Microbial glycosylation of daidzein, genistein and biochanin a: Two new glucosides of biochanin A

Article abstract of DOI:10.3390/molecules22010081

Biotransformation of daidzein, genistein and Biochanin A by three selected filamentous fungi was investigated. As a result of biotransformations, six glycosylation products were obtained. Fungus Beauveria bassiana converted all tested isoflavones to 4″-O-methyl-7-O-glucosyl derivatives, whereas Absidia coerulea and Absidia glauca were able to transform genistein and Biochanin A to genistin and sissotrin, respectively. In the culture of Absidia coerulea, in addition to the sissotrin, the product of glucosylation at position 5 was formed. Two of the obtained compounds have not been published so far: 4″-O-methyl-7-O-glucosyl Biochanin A and 5-O-glucosyl Biochanin A (isosissotrin). Biotransformation products were obtained with 22%-40% isolated yield.

Full text of DOI:10.3390/molecules22010081

molecules
Article
Microbial Glycosylation of Daidzein, Genistein and
Biochanin A: Two New Glucosides of Biochanin A
Sandra Sordon *, Jarosław Popłon´ski, Tomasz Tronina and Ewa Huszcza
Department of Chemistry, Wrocław University of Environmental and Life Sciences, Norwida 25,
50-375 Wrocław, Poland; jaroslaw.poplonski@up.wroc.pl; (J.P.); tomasz.tronina@up.wroc.pl (T.T.);
ewa.huszcza@up.wroc.pl (E.H.)
* Correspondence: sandra.sordon@wp.pl; Tel.: +48-71-320-5197
Academic Editor: Derek J. McPhee
Received: 27 November 2016; Accepted: 30 December 2016; Published: 3 January 2017
Abstract: Biotransformation of daidzein, genistein and Biochanin A by three selected filamentous
fungi was investigated. As a result of biotransformations, six glycosylation products were obtained.
Fungus Beauveria bassiana converted all tested isoflavones to 4”-O-methyl-7-O-glucosyl derivatives,
whereas Absidia coerulea and Absidia glauca were able to transform genistein and Biochanin A to
genistin and sissotrin, respectively. In the culture of Absidia coerulea, in addition to the sissotrin,
the product of glucosylation at position 5 was formed. Two of the obtained compounds have not been
published so far: 4”-O-methyl-7-O-glucosyl Biochanin A and 5-O-glucosyl Biochanin A (isosissotrin).
Biotransformation products were obtained with 22%–40% isolated yield.
Keywords: isoflavones; daidzein; genistein; Biochanin A; microbial glycosylation; fungi
1. Introduction
Isoflavones are the most well-known phytoestrogens [1]. They are secondary metabolites of the
very restricted distribution in the plant kingdom, found exclusively in legumes (Leguminosae family).
The major dietary sources of this most common form of phytoestrogens are soybeans, chickpeas and
lentils [
as: cancers (including breast, prostate and colon carcinoma) [
bone health problems [ ] and postmenopausal symptoms [10 11]. Therefore, there is considerable
2
]. Isoflavones have been implicated in the prevention of hormone-dependent diseases, such
3–
6
], cardiovascular disorders [ ],
7,8
9
,
interest in the use of isoflavones in the prevention of estrogen-related cancers and certain diseases
caused by estrogen deficiency.
Genistein and daidzein, the major isoflavones present in legumes, exist predominantly in the
0
glycoside form, i.e., genistin and daidzin or 4 -methoxy derivatives, i.e., Biochanin A and formononetin,
respectively [2].
Due to better solubility, greater stability and functionality compared to aglycones, glycosylated
forms of flavonoids have recently been gaining increasing attention. Biotransformations of flavonoids
is a useful tool to obtain their glycosylated derivatives. Application of microorganisms as biocatalysts
allows us to obtain these compounds in sufficient amounts for research, e.g., the effect of a glycoside
group on compound properties and for further application as ingredients of dietary supplements and
pharmaceuticals [12].
