeOH (55/45) at a flow rate of 2.4 ml/min at ambi-
HPLC analysis of O-DMA was performed with an i.d.
4.6 Â 250 mm column (Wakogel-II 5C18HG; Wako). An aqueous
40% MeOH solution was eluted at 1.0 ml/min at 40 °C with moni-
toring at 254 nm.
ent temperature was used to elute the compound. O-DMA was col-
lected by monitoring the absorbance at 254 nm.
2.3. Synthesis of 2-HPPA from O-DMA
2.8. Chiral analysis of O-DMA obtained from human urine
2 3
To a reaction mixture of O-DMA (4.0 mg), K CO (5.6 mg) and
NaHCO
CHCl (1.5 ml) was added (Kiyota, Nakashima, & Oritani, 1999).
The reaction was performed by stirring at ambient temperature
for 25 h. The reactant was mixed with 5% NaHCO (50 ml), and
3
(34 mg) in CHCl
3
(3 ml), mCPBA (44 mg) dissolved in
An O-DMA producer drank soy milk and urine samples were
collected. The sample (200 ml) was treated with the enzymes
described above. O-DMA was purified in a similar manner to that
used in the isolation from the bacterial fermentation. Finally,
0.2 mg of O-DMA was isolated and subjected to chiral separation
as described in Section 2.4.
3
3
the organic layer was discarded. The aqueous solution was acidi-
fied by the addition of 1 M HCl (50 ml) and the organic components
extracted with EtOAc (80 ml). The EtOAc solution was washed with
distilled water and saturated brine. The solvent was removed
3
. Results and discussion
2 4
under reduced pressure after dried over anhydrous Na SO . The
crude product was added to 35% MeOH (2 ml) and centrifuged.
The supernatant was filtered and then subjected to semi-prepara-
tive HPLC as previously described (Niwa et al., 2013). Finally,
Natural products often exhibit optical activity. Daidzein and
genistein do not have enantiomers due to their structures. How-
ever, these compounds will have an asymmetric carbon after
C-ring reduction. Therefore, some metabolites of soy isoflavonoids
have enantiomers. In a previous study, Wang et al. (2004) reported
that O-DMA produced by a bacterium is optically active. However,
the stereochemistry has not yet been determined.
0
.5 mg of 2-HPPA was obtained.
2.4. Chiral analysis using HPLC
Chiral analysis of dihydrodaidzein and 2-HPPA using HPLC was
Strain SY8519 was identified as an O-DMA-producing bacte-
rium by our group. We then analysed SY8519-produced O-DMA
using a SUMICHIRAL OA-7000 column. The SY8519-produced
O-DMA was also highly optically active (e.e. 90%). Next, we tried
to determine the stereochemistry of the 2-position of O-DMA by
derivatization to 2-HPPA. We achieved this by synthesising
2-HPPA, as shown in Fig. 1. The Baeyer–Villiger reaction would
produce an ester of 2-HPPA from O-DMA. The resulting ester would
be converted to 2-HPPA by alkaline hydrolysis. Then, we reacted
performed according to previously described methods (Wang,
Shin, Hur, & Kim, 2005; Niwa et al., 2013). The chiral separation
of O-DMA was performed with a slight modification of the reported
conditions (Wang et al., 2004). Briefly, we used an eluent (20 mM
3
phosphate buffer (pH 3.0):CH CN, 75:25) on a SUMICHIRAL OA-
7
000 column (i.d. 4.6 Â 250 mm: Sumika Chemical Analysis Ser-
vice, Osaka, Japan) at a flow rate of 1.0 ml/min at 40 °C. The UV
detector was set to 254 nm.
