Biotransformations of flavones and an isoflavone (daidzein) in cultures of entomopathogenic filamentous fungi

Article abstract of DOI:10.3390/molecules23061356

Entomopathogenic filamentous fungi of the genus Isaria are effective biocatalysts in the biotransformation of flavonoids as well as steroids. In the present study, the species Isaria fumosorosea and Isaria farinosa isolated from the environment were used. Their catalytic capacity to carry out biotransformations of flavones—unsubstituted, with hydroxy- and amino-substituents as well as a hydroxylated isoflavone—was investigated. Biotransformations of flavone, 5-hydroxyflavone, 6-hydroxyflavone, 7-hydroxyflavone, and daidzein resulted in the formation of O-methylglucosides, in the case of flavone and 5-hydroxyflavone with additional hydroxylations. 7-Aminoflavone was transformed into two acetamido derivatives. The following products were obtained: From flavone–flavone 2-O-β-D-(4-O-methyl)-glucopyranoside, flavone 4-O-β-D-(4-O-methyl)-glucopyranoside and 3-hydroxyflavone 4-O-β-D-(4-O-methyl)-glucopyranoside; from 5-hydroxyflavone–5-hydroxyflavone 4-O-β-D-(4-O-methyl)-glucopyranoside; from 6-hydroxyflavone–flavone 6-O-β-D-(4-O-methyl)-glucopyranoside; from 7-hydroxyflavone–flavone 7-O-β-D-(4-O-methyl)-glucopyranoside; from daidzein–daidzein 7-O-β-D-(4-O-methyl)-glucopyranoside; and from 7-aminoflavone–7-acetamidoflavone and 7-acetamido-4-hydroxyflavone. Seven of the products obtained by us have not been previously reported in the literature.

Full text of DOI:10.3390/molecules23061356

molecules
Article
Biotransformations of Flavones and an Isoflavone
(Daidzein) in Cultures of Entomopathogenic
Filamentous Fungi
ID
Monika Dymarska * ID , Tomasz Janeczko and Edyta Kostrzewa-Susłow
Department of Chemistry, Faculty of Biotechnology and Food Science, Wrocław University of Environmental
and Life Sciences, 50-375 Wrocław, Poland; janeczko13@interia.pl (T.J.); ekostrzew@gmail.com (E.K.-S.)
* Correspondence: monika.dymarska@gmail.com
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 20 April 2018; Accepted: 1 June 2018; Published: 5 June 2018
Abstract: Entomopathogenic filamentous fungi of the genus Isaria are effective biocatalysts
in the biotransformation of flavonoids as well as steroids. In the present study, the species
Isaria fumosorosea and Isaria farinosa isolated from the environment were used. Their catalytic
capacity to carry out biotransformations of flavones—unsubstituted, with hydroxy- and
amino-substituents as well as a hydroxylated isoflavone—was investigated. Biotransformations
of flavone, 5-hydroxyflavone, 6-hydroxyflavone, 7-hydroxyflavone, and daidzein resulted in the
formation of O-methylglucosides, in the case of flavone and 5-hydroxyflavone with additional
hydroxylations. 7-Aminoflavone was transformed into two acetamido derivatives. The following
products were obtained: From flavone–flavone 20-O-
β
-D-(4000-O-methyl)-glucopyranoside, flavone
0
00
0
00
4 -O-
from
β
-D-(4 -O-methyl)-glucopyranoside and 3 -hydroxyflavone 4 -O-
β
-D-(4 -O-methyl)-glucopyranoside;
5-hydroxyflavone–5-hydroxyflavone
40-O-
β
-D-(400-O-methyl)-glucopyranoside;
from
6-hydroxyflavone–flavone 6-O-
β
-D-(400-O-methyl)-glucopyranoside; from 7-hydroxyflavone–flavone
00
00
7-O-β
-D-(4 -O-methyl)-glucopyranoside; from daidzein–daidzein 7-O-
β
-D-(4 -O-methyl)-glucopyranoside;
and from 7-aminoflavone–7-acetamidoflavone and 7-acetamido-40-hydroxyflavone. Seven of the products
obtained by us have not been previously reported in the literature.
Keywords: biotransformations; flavone; daidzein; 5-hydroxyflavone; 6-hydroxyflavone;
7-hydroxyflavone; 7-aminoflavone; Isaria fumosorosea; Isaria farinosa
1. Introduction
Flavonoid compounds are used in pharmacy [
in the prevention and treatment of a number of diseases, including civilization diseases, is confirmed
by many studies both in vitro on cell lines and in vivo ]. However, it is not certain whether
1] and the food industry [2], and their effectiveness
[3–8
the substance taken orally reaches its destination in an unchanged form, whether it undergoes
biotransformation in the body and whether the acting therapeutic compound is its metabolite [8,9].