The sugar moiety of flavonoids was proposed to be the major determinant of their absorption
in humans [13]. It is generally believed that flavonoid glycosides are converted to the corresponding
aglycones by the intestinal microflora and/or by the intestinal glucosidases, and as such absorbed
from the small intestine [14]. Many studies have shown that flavonoid glycosides are poorly absorbed
compared with their aglycones [13
,15,16]. This phenomenon was partially revised recently as a result
of studies on the bioavailability of flavonol quercetin. It turned out that quercetin-3-O-β-D-glucoside
Molecules 2017, 22, 81; doi:10.3390/molecules22010081
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has higher bioavailability than its aglycone in humans, what suggests that conjugation with glucose
would enhance quercetin absorption in the small intestine [17 19]. However, in the case of isoflavones,
there are many evidences that aglycones have more biological effects than related glycosides, due
to faster absorption in a greater amount [20 21]. These studies were focused on the most common
–
,
isoflavone 7-O-glucosides, genistin and daidzin.
In this paper we described fungal biotransformation of three isoflavones: daidzein, genistein and
Biochanin A. We found that substituents in the B-ring of tested flavonoids showed the impact on the
regioselectivity of sugar moiety coupling.
2. Results and Discussion
Our previous research on microbial metabolism of flavonoids revealed that the fungal strains
Beauveria bassiana AM 278, Absidia glauca AM 177 and Absidia coerulea AM 93 are able to attach sugar
moiety to the chalcones and flavanones [22–24]. Therefore, biotransformation of daidzein, genistein and
Biochanin A, the aglycones of the most commonly occurring isoflavones, using those microorganisms
was investigated.
Screening tests have shown that genistein and Biochanin A were transformed by all of the tested
strains, while daidzein was metabolized only by the fungus Beauveria bassiana. Experiments conducted
on a larger scale enabled us to determine the chemical structure of the resulting products and their
isolated yields (Figure 1).
Figure 1. Transformation of isoflavonoids by selected fungi.

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Daidzein was converted to 4”-O-methyldaidzin by Beauveria bassiana with a yield of 33.3% (33.8 mg).
This compound was earlier identified only in a soybean broth fermented by Paecilomyces militaris [25].
Genistein was metabolized by Absidia glauca and Absidia coerulea to genistin with a yield of 21.9%
(21.0 mg) and 12.5% (12.0 mg), respectively. Glucosylation at C-7 position of genistein was also observed
for solvent-tolerant bacterium Staphylococcus saprophyticus CQ16 [26], as well as for plant cultured
cells of Eucalyptus perriniana [27]. As it was expected based on our previous studies [22–24], fungus
Beauveria bassiana transformed genistein to the typical for this species derivative-4”-O-methylgenistin
(27.0 mg, 27.2%).
Incubation of Biochanin A with Beauveria bassiana led to the new metabolite 4”-O-methylBiochanin
A (29.9 mg, 30.8%). Seven proton and six carbon signals were observed in the regions from 3.21 to
5.03 ppm in the 1H-NMR and from 60.9 to 101.5 ppm in the 13C-NMR spectra. HSQC, HMBC and
COSY spectra proved that the signals were corresponding to a glucose molecule. The methyl carbon
(C-4”OCH3) resonating at
δ
60.9 showed a one-bond correlation with three protons at
δ
3.60 on HMQC
spectrum. These protons also showed three-bond correlation with carbon at
δ 80.5 (C4”) on HMBC
−
spectrum. Discussed metabolite had the molecular formula (C23H24O10-H) as indicated by HR-ESIMS
of the [M − H]-ion peak at m/z 459.1297 (calcd. 459.1296).
When Biochanin A was transformed by Absidia glauca as a bioreagent, the sissotrin was observed as
the only product (38.0 mg, 40.3%). Glucosylation of Biochanin A at C-7 position was also performed by
Absidia coerulea, however with a much lower yield (4.5 mg, 4.6%). This phenomenon can be attributed
to concurrent formation of the second metabolite, which was recognized as 5-O-β-glucosylBiochanin
A. The yield of this positional isomer of sissotrin, named isosissotrin by us, was 23.2% (22.5 mg).
The composition of the products mixture was determined based on the NMR spectra analyses.