2 3 3
O-DMA with mCPBA in the presence of K CO and NaHCO . TLC
analysis of the reaction product yielded a spot corresponding to
the starting material, as well as an additional spot. In spite of our
scheme, the product was 2-HPPA, which was identified by HPLC
2
.5. Metabolism of dihydrodaidzein to O-DMA by SY8519
Commercially available dihydrodaidzein (20 mg) was metabo-
lized by SY8519 in a similar manner to daidzein (Yokoyama
et al., 2010). The culture medium was extracted with EtOAc similar
to the method used in the preparation of the metabolites
Baeyer-Villiger
reaction
alkaline
HO
OH
O
OH
hydrolysis
O
HO
(Yokoyama et al., 2010; Niwa et al., 2013). The resulting residue
O
O
was applied to a Merck preparative TLC plate (0.5 mm thickness)
and eluted with a solvent mixture of n-hexane/EtOAc (1:1). Finally,
OH
HO
OH
OH
O-desmethylangolensin
O-DMA)
2-(4-hydroxyphenyl)propionic acid
(
(2-HPPA)
2
.5 mg of O-DMA was obtained along with the recovery of 8.8 mg
of dihydrodaidzein.
Fig. 1. Synthetic strategy for the preparation of 2-HPPA from O-DMA.

T. Niwa et al. / Food Chemistry 171 (2015) 153–156
155
analysis and 1H NMR spectroscopy. In a previous study, Corma,
b)
Abs. 254 nm
Fornés, Iborra, Mifsud, and Renz (2004) described a one-pot phenol
production using the Baeyer–Villiger reaction. We have not identi-
fied the precise mechanism of this direct reaction. The optical
activity of synthesized 2-HPPA was analysed by HPLC, and com-
pared to that of commercially available racemic 2-HPPA and
SY8519-produced (R)-2-HPPA (Fig. 2). Therefore, the predominant
peak for the product was identified as (R)-2-HPPA. These results
indicated that SY8519 produces (R)-O-DMA from daidzein, with a
high selectivity. This result was reasonable as the same selectivity
of the asymmetric reduction of the C-ring in the metabolism of
daidzein and genistein is observed by the same bacterium.
(S)
(R)
biosynthetic O-DMA
synthetic O-DMA
a)
(racemate)
Wang, Hur, Lee, Kim, and Kim (2005) reported that (S)-equol is
produced from dihydrodaidzein by human intestinal bacterium.
Recently, Shimada et al. (2012) identified a dihydrodaidzein race-
mase and described a metabolic pathway that produces optically
active (S)-equol from racemic dihydrodaidzein. We also metabo-
lized commercially available dihydrodaidzein by SY8519 and iso-
lated the O-DMA produced and remaining dihydrodaidzein.
Identification was performed by co-injection into an authentic
sample for reverse-phase HPLC, and then, the isolated samples
were subjected to HPLC equipped with a SUMICHIRAL OA-7000
column. The optical activity (e.e. 88%) of O-DMA (Fig. 3) was sim-
ilar to that of the product from daidzein. The unreacted dihydrod-
aidzein exhibited no apparent difference between the enantiomers
7
.5
22.5
Retention time [min]
Fig. 3. Chiral separation of O-DMA produced from racemic dihydrodaidzein by a
SY8519 strain. Dihydrodaidzein (20 mg) was metabolized by SY8519 for 2 days. O-
DMA (2.5 mg) was isolated from the culture medium where 8.8 mg of dihydrod-
aidzein was recovered as described in Section 2.
phosphate buffer (pH 3.0):CH CN, 75:25) was eluted on a SUMICHIRAL OA-7000
A solvent mixture (20 mM
3
column at 1.0 ml/min at 40 °C. (a) Synthetic racemic sample and (b) isolated
dihydrodaidzein purified from SY8519 strain product.