The limitation in the use of many flavonoid compounds with high biological activity proved
in vitro is their low bioavailability resulting inter alia from poor solubility in water [
compounds in the small intestine are absorbed only to a small extent (5–10%) [
flavonoid compounds that are taken along with the diet pass to the large intestine, where they undergo
intense bacterial metabolism [ 12]. The effect of these transformations is often the creation of products
7,
10
,11]. Flavonoid
9
]. The majority of
9
,
with higher activity than the starting compound. This is the case of soy isoflavones, which can be
metabolized to equol—a compound with significantly higher estrogenic activity [13].
Flavonoids in plants are mainly in the form of glycosides. Thermal processing of food does not
damage glycosidic bonds, and therefore flavonoid glycosides predominate in products of plant origin
Molecules 2018, 23, 1356; doi:10.3390/molecules23061356
www.mdpi.com/journal/molecules

Molecules 2018, 23, 1356
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taken with the diet. The issue of absorption of sugar glycoside combinations remains unexplained.
There are reports of possible absorption of flavonoid glycosides in the small intestine. However,
many studies have shown that these compounds reach the large intestine in unchanged form and only
after glycosidic bonds are broken by the intestinal microflora can they be absorbed [14–18].
The preparation of flavonoid glycosides by chemical synthesis is often impossible due to
the drastic conditions leading to the degradation of substrates, the yields of the process are low,
and undesirable anomers are formed [19]. Biotransformations are considered to be an environmentally
friendly alternative that allows modification of the structure of compounds to improve their
properties [20]. The number of literature reports on the microbiological production of flavonoid
sugar derivatives is on the rise. The use of microorganisms makes it possible to obtain compounds
with high regio- and stereoselectivity. Many flavonoid glycosides that are naturally present in plants
in small amounts and are difficult to isolate along with novel compounds can already be effectively
obtained from flavonoid aglycones by using microorganisms as biocatalysts [21–25].
Isaria fumosorosea is an entomopathogenic filamentous fungus with a wide range of possible
applications. Microorganisms of the species I. fumosorosea are used in agriculture as a natural
alternative to chemical pesticides [26,27]. Fumosorinone synthesized by I. fumosorosea has an
anti-diabetic effect [28]. In addition, research carried out by our team has shown that the strain
of this fungus, isolated from a spider from urban green areas in Wroclaw, is an excellent biocatalyst
in the biotransformations of flavonoid compounds and steroids by carrying out reactions not known
in the literature [
1
,
29]. For the first time, we described the transformation in which the sugar unit is
]. Additionally, we had
attached to the flavonoid aglycone lacking hydroxyl groups in its structure [
1
13 strains of Isaria farinosa fungi isolated from Lower Silesia adits. So far, we have described catalytic
abilities in steroid biotransformations of several strains from the I. farinosa species [30]. Initial studies
indicate that these strains were also efficient biocatalysts in flavonoid biotransformations.
The main aim of this study was to evaluate the catalytic capacity of Isaria fumosorosea KCH J2
I. farinosa J1.4, I. farinosa J1.6 and I. farinosa KW1.2 to carry out biotransformations of flavone,
5-hydroxyflavone, 6-hydroxyflavone, 7-hydroxyflavone, daidzein, and 7-aminoflavone. For all
substrates used, flavonoid O-methylglucosides were formed, but not for 7-aminoflavone which resulted
in formation of acetamido derivatives.
,
2. Results and Discussion
The present results are a continuation of research into the microbiological transformations
of flavonoid compounds in cultures of entomopathogenic filamentous fungi.
In our
previous publication, we presented a biotransformation of 6-methylflavone in I. fumosorosea
KCH J2 culture. As a result of 7-day biotransformation, we obtained two new products:
6-methylflavone 8-O-β
-D-(400-O-methyl)-glucopyranoside (6.6% yield) and 6-methylflavone
40-O-β-D-(400-O-methyl)-glucopyranoside (13.2% yield) [1].
In the present paper, we used six flavonoid substrates and four strains of entomopathogenic
filamentous fungi that were isolated from the environment. Detailed characteristics of the strains we
used can be found in our previous publications [1,30].
In screening, the only strain that carried out biotransformations of all selected substrates was
I. fumosorosea KCH J2. The yields obtained as a result of the use of this strain were the highest;
therefore, it was chosen as a biocatalyst for scaled-up biotransformations. Experiments conducted on
a larger scale enabled us to determine the chemical structures of products and their isolated yields.