The NMR spectra of products obtained by biotransformation by Absidia coerulea AM 93 showed
characteristic doubled signals (Figures S1–S12, Supplementary Materials). HMQC, HMBC and COSY
experiments allowed us to establish unambiguously the flavonoid glycosides structure. The presence
of doubled signals typical for a glucose moiety suggested the formation of two glucosylation
1
products. In the H-NMR and 13C-NMR spectrum, doubled six proton and doubled six carbon
signals corresponding to a glucose moiety structure were observed. The proton signal of H-1” of
glucosylation product at C-5 position was overlapped with the artifact peak (likely of water) (Figures S1,
S3, S5, S7, S8 and S10 Supplementary Materials).
Correlations observed in the HMBC spectrum permitted the assignment of the H-6 and H-8
protons of the two flavonoid products (Figure S9, Supplementary Materials). The aromatic A-ring
protons H-6 appearing as doublets at
carbons (C-10: 110.3 ppm; C-5: 160.6 ppm; C-7: 164.7 ppm) and (C-10: 108.0 ppm; C-5: 163.5 ppm;
C-7: 164.8 ppm) respectively. In turn, the other A-ring protons H-8 appearing as doublets at 6.58
J = 2.2 Hz) and 6.70 (J = 2.2 Hz) resonated with four carbons (C-6: 104.5 ppm; C-10: 110.3 ppm;
δ 6.83 (J = 2.2 Hz) and 6.51 (J = 2.2 Hz) resonated with three
δ
(
C-9: 160.9 ppm; C-7: 164.7 ppm) and (C-6: 101.2 ppm; C-10: 108.0 ppm; C-9: 159.2 ppm; C-7:
164.8 ppm), respectively.
The HMBC correlation between H-1” and C-7 and between H-1” and C-5 confirmed the location
of sugar molecules. Noteworthy is that no significant signal shifts of both protons and carbons of
the ring A of this product were observed, thus providing further confirmation of glucosyl moiety
attachment. The glucose moieties were connected by 1,7-glycosidic linkages in sissotrin and by a
1,5-glycosidic linkage in isosissotrin (Figure S10, Supplementary Materials).
The products showed the [M
The exact molecular mass was consistent with a molecular formula of C22H22O10 and confirmed the
formation of two positional isomers (Figure S13, Supplementary Materials).
−
M]-ion peak at m/z 445.1151 (calcd. 445.1140) in HR-ESIMS.
Lewis et al.
the phase transfer catalyzed reaction between isoflavone aglycones of obtained products and
1-bromo-2,3,4,6-tetra-O-acetyl- -glucopyranose in the presence of tetrabutylammonium bromide
as a catalyst [28]. This method has some disadvantages: the catalyst used in the reaction is harmful
described the chemical synthesis of daidzin, genistin and sissotrin by
α

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for the environment and the protection of hydroxyl groups that are not meant to conjugate with
saccharide is necessary. Moreover, chemical synthesis does not solve the synthetic challenges of
flavonoid 5-O-glucosides [29].
The use of filamentous fungi as a biocatalyst is the relevant method for obtaining isoflavonoid
glucosides in simple and cheap one-step biotransformation process. Furthermore, according to the
European Union Law, the products obtained by biotransformation are classified as natural compounds
(EU Directive 88/388/EEC).
Summarizing, the course of the biotransformation of isoflavones by three selected filamentous
fungi known from their ability to O-glycosylation was investigated. High regioselectivity maintaining
low substrate specificity was observed in biotransformation conducted by Beauveria bassiana, which is
typical for this species and has been shown in previous studies [22–24,30–32]. Whilst, the substrate
specificity of enzymes from Absidia genus is higher. In the presented studies it was observed that
isoflavones without a hydroxyl group at the C-5 position do not undergo biotransformation by Absidia
species. In addition, presence of the methoxy group at C-40 position change the regioselectivity of
biotransformations by Absidia coerulea, which may be associated with different substrate orientation in
the catalytic pocket of the enzyme or with different enzyme catalyzing conversion of this substrates.
Fungus Absidia coerulea glucosylates Biochanin A mainly at the 5-OH position, whereas Absidia glauca
metabolizes Biochanin A only to one glucosylation product at the 7-OH position.