(Fig. 4). This result suggested that SY8519 produced (R)-O-DMA
b) Abs. 254 nm
equally from racemic dihydrodaidzein. Our results were also con-
sistent with previous results concerning bacterial production of
equol from dihydrodaidzein racemate (Shimada et al., 2012). In
addition, we proposed another mechanism that inverts the stereo-
chemistry of (S)-O-DMA to the R-form, as shown in Fig. 5. Then, we
applied commercially available racemic 2-HPPA to SY8519 and
analysed the chirality. However, the recovered 2-HPPA still exhib-
ited no optical purity (data not shown). These results suggested
that very little (S)-O-DMA was produced from daidzein and race-
mic dihydrodaidzein. Therefore, we concluded that SY8519 has
dihydrodaidzein recovered
from culture medium
a)
synthetic dihydrodaidzein
(racemate)
7.5
22.5
Retention time [min]
c) Abs. 235 nm
SY8519-producing
biosynthetic (R)-2-HPPA
Fig. 4. Chiral separation of recovered dihydrodaidzein, which was obtained in
Fig. 3. The samples were analysed by a previously published protocol (Wang et al.,
2004). (a) Synthetic racemic sample and (b) an isolated sample recovered from the
culture medium.
b)
synthetic 2-HPPA obtained
from biosynthetic O-DMA
a)
authentic 2-HPPA
(
racemate)
Fig. 5. Proposed mechanism for the production of (R)-O-DMA from raceme
dihydrodaidzein by SY8519.
5
15
Retention time [min]
dihydrodaidzein racemase activity and produces (R)-O-DMA from
racemic dihydrodaidzein, as shown in Fig. 5. The results in Figs. 3
and 4 suggested that the reaction of the racemase is faster than
the cleavage of the C-ring of (S)-dihydrodaidzein.
Several O-DMA producing bacteria have been isolated (Hur
et al., 2002; Schoefer et al., 2002; Yokoyama et al., 2010), and many
Fig. 2. Chiral separation of 2-HPPA obtained from SY8519-producing O-DMA. HPLC
analysis was performed using a CHIRALPAK IB column as described previously
Niwa et al., 2013). (a) authentic racemic sample, (b) synthetic 2-HPPA from
SY8519-produced O-DMA, and (c) strain SY8519 produced (R)-2-HPPA.
(

1
56
T. Niwa et al. / Food Chemistry 171 (2015) 153–156
more unidentified O-DMA producing bacteria still exist, including a
number in the human intestine. Next, we examined whether the
SY8519-produced O-DMA structure is common. We analysed the
stereochemistry of O-DMA obtained from human urine. Five
female students in our laboratory drank soy milk prior to going
to sleep and their urine was collected the next morning. Soy isofl-
avonoids are typically present as conjugates of glucuronide and
sulfate in human urine (Clarke et al., 2002). Therefore, the five
urine samples were treated with glucuronidase/sulfatase and sub-
jected to reversed-phase HPLC. Although we did not perform a
quantitative analysis, such as the recovery of daidzein or O-DMA
concentration, only one sample had an apparent O-DMA signal
on the chromatogram. Next, we isolated O-DMA from human urine
and analysed the optical purity using HPLC. The isolated O-DMA
also predominantly contained the R-enantiomer on the chromato-
gram. However, the optical purity (e.e. 80%) was less than that of
the SY8519-produced one. We believe that the optical purity of
conjugates in human urine using isotope dilution liquid chromatography-
tandem mass spectrometry with [13C3]isoflavone internal standards. Analytical
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the fragile
a-position of the carbonyl was reduced by some pro-
cess, such as glucuronidase/sulfatase treatment or circulation in
the body. Because only one result was available for determination
of the stereochemistry of O-DMA obtained from human urine, we
were unable to determine if this was the case. However, our result
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metabolites is required to understand the precise role of the activ-
ity of soy isoflavones in vivo. In these studies, we must also con-
sider the stereochemistry of the soy metabolites, not only O-DMA
but also 2-HPPA, to determine their precise activity, as has been
performed for equol (Muthyala et al., 2004; Brown et al., 2010).
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Acknowledgements
We wish to thank Prof. Hirokazu Arimoto (Tohoku University),
Prof. Shu Xu (Tohoku University) and Prof. Kazuaki Akasaka (Sho-
kei Gakuin University) for their assistance in measuring the NMR.
2
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a
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