In the course of our study, we obtained nine biotransformation products, seven of which (flavone
20-O-
β
-D-(400-O-methyl)-glucopyranoside (1a), flavone 40-O-
-D-(400-O-methyl)-glucopyranoside
-D-(400-O-methyl)-glucopyranoside (2a), flavone 6-O- -D-(400-O-methyl)-glucopyranoside (3a),
-D-(400-O-methyl)-glucopyranoside (4a), and 40-hydroxy-7-acetamidoflavone (6b) have
β
-D-(400-O-methyl)-glucopyranoside
(
1b), 30-hydroxyflavone 40-O-
β
(1c), 5-hydroxyflavone
40-O-
β
β
flavone 7-O-
β
not been previously described in the literature.

Molecules 2018, 23, 1356
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2.1. Biotransformations of Flavone (1)
As a result of 6-day biotransformation of flavone (
1
) in the culture of I. fumosorosea KCH
J2 we obtained three products: Flavone 20-O- -D-(400-O-methyl)-glucopyranoside (1a) with 5.5%
β
yield, flavone 40-O-
β
-D-(400-O-methyl)-glucopyranoside (1b) with 14% yield, and 30-hydroxyflavone
40-O- -D-(400-O-methyl)-glucopyranoside (1c) with 4% yield (Scheme 1). Compound 1b was also
β
formed when we used I. farinosa J1.6 as a biocatalyst.
OH
H3C
O
O
HO
O
OH
O
HO
HO
O
CH3
3'
6'
O
O
(
1a
)
O
2'
4'
5'
OH
HO
8
5
O
O
O
7
6
I. fumosorosea KCH J2
3
6 days
OH
O
CH3
O
O
(1)
O
(
1b
)
OH
HO
O
O
(1c)
Scheme 1. Microbial transformation of flavone (1) in I. fumosorosea KCH J2 culture.
The presence of a glucose unit in molecules 1a
carbon signals observed in the region from about = 80.0 ppm to about
spectra, along with proton signals of H ranging from about = 3.90 ppm to
1H-NMR spectra. Additionally, the attachment of a sugar unit to substrate
one-proton doublet visible at
= 5.23 ppm in the 1H-NMR spectrum of 1a = 5.14 ppm in the 1H-NMR
spectrum of 1b and
= 5.00 ppm in the 1H-NMR spectrum of 1c, which are due to protons at the
hemiacetal carbon atoms. The -configuration of the glucose unit was proved by the coupling constant
(J = 7.6 Hz/J = 7.7 Hz/J = 7.8 Hz) for the anomeric proton. A three-proton singlet at about δ = 3.60 ppm
in the 1H-NMR and the corresponding signal at about = 60.6 ppm in the 13C-NMR proved that one
,
1b, and 1c was confirmed by five characteristic
= 62.0 ppm in the 13C-NMR
= 3.29 ppm in the
was confirmed by a
δ
δ
δ
δ
δ
1
δ
, δ
δ
β
δ
of the hydroxyl groups had been methylated. O-methylation occurred in the C-4 hydroxyl group of
the glucose moieties, which was confirmed in the correlation spectra, where the proton signals due
to -OCH3 were coupled with the signal of C-4 (about
of compound 1a, the sugar unit was attached to C-20, because in the correlation spectrum the signal
due to the proton at the hemiacetal carbon atom (
= 5.23 ppm) was coupled with the C-20 signal
δ = 156.7 ppm), which was shifted from = 127.2 ppm, indicating the attachment of an electronegative
δ = 80.0 ppm) in the glucose unit. In the case
δ
(
δ
atom. In addition, protons at C-30 and C-50 become non-equivalent, two triplets can be observed
from protons at C-40 ( 0
δ
= 7.57 ppm) and C-50 (
δ
= 7.27 ppm) and two doublets from protons at C-30
0
(δ = 7.44 ppm) and C-6 (
δ
= 7.99 ppm). In the case of 1b, the sugar unit was attached to C-4 , because in
the correlation spectrum the signal due to the proton at the hemiacetal carbon atom (
δ = 5.14 ppm) was
coupled with the C-40 signal (
δ
= 161.4 ppm), which was shifted from = 132.5 ppm. The substitution
δ
in position 40 was also evidenced by the characteristic two doublets in the 1H-NMR spectrum from
protons at C-20 and C-60 (
δ
= 8.09 ppm) as well as C-30 and C-50 (
δ
= 7.28 ppm). In the case of 1c
,
0
the sugar unit was attached to C-4 , because in the correlation spectrum the signal due to the proton at
the hemiacetal carbon atom (
was shifted from
= 132.5 ppm. The substitution at the carbon 30 was evidenced by the 1H-NMR
δ δ = 148.9 ppm), which
= 5.14 ppm) was coupled with the C-40 signal (
δ

Molecules 2018, 23, 1356
4 of 15
spectrum of 1c: The signal from the proton at C-20 was visible as a singlet and there is no signal from
the proton at C-30. Hydroxylation was assumed because of the shift of the C-30 carbon signal from
δ = 129.0 to δ
= 148.7. Chemical shifts of the other signals in the 1H and 13C-NMR spectra have only
slightly changed, which indicates that the flavone skeleton remained intact.