As we expected, the microorganisms used in the studies carried out conjugation of isoflavones
with the sugar molecule. Daidzin and genistin occur naturally in relatively large amounts; however,
sissotrin, isolated mainly from red clover, occurs in amounts more than 10-fold less than its aglycone
form Biochanin A [33]. Therefore, our biotechnological methods of obtaining sissotrin as well as of
isosissotrin may have practical significance. The fungal transformations presented in this article turned
out to be a useful tool for obtaining new isoflavone glucosides as well as these, which naturally occur
in small quantities. Due to relatively high yields (compared to chemical methods) the filamentous
fungi biocatalysis can be used as a method of choice for bioavailability studies. There are many
studies on bioavailability of genistein and its glucoside genistin and also daidzein and its glucoside
daidzin, although absorption of isoflavonoid glycosides and their respective aglycones that form in
humans is a subject of controversy [34]. Although there are reports in the literature on bioavailability
of Biochanin A [35–37], there is no data about Biochanin A glucoside bioavailability, possibly due to
the rarer occurrence of sissotrin in nature. Described biotransformation of Biochanin A is the method
that allows us to obtain a glucoside in an amount sufficient for the research concerning the influence of
glucose moiety on biological activity.
3. Materials and Methods
3.1. Compounds
0
Biochanin A (5,7-dihydroxy-4 -methoxyisoflavone) was purchased from Sigma-Aldrich (St. Louis,
MO, USA). Daidzein (40,7-dihydroxyisoflavone) and genistein (40,5,7-trihydroxyisoflavone) were
isolated from soy extract. Flavonoid glycosides included in the dry extract were hydrolyzed and the
aglycones of daidzein and genistein were isolated from the reaction mixture according to the method
described by Utkina et al. [38] and were of high purity (>98% by HPLC).
1
Daidzein: H-NMR (600 MHz, DMSO-d6) δ: 6.80 (2H, m, H-30, H-50), 6.85 (1H, d, J = 2.2 Hz, H-8), 6.93
(1H, dd, J = 8.7, 2.2 Hz, H-6), 7.37 (2H, m, H-20, H-60), 7.96 (1H, d, J = 8.7, H-5), 8.29 (1H, s, H-2), 9.51
(1H, s, 40-OH), 10.77 (1H, s, 7-OH); 13C-NMR (150 MHz, DMSO-d6) δ0: 102.8 (C-8), 114.1 (C-6), 116.6
0
0
0
0
0
(C-10), 115.1 (C-3 , C-5 ), 122.5 (C-1 ), 123.5 (C-3), 127.3 (C-5), 130.1 (C-2 , C-6 ), 152.8 (C-2), 157.2 (C-4 ),
157.4 (C-9), 162.5 (C-7), 174.7 (C-4).
1
Genistein: H-NMR (600 MHz, DMSO-d6)
δ
: 6.26 (1H, d, J = 2.1 Hz, H-6), 6.42 (1H, d, J = 2.1 Hz, H-8),
6.82 (2H, m, H-30, H-50), 7.37 (2H, m, H-20, H-60), 8.32 (1H, s, H-2), 9.65 (1H, s, 40-OH), 11.04 (1H, s,

Molecules 2017, 22, 81
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7-OH), 12.94 (1H, s, 5-OH); 13C-NMR (150 MHz, DMSO-d6)
δ
: 94.2 (C-8), 99.5 (C-6), 104.9 (C-10), 115.5
(C-30, C-50), 121.4 (C-10), 122.7 (C-3), 130.6 (C-20, C-60), 154.5 (C-2), 157.9 (C-40), 158.0 (C-9), 162.4 (C-5),
164.9 (C-7), 180.8 (C-4).
1H- and 13C-NMR data were found to be identical to that of daidzein and genistein reported in
the literature [39,40].
3.2. Biotransformation
3.2.1. Microorganisms
The microorganisms used in this work, i.e., Beauveria bassiana AM 278, Absidia glauca AM 177
and Absidia coerulea AM 93, were obtained from the collection of the Department of Biology and
Pharmaceutical Botany, Medical University of Wrocław, Poland.