2.2. Biotransformations of 5-Hydroxyflavone (2)
As a result of 7-day biotransformation of 5-hydroxyflavone (
2) in the culture of I. fumosorosea
KCH J2, we obtained 5-hydroxyflavone 40-O- -D-(400-O-methyl)-glucopyranoside (2a) with 29% yield
β
(Scheme 2). Compound 2a was also formed when we used I. farinosa J1.4 as a biocatalyst.
HO
CH3
O
O
OH
O
HO
O
O
O
I. fumosorosea KCH J2
7 days
OH
OH
O
(2)
(
2a)
Scheme 2. Microbial transformation of 5-hydroxyflavone (2) in I. fumosorosea KCH J2 culture.
The presence of a glucose unit in molecule 2a was confirmed by five characteristic carbon signals
observed in the region from
δ
= 80.1 ppm to
δ
= 62.0 ppm in the 13C-NMR spectra, along with
1
proton signals of H ranging from about
δ
δ
= 3.89 ppm to
δ
= 3.27 ppm in the H-NMR spectra.
The attachment of a sugar unit to substrate
2
was also confirmed by a one-proton doublet visible at
1
δ
= 5.15 ppm in the H-NMR spectrum of 2a, which was due to protons at the hemiacetal carbon
atoms. The
anomeric proton. A three-proton singlet at
at
= 60.6 ppm in the 13C-NMR indicates the O-methylation of the hydroxyl group at C-4 of glucose,
which was confirmed on the basis of the analysis of the correlation spectra of compound 2a, where
the proton signals due to -OCH3 ( = 3.61 ppm) were coupled with the signal of C-4 (δ = 80.1 ppm) in
the glucose unit. The sugar unit was attached to C-40, because in the correlation spectrum the signal
due to the proton at the hemiacetal carbon atom (
= 5.15 ppm) was coupled with the C-40 signal
β
-configuration of the glucose unit was proved by the coupling constant (J = 7.7 Hz) for the
δ
= 3.61 ppm in the 1H-NMR and the corresponding signal
δ
δ
δ
(
δ = 161.8 ppm). There was no signal from the proton at C-40 in the 1H-NMR spectrum. Two doublets
from the protons at C-20 and C-60 (δ = 8.10 ppm) and C-30 and C-50 (δ = 7.29 ppm) can be observed.
2.3. Biotransformations of 6-Hydroxyflavone (3)
As a result of 5-day biotransformation of 6-hydroxyflavone (
we obtained 6-O-
-D-(400-O-methyl)-glucopyranoside (3a) with 13% yield (Scheme 3). Product 3a was
also formed when we used I. farinosa J1.6 and I. farinosa KW1.2 as biocatalysts.
3) in the culture of I. fumosorosea KCH J2,
β
OH
O
O
H3C
O
O
HO
I. fumosorosea KCH J2
O
5 days
OH
HO
O
O
(3)
(3a
)
Scheme 3. Microbial transformation of 6-hydroxyflavone (3) in I. fumosorosea KCH J2 culture.
The presence of a glucose unit in molecule 3a was confirmed by five characteristic carbon signals
observed in the region from = 79.9 ppm to
= 61.9 ppm in the 13C-NMR spectra and proton signals
δ
δ

Molecules 2018, 23, 1356
5 of 15
from about
a sugar unit to substrate
spectrum of 3a, which was due to protons at the hemiacetal carbon atoms. The
glucose unit was proved by the coupling constant (J = 7.8 Hz) for the anomeric proton. A three-proton
singlet at
= 3.62 ppm in the 1H-NMR and the corresponding signal at = 60.6 ppm in the 13C-NMR
δ
= 3.89 ppm to
δ
= 3.31 ppm in the 1H-NMR spectra. Additionally, the attachment of
3
was confirmed by a one-proton doublet at δ
= 5.11 ppm in the 1H-NMR
β-configuration of the
δ
δ
prove that one of the hydroxyl groups was methylated. O-methylation occurred in the C-4 hydroxyl
group of the glucose moiety, which was confirmed in the correlation spectra of compound 3a, where
the proton signals due to -OCH3 (
δ = 3.62 ppm) were coupled with the signal of C-4 (δ = 79.9 ppm)
in the glucose unit. The sugar unit was attached to C-6, because in the correlation spectrum the
signal due to the proton at the hemiacetal carbon atom (
δ
= 5.11 ppm) was coupled with the C-6
1
signal (δ = 156.0 ppm). The signals in the H spectrum from the protons at C-5, C-7 and C-8 were
shifted towards lower field values, indicating the appearance of a deactivating (electron-withdrawing)
substituent in the A ring of flavonoid.
2.4. Biotransformations of 7-Hydroxyflavone (4)
As a result of 7-day biotransformation of 7-hydroxyflavone (
4
) in the culture of I. fumosorosea
00
KCH J2, we obtained flavone 7-O-β-D-(4 -O-methyl)-glucopyranoside (4a) with 16% yield (Scheme 4).
Product 4a was also formed when we used I. farinosa J1.4 and I. farinosa J1.6 as biocatalysts.
OH
H3C
O
O
HO
O
HO
O
O
I. fumosorosea KCH J2
OH
7 days
O
O
(4)
(4a)
Scheme 4. Microbial transformation of 7-hydroxyflavone (4) in I. fumosorosea KCH J2 culture.
The presence of a glucose unit in molecule 4a was confirmed by five characteristic carbon signals
observed in the region from
signals of δH ranging from about δ = 3.92 ppm to δ = 3.30 ppm in the 1H-NMR spectra. Additionally,
the attachment of a sugar unit to substrate was confirmed by a one-proton doublet at = 5.24 ppm in
the 1H-NMR spectrum of 4a from protons at the hemiacetal carbon atoms. The
-configuration of the
glucose unit was proved by the coupling constant (J = 7.7 Hz) for the anomeric proton. A three-proton
singlet at
= 3.62 ppm in the 1H-NMR and the corresponding signal at = 60.6 ppm in the 13C-NMR
δ = 80.0 ppm to δ
= 61.9 ppm in the 13C-NMR spectra, along with proton
4
δ
β
δ
δ
proved that one of the hydroxyl groups were methylated. O-methylation occurred in the C-4 hydroxyl
group of the glucose moiety, which was confirmed in the correlation spectra of compound 4a, where
the proton signals due to -OCH3 (
δ = 3.62 ppm) were coupled with the signal of C-4 (δ = 80.0 ppm)
in the glucose unit. The attachment of a sugar unit to C-7 was proved in the correlation spectrum.
The signal due to the proton at the hemiacetal carbon atom (
δ
= 5.24 ppm) was coupled with the
1
C-7 signal (
δ
= 162.9 ppm). The signals in the H spectrum from the protons at C-6 and C-8 were
shifted towards lower field values, suggesting the appearance of a strongly electronegative substitute
in their vicinity.
2.5. Biotransformations of Daidzein (5)
As a result of 7-day biotransformation of daidzein (
5) in the culture of I. fumosorosea KCH J2 we
obtained 40-hydroxyisoflavone 7-O- -D-(400-O-methyl)-glucopyranoside (400-O-methyldaidzin) (5a
β
)
with 14.8% yield (Scheme 5). Product 5a was also formed when we used I. farinosa J1.6 and I. farinosa
KW1.2 as biocatalysts.

Molecules 2018, 23, 1356
6 of 15
OH
H3C
O
O
O
HO
O
O
O
HO
KCH J2
I. fumosorosea
7 days
OH
O
OH
OH
(5)
(
5a
)
Scheme 5. Microbial transformation of daidzein (5) in I. fumosorosea KCH J2 culture.
The presence of a glucose unit in molecule 5a was confirmed by five characteristic carbon signals
observed in the region from
proton signals of H ranging from about
Additionally, the attachment of a sugar unit to substrate
visible at δ = 5.21 ppm in the 1H-NMR spectrum of 5a that came from protons at the hemiacetal carbon
atoms. The -configuration of the glucose unit was proved by the coupling constant (J = 7.7 Hz) for
the anomeric proton. A three-proton singlet at
= 3.62 ppm in the 1H-NMR and the corresponding
signal at
= 60.6 ppm in the 13C-NMR proved that one of the hydroxyl groups had been methylated.
O-methylation occurred in the C-4 hydroxyl group of the glucose moiety, which was confirmed in the
correlation spectra of compound 5a, where the proton signals coming from-OCH3 ( = 3.62 ppm) were
coupled with the signal of C-4 ( = 80.0 ppm) in the glucose molecule. The sugar unit was attached
δ
= 80.0 ppm to
δ
= 62.0 ppm in the 13C-NMR spectra, along with
1
δ
δ
= 3.92 ppm to
δ
= 3.28 ppm in the H-NMR spectra.