3.2.2. Screening Procedure
All fermentation experiments were carried out in Sabouraud medium (D-glucose 30 g and peptone
◦
10 g per liter of distilled water) on rotary shakers (130 rpm, 6.5 amplitude) at 28 C. Agar slant cultures
were used to obtain the inoculation culture. Portions of 0.5 mL of the 3-day inoculum were transferred
to 100 mL Erlenmeyer flasks, each containing 30 mL of the medium. After cultivation, 5 mg of a
substrate dissolved in 0.5 mL of dimethyl sulfoxide (DMSO) was added to the grown culture and the
reaction mixture was incubated for 7 days. After incubation the cultures were acidified with 1 M HCl
to pH around 5 and extracted with ethyl acetate (3
residue dissolved in methanol and analyzed by TLC and HPLC. All experiments were performed with
appropriate controls (the substrate in a sterile growth medium and incubation with no substrate).
×
15 mL). The extracts were evaporated, and the
3.2.3. Preparative (Large) Scale Biotransformation
Portions of 1.5 mL of the 3-day inoculation culture were transferred to 300 mL Erlenmeyer flasks,
each containing 100 mL of the medium. In the preparative (large scale) biotransformation, a total of
60 mg of each substrate dissolved in 6 mL of DMSO and was equally distributed among four flasks with
4-day fungal cultures to give a final concentration of 150 mg/L. After 10 days of incubation, the cultures
were acidified with 1 M HCl to pH around 5 and three time extracted with the same volume of ethyl
acetate. The other procedures and culture conditions were the same as those of the screening experiments.
The collected organic phase was dried over anhydrous MgSO4, the solvent was filtered, evaporated
in a vacuum and analyzed using TLC and HPLC. The biotransformation products were separated by
column chromatography on silica gel 60 (230–400 mesh, Merck, Darmstadt, Germany) using chloroform:
methanol (3:1 v/v) as eluent. TLC was carried out with Merck silica gel 60, F254 (0.2 mm thick)
plates using the same eluents. Products were detected by inspecting plates under UV irradiation.
Rf: 0.53 (genistin); 0.64 (sissotrin, isosissotrin); 0.77 (4”-O-methyldaidzin); 0.79 (4”-O-methylgenistin);
0.84 (4”-O-methylsissotrin); 0.85 (genistein); 0.87 (daidzein); 0.89 (biochanin A).
3.3. Analysis of Products with High Performance Liquid Chromatography (HPLC)
HPLC was carried out on a Waters 2695 Alliance instrument with the photodiode array detector
Waters 2996 (detection from 220 to 500 nm wavelength) using the analytical HPLC column Agilent C-18
ZORBAX Eclipse XDB 5
µ
m (46 mm
×
250 mm) at the flow rate of 1 mL/min and injection volume
10 L. A linear solvent gradient composed of 0.05% formic acid in water (A) and methanol containing
µ
0.05% formic acid (B) was used. Chromatographic separation was achieved using the isocratic elution
of 50% A and 50% B for 2 min, then linear gradient of B from 50% to 95% for 10 min and isocratic elution
of 95% B for 2 min. RT (min): 3.79 (4”-O-methyldaidzin); 3.90 (genistin); 5.03 (4”-O-methylgenistin);
7.87 (daidzein); 8.51 (sissotrin); 8.82 (isosissotrin); 9.14 (genistein); 9.22 (4”-O-methylsissotrin);
12.15 (Biochanin A).

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3.4. Identification of the Products
1
Purified products were identified by NMR spectra analysis. H-NMR, 13C-NMR, 1H-1H-NMR
(COSY) and 1H-13C-NMR (HSQC, HMBC) were recorded on a DRX Bruker Avance TM 600 (600 MHz)
instrument. Negative-ion HR-ESIMS spectra were measured on a Bruker ESI-TOF Mass Spectrometer
micrOTOF-Q. The direct infusion of ESI-MS parameters: The mass spectrometer was operated in
negative ion mode with the potential between the spray needle and the orifice 4, 5 kV, nebulizer
◦
pressure of 0.4 bar, and a drying gas flow rate of 4 L/min at 200 C. The sample flow rate was
180 µL/min. Ionization mass spectra were collected at the ranges m/z 150–3000. The instrument
was calibrated with an Agilent electrospray calibration solution (ESI-L low concentration Tuning
Mix-Agilent Technologies, Agilent Product Number: G1969-85000) that was diluted with acetonitrile.