5
was confirmed by a one-proton doublet
β
δ
δ
δ
δ
to C-7, because in the correlation spectrum, the signal due to the proton at the hemiacetal carbon atom
(δ = 5.21 ppm) was coupled with the C-7 signal (δ = 162.6 ppm).
2.6. Biotransformations of 7-Aminoflavone (6)
As a result of 7-day biotransformation of 7-aminoflavone (
we obtained two products: 7-acetamidoflavone (6a) with 10% yield and 4 -hydroxy-7-acetamidoflavone
(6b) with 3% yield (Scheme 6).
6
) in the culture of I. fumosorosea KCH J2
0
O
CH3
N
O
H
O
H2N
O
O
(
6a
)
KCH J2
I. fumosorosea
7 days
O
H
CH3
OH
N
O
(6)
O
(6b)
Scheme 6. Microbial transformation of 7-aminoflavone (6) in I. fumosorosea KCH J2 culture.
1
In the case of compound 6a
corresponds to the -NH proton attached to C-7 and was not present in the spectrum of the substrate (6)
For compound , the signal of the two amino protons was observed at
= 5.82 ppm in the 1H-NMR
spectrum. In the HMBC spectrum, a -CH3 signal, absent in the case of the substrate (singlet at
δ = 2.21 ppm on 1H-NMR and = 24.5 ppm for 6b) on 13C-NMR) correlated with the
= 24.5 ppm (
signal at = 169.9 ppm ( = 169.8 ppm for 6b) from the carbonyl carbon of the acetamide group.
(6b), a broad singlet at δ = 9.78 ppm (δ = 9.73) in the H-NMR
.
6
δ
δ
δ
δ
δ
Carbonyl carbon from the acetamide group correlated with the signal at
δ = 7.46 ppm that came
from the proton at C-6. The signals in the 1H spectrum from the protons at C-5, C-6 and C-8 were
shifted towards lower field values, suggesting the appearance of a deactivating substituent in their

Molecules 2018, 23, 1356
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vicinity. In addition, the 1H-NMR spectrum of compound 6b showed a characteristic pattern of signals
indicating substitution at position 40: Two two-proton signals at
= 8.00 and = 7.08, from protons
at C-30 and C-50 and at C-20 and C-60, the signal from the proton at C-40 was also no longer visible.
The C-40 carbon signal was shifted from = 161.6 in the 13C-NMR spectrum, which
= 131.9 to
indicates the attachment of a strongly electronegative atom. A broad singlet at = 9.27 ppm from the
δ
δ
δ
δ
δ
proton of the hydroxyl group can be observed in the 1H-NMR spectrum. Chemical shifts of the other
signals in the 1H and 13C-NMR spectra have only slightly changed, which indicates that the flavone
skeleton remained intact.
The I. fumosorosea KCH J2 strain was characterized by low substrate specificity; it was capable
of catalyzing biotransformations of flavones with hydroxyl, methyl, and amino substituents as well
as unsubstituted flavone and hydroxylated isoflavone. Biotransformations of flavone confirmed our
earlier observations: the sugar derivative of the flavonoid compound can be formed despite the lack of
a hydroxyl group in its structure [1]. I. fumosorosea KCH J2 was to date the only strain found to possess
this unique capability. Our present study proves that I. farinosa J1.6 is also capable of performing
such bioconversion.
In the case of hydroxy derivatives of flavonoids, attachment of the sugar unit to the
hydroxyl group takes place, but not for 5-hydroxyflavone. This may be due to the proximity
of the carbonyl oxygen, which is a steric hindrance and prevents the flavonoid substrate
fitting into a pocket in the enzyme’s active site so that the compound can be functionalized
in position 5. It may also be due to the formation of hydrogen bonds between the hydroxyl
group present in the 5-position and the oxygen from the carbonyl group of the substrate.
In the available literature, there are no reports on microbial glycosylation in the 5-position of
popular flavones, such as apigenin (5,7,40-trihydroxyflavone), chrysin (5,7-dihydroxyflavone),
baicalein (5,6,7-trihydroxyflavone), or wogonin (5,7-dihydroxy-8-methoxyflavone).
In the
case of quercetin (3,5,7,30,40-pentahydroxyflavone) in the culture of Cunninghamella elegans
ATCC 9245 the glucose unit was attached in the 3 position [31], the same as when
Gliocladium deliquescens NRRL 1086 was used as a biocatalyst in biotransformations of
0
0
0
quercetin, kaempferol (3,5,7,4 -tetrahydroxyflavone), morin (3,5,7,2 ,4 -pentahydroxyflavone), galangin
(3,5,7-trihydroxyflavone) and myricetin (3,5,7,30,40,50-hexahydroxyflavone) [23]. Usually when a
microbiological catalyst is used, the sugar unit is attached to the flavonoid aglycone in position
3 or 7 [23,25]. A rare example of attaching glucose in the 5 position of flavonoid aglycone was
biotransformation of naringenin (5,7,40-trihydroxyflavanone) in the plant culture of Eucalyptus
perriniana. The research reports the formation of several glycosides, e.g., when the sugar is attached to
hydroxyl groups in positions 5 and 7 [32]. Flavones, unlike flavanones, have a double bond between
C2 and C3. The presence of this bond provides the molecule with planarity and is of great importance
for interaction with enzymes and the biological activity exhibited by the compounds [7,33].
400-O-methyldaidzin (5a) was obtained earlier as a result of biotransformation of daidzein in the
culture of Beauveria bassiana AM 278 [34] as well as from a soybean broth fermented by Paecilomyces
militaris [35]. Both strains used as biocatalysts belong to entomopathogenic filamentous fungi.
7-Aminoflavone was the only substrate for which we did not observe the formation of a glucoside
derivative. The only example of biotransformation of 7-aminoflavone available in the literature to
date is the transformation of the substrate into 7-hydroxyflavone in the filamentous fungi cultures
of Aspergillus niger SBJ and Aspergillus oryzae 13/5 described by us [36]. The acetamido derivative
of aminoflavone was obtained through the microbiological transformation of 6-aminoflavone in the
cultures of Rhodococcus sp. DSM 364 and Gordonia sp. DSM 44456 [37]. The method for the preparation
of 7-acetamidoflavone is known by chemical synthesis from 4-acetamido-2-hydroxydibenzoylmethane.
The substrate dissolved in cold concentrated sulfuric acid was left for two hours. The product obtained
after the addition of ice crystallized from the alcohol in the form of white needles [38].
Flavonoid glycosides are usually far more expensive than their parent aglycones [39]. This is due
to the lack of economically viable methods for obtaining those derivatives via chemical or enzymatic

Molecules 2018, 23, 1356
8 of 15
synthesis, which we have discussed more broadly in our previous paper [
1]. In this study we present a
cheap, efficient, and environmentally friendly method for obtaining flavonoid methylglucosides from
various flavonoid substrates. The next goal we set for ourselves is to attempt to increase the efficiency
of the biotransformations described by us. We plan to check how the earlier addition of substrate to
the culture of the microorganism will change the amount of products being formed, as was the case
with the cultures of the genera Aspergillus and Penicillium [20,40]. We will also determine the optimal
temperature and composition of the culture medium for the process, in order to conduct large-scale
cultures in the bioreactor at the final stage. The next step will be an attempt to thoroughly examine the
enzymes involved in the biotransformations described by us.
3. Materials and Methods
3.1. Chemicals
The substrates for biotransformations except for daidzein were purchased from Sigma Chemical
Company (St. Louis, MO, USA).
Daidzein was obtained via column chromatography of soybean (Glycine max) extract. Column
chromatography (SiO2, Kieselgel 60, 230–400 mesh, 40–63 µm, Merck, Darmstadt, Germany) was
performed using ethyl acetate: methylene chloride (1:1) mixture as an eluent.
3.1.1. Flavone (1)
C15H10O2, tR 18.99; 1H-NMR, see Table 1; 13C-NMR, see Table 2.
3.1.2. 5-Hydroxyflavone (2)
C15H10O3, tR 22.98; 1H-NMR, see Table 3; 13C-NMR, see Table 4.
3.1.3. 6-Hydroxyflavone (3)
C15H10O3, tR 15.25; 1H-NMR, see Table 3; 13C-NMR, see Table 4.
3.1.4. 7-Hydroxyflavone (4)
C15H10O3, tR 14.33; 1H-NMR, see Table 3; 13C-NMR, see Table 4.
3.1.5. 40,7-Dihydroxyisoflavone (Daidzein) (5)
C15H10O4, tR 11.32; 1H-NMR, see Table 3; 13C-NMR, see Table 4.
3.1.6. 7-Aminoflavone (6)
C15H11NO2, tR 13.56; 1H-NMR, see Table 1; 13C-NMR, see Table 2.
Table 1. 1H-NMR shifts (
δ) of flavone (1) and 7-aminoflavone (6) and products of their
biotransformations (1a, 1b, 1c, 6a, 6b) in Acetone-d6, 600 MHz (Supplementary Materials).