3.5. Obtained Biotransformation Products
7-O-
β
-D-4”-O-Methyl-glucopyranosyl-40-hydroxyisoflavone (4”-O-methyldaidzin), (biocatalyst: Beauveria
1
bassiana AM 278, yield: 33.3%): H-NMR (600 MHz, DMSO-d6)
δ: 3.06 (1H, m, H-4”), 3.31 (1H, m,
H-2”), 3.45 (1H, m, H-3”), 3.47 (3H, s, 4”-OCH3) 3.50 (1H, m, Ha-6”), 3.52 (1H, m, H-5”), 3.65 (1H, m,
Hb-6”), 4.75 (1H, m, 6”-OH), 5.13 (1H, d, J = 7.8 Hz, H-1”), 5.32 (1H, d, J = 5.5 Hz, 3”-OH), 5.52
(1H, d, J = 5.3 Hz, 2”-OH), 6.82 (2H, m, H-30, H-50), 7.14 (1H, dd, J = 2.3; 8.9 Hz, H-6), 7.22 (1H, d,
J = 2.3 Hz, H-8), 7.40 (2H, m, H-20, H-60), 8.05 (1H, d, J = 8.9 Hz, H-5), 8.38 (1H, s, H-2), 9.56 (1H, s,
40-OH); 13C-NMR (150 MHz, DMSO-d6)
δ: 60.2 (C-6”), 73.3 (C-2”), 75.7 (C-5”), 59.7 (C-4”OCH3), 76.2
(C-3”), 78.9 (C-4”), 99.6 (C-1”), 103.3 (C-8), 115.0 (C-30, 50), 115.5 (C-6), 118.5 (C-10), 122.3 (C-10), 123.7
1
(C-3), 127.0 (C-5), 130.1 (C-20, 60), 153.4 (C-2), 157.0 (C-9), 157.3 (C-40), 161.3 (C-7), 174.8 (C-4). H- and
13C-NMR data were found to be identical to that reported in the literature [25].
7-O-
β
-D-Glucopyranosyl-5,40-dihydroxyisoflavone (genistin), (biocatalysts: Absidia coerulea AM 93, yield:
1
12.5%; Absidia glauca AM 177, yield: 21.9%): H-NMR (600 MHz, DMSO-d6)
δ: 3.17 (1H, m, H-4”), 3.26
(1H, m, H-2”), 3.30 (1H, m, H-3”), 3.44 (1H, m, H-5”), 3.47 (1H, m, Ha-6”), 3.71 (1H, m, Hb-6”), 4.61
(1H, m, 6”-OH), 5.06 (1H, d, J = 7.5 Hz, H-1”), 5.08 (1H, m, 4”-OH), 5.15 (1H, m, 3”-OH), 5.41 (1H, m,
0
0
2”-OH), 6.47 (1H, d, J = 2.2 Hz, H-6), 6.72 (1H, d, J = 2.2 Hz, H-8), 6.83 (2H, m, H-3 , H-5 ), 7.40 (2H, m,
H-20, H-60), 8.43 (1H, s, H-2), 9.63 (1H, s, 40-OH), 12.94 (1H, s, 5-OH); 13C-NMR (150 MHz, DMSO-d6)
δ
: 60.6 (C-6”), 73.1 (C-2”), 76.4 (C3”), 69.6 (C-4”), 77.2 (C-5”), 94.5 (C-8), 99.6 (C-6), 99.8 (C-1”), 106.1
(C-10), 115.1 (C-30, 50), 121.0 (C-10), 122.6 (C-3), 130.2 (C-20, 60), 154.6 (C-2), 157.2 (C-9), 157.5 (C-40),
161.6 (C-5), 163.0 (C-7), 180.5 (C-4). H- and 13C-NMR data were found to be identical to that reported
1
in the literature [41,42].
7-O-
β
-D-4”-O-Methyl-glucopyranosyl-5,40-dihydroxyisoflavone (4”-O-methylgenistin) (biocatalyst:
1
Beauveria bassiana AM 278, yield: 27.2%): H-NMR (600 MHz, DMSO-d6)
δ: 3.0 (1H, m, H-4”), 3.27 (1H,
m, H-2”), 3.44 (1H, m, H-3”), 3.46 (3H, s, 4”-OCH3) 3.49 (1H, m, Ha-6”), 3.52 (1H, m, H-5”), 3.63 (1H,
m, Hb-6”), 4.73 (1H, m, 6”-OH), 5.09 (1H, d, J = 7.8 Hz, H-1”), 5.30 (1H, d, J = 5.5 Hz, 3”-OH), 5.48 (1H,
0
0
d, J = 5.2 Hz, 2”-OH), 6.47 (1H, d, J = 2.2 Hz, H-6), 6.71 (1H, d, J = 2.2 Hz, H-8), 6.83 (2H, m, H-3 , H-5 ),
7.40 (2H, m, H-2 , H-6 ), 8.43 (1H, s, H-2), 9.62 (1H, s, 4 -OH), 12.94 (1H, s, 5-OH); 13C-NMR (150 MHz,
0
0
0
DMSO-d6) δ: 59.7 (C-4”-OCH3), 60.2 (C-6”), 73.2 (C-2”), 75.7 (C-5”), 76.1 (C3”), 78.9 (C-4”), 94.5 (C-8),
99.4 (C-1”), 99.5 (C-6), 106.1 (C-10), 115.1 (C-30, 50), 121.0 (C-10), 122.6 (C-3), 130.2 (C-20, 60), 154.6 (C-2),
1
157.2 (C-9), 157.5 (C-40), 161.7 (C-5), 162.9 (C-7), 180.5 (C-4). H- and 13C-NMR data were found to be
identical to that reported in the literature [25,30].
7-O-
β
-D-Glucopyranosyl-5-hydroxy-40-methoxyisoflavone (sissotrin), (biocatalysts: Absidia coerulea AM
1
93, yield: 4.6%; Absidia glauca AM 177, yield: 40.3%): H-NMR (600 MHz, CD3OD)
δ: 3.44–3.48 (1H,
m, H-4”), 3.47–3.51 (1H, m, H-2”), 3.71–3.74 (1H, m, Ha-6”), 3.83 (3H, s, 40-OCH3), 3.90–3.93 (1H, m,
Hb-6”), 5.05 (1H, m, H-1”), 6.51 (1H, d, J = 2.2 Hz, H-6), 6.70 (1H, d, J = 2.2 Hz, H-8), 6.98 (2H, pd,
J = 8.7 Hz, H-30, H-50), 7.48 (2H, pd, J = 8.7 Hz, H-20, H-60), 8.16 (1H, s, H-2); 13C-NMR (150 MHz,
CD3OD) δ
: 55.8 (C-40OCH3), 62.4 (C-6”), 71.2 (C-4”), 74.7 (C-2”), 77.8 (C-3”), 78.4 (C5”), 95.9 (C-8),

Molecules 2017, 22, 81
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101.2 (C-6), 101.6 (C-1”), 108.0 (C-10), 114.9 (C-30, C-50), 124.3 (C-10), 124.8 (C-3), 131.3 (C-20, C-60),
1
155.5 (C-2), 159.2 (C-9), 161.3 (C-40), 163.5 (C-5), 164.8 (C-7), 182.4 (C-4). H- and 13C-NMR data were
found to be identical to that reported in the literature [43]. HRESI-MS [M
(m/z 445.1140/445.1151).
−
H]− (calculated/found)
5-O-
93, yield: 23.2%): H-NMR (600 MHz, CD3OD)
β
-D-Glucopyranosyl-7-hydroxy-40-methoxyisoflavone (isosissotrin), (biocatalyst: Absidia coerulea AM
1
δ
: 3.40–3.44 (1H, m, H-4”), 3.41–3.51 (2H, m, H-3”,
0
H-5”), 3.60–3.68 (1H, m, H-2”), 3.74-3.77 (1H, m, Ha-6”), 3.82 (3H, s, 4 -OCH3), 3.93–3.96 (1H, m, H-6b”),
4.86 (1H, signal obscured by water, H-1”), 6.58 (1H, d, J = 2.2 Hz, H-8), 6.83 (1H, d, J = 2.2 Hz, H-6),
6.95 (2H, pd, J = 8.7 Hz, H-30, H-50), 7.42 (2H, pd, J = 8.7 Hz, H-20, H-60), 8.02 (1H, s, H-2); 13C-NMR
(150 MHz, CD3OD) δ
: 55.7 (C-40OCH3), 62.5 (C-6”), 71.2 (C-4”), 74.7 (C-2”), 77.3 (C-3”), 78.6 (C-5”),
98.9 (C-8), 104.5 (C-6), 104.9 (C-1”), 110.3 (C-10), 114.7 (C-30, C-50), 125.5 (C-10), 126.7 (C-3), 131.6 (C-20,
C-60), 153.3 (C-2), 160.6 (C-5), 160.9 (C-9), 161.1 (C-40), 164.7 (C-7), 178.1 (C-4). HRESI-MS [M
−
H]−
(calculated/found) (m/z 445.1140/445.1151).
0
7-O-
β
-D-4”-O-Methyl-glucopyranosyl-5-hydroxy-4 -methoxyisoflavone (4”-O-methylsissotrin), (biocatalyst:
1
Beauveria bassiana AM 278, yield: 30.8%): H-NMR (600 MHz, CD3OD) δ: 3.21 (1H, m, H-4”), 3.46–3.53
(2H, m, H-2”, H-5”), 3.60 (1H, m, H-3”), 3.60 (3H, s, 4”-OCH3), 3.72 (1H, m, Hb-6”), 3.83 (3H, s,
40-OCH3), 3.87 (1H, m, Ha-6”), 5.03 (1H, d, J = 7.7 Hz, H-1”), 6.52 (1H, m, H-6), 6.70 (1H, m, H-8), 6.99
(2H, m, H-30, H-50), 7.49 (2H, m, H-20, H-60), 8.17 (1H, s, H-2); 13C-NMR (150 MHz, CD3OD)
δ: 55.8
(C-40OCH3), 60.9 (C-4”OCH3), 62.0 (C-6”), 74.8 (C-2”), 77.4 (C-5”), 77.9 (C3”), 80.5 (C-40”), 95.9 (C-8),
0
0
0
0
101.1 (C-6), 101.5 (C-1”), 108.1 (C-10), 115.0 (C-3 , 5 ), 124.4 (C-1 ), 124.9 (C-3), 131.4 (C-2 , 6 ), 155.5 (C-2),
157.3 (C-9), 161.4 (C-40), 163.6 (C-5), 164.8 (C-7), 182.4 (C-4). HRESI-MS [M
−
H]− (calculated/found)
(m/z 459.1296/459.1297).
4. Conclusions
The aim of this paper was to develop a simple and efficient method for the preparation
isoflavonoid glycosides, which can serve as analytical standards or model compounds for various
biological tests.
All the microorganisms that we used performed conjugation of isoflavones with the sugar
molecule. Fungi Absidia glauca AM 177 and Absidia coerulea AM 93 attached glucose, while the
fungus Beauveria bassiana AM 278 4-O-methylglucose. With one exception, the preferred site of
glycosylation was the C-7 hydroxyl group of substrates. Thus, as a result of glucosylation of genistein
and Biochanin A, we obtained genistin and sissotrin, respectively. In the culture of fungi Absidia coerulea,
Biochanin A was converted to a new derivative with glucose residue at the C-5 position. To our best
knowledge, this compound has not been published so far. The product of the biotransformation of
Biochanin A by Beauveria bassiana AM 278 is also reported for the first time.
Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/21/
1/81/s1.
Acknowledgments: Publication supported by Wrocław Centre of Biotechnology, programme the Leading
Notional Research Centre (KNOW) for years 2014–2018.
Author Contributions: Ewa Huszcza and Sandra Sordon conceived and designed the experiments; Sandra Sordon
performed the experiments; Jarosław Popłon´ski analyzed the data and interpreted the NMR spectra;
Tomasz Tronina isolated the isoflavonoids from soy extract; Sandra Sordon and Ewa Huszcza wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds daidzein, 4”-O-methyldaidzin, Biochanin A, sissotrin and
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2017 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
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(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).