Compound
Proton
1
1a
1b
1c
6
6a
6b
H-3
H-5
6.91 (s)
7.15 (s)
6.83 (s)
6.81 (s)
6.69 (s)
6.84 (s)
6.70 (s)
8.17 (dd)
J5,6 = 7.9,
J = 1.6
8.16 (d)
J5,6 = 8.0
8.15 (d)
J5,6 = 7.8
8.15 (d)
J5,6 = 7.8
7.86 (d)
J5,6 = 8.4
8.05 (d)
J5,6 = 8.6
8.03 (d)
J5,6 = 8.6
7.46 (dd)
J6,5 = 8.6,
J6,8 = 2.0
7.42 (dd)
J6,5 = 8.6,
J6,8 = 1.9
7.54 (t)
J = 7.5
7.52 (t)
J = 7.5
7.52 (t)
J = 7.4
7.52 (t)
J = 7.4
6.83 (d)
J8,6 = 2.0
H-6
H-7
7.84 (t)
J = 7.7
7.84 (t)
J = 7.7
7.85 (t)
J = 7.8
7.86 (m)
-
-
-

Molecules 2018, 23, 1356
9 of 15
Table 1. Cont.
Compound
Proton
1
1a
1b
1c
6
6a
6b
7.79 (d)
J8,7 = 8.4
7.73 (d)
J8,7 = 8.4
7.76 (d)
J8,7 = 8.4
7.79 (d)
J8,7 = 8.4
6.82 (t)
J = 2.7
8.40 (d)
J8,6 = 1.9
8.37 (d)
J8,6 = 1.8
H-8
8.09 (d)
8.05 (dd)
J = 6.6, 3.0
0
H-2
8.14 (m)
-
7.63 (s)
-
8.13 (m)
7.08 (m)
0
0
J2 ,3 = 7.9
7.44 (d)
J = 8.3
7.28 (d)
7.61 (dd)
J = 6.5, 3.5
0
H-3
7.65 (m)
7.64 (m)
8.00 (m)
0
0
J3 ,2 = 7.9
7.57 (t)
J = 7.7
7.61 (dd)
J = 6.5, 3.5
0
H-4
7.65 (m)
-
-
7.64 (m)
-
7.28 (d)
7.27 (t)
J = 7.5
7.38 (d)
7.61 (dd)
J = 6.5, 3.5
0
H-5
7.65 (m)
7.64 (m)
8.00 (m)
0
J5 = 7.9
0
0
J5 ,6 = 8.3
0
,6
7.99 (d)
J = 7.8
8.09 (d)
7.60 (d)
8.05 (dd)
J = 6.6, 3.0
0
H-6
8.14 (m)
8.13 (m)
7.08 (m)
0
0
0
J6,5 = 7.9
J6 ,5 = 8.4
5.23 (d)
J = 7.6
5.14 (d)
J = 7.7
5.00 (d)
J = 7.8
00
H-1
-
-
-
-
-
-
-
-
-
-
-
-
3.56 (t)
J = 8.4
00
H-2
3.61 (m)
3.69 (m)
3.58 (m)
3.70 (t)
J = 9.0
3.71 (t)
J = 9.1
00
H-3
3.29 (t)
J = 9.2
3.28 (t)
J = 9.3
3.29 (t)
J = 9.1
00
H-4
-
-
-
-
-
-
-
-
00
H-5
3.61 (m)
3.59 (m)
3.58 (m)
3.89 (d)
J = 12.2,
3.74 (dd)
3.90 (d)
J = 11.9,
3.75 (dd)
3.91 (d)
J = 11.5,
3.76 (dd)
00
H-6
-
-
-
-
J = 13.6, 3.9 J = 11.6, 4.0 J = 11.3, 3.8
00
C-4 -O
CH3
-
-
-
-
-
3.61 (m)
3.62 (s)
3.61 (s)
-
-
-
-COCH3
-NH2
-NH-
-
-
-
-
-
-
-
-
-
-
-
-
-
2.21 (s)
2.21 (s)
-
5.82 (s)
-
-
-
9.78 (s)
-
9.73 (s)
9.27 (s)
-OH
Table 2. 13C-NMR shifts (
δ) of flavone (1) and 7-aminoflavone (6) and products of their
biotransformations (1a, 1b, 1c, 6a, 6b) in Acetone-d6, 600 MHz (Supplementary Materials).
Compound
Carbon
1
1a
1b
1c
6
6a
6b
C-2
C-3
C-4
C-4a
C-5
C-6
C-7
C-8
C-8a
163.9
108.0
178.0
124.8
126.0
126.2
134.9
119.3
157.1
132.8
127.2
130.0
132.5
130.0
127.2
-
161.6
113.4
178.3
124.7
125.9
125.9
134.7
119.3
157.5
122.6
156.7
116.7
133.3
123.0
130.1
101.6
74.7
163.8
106.8
177.8
124.8
126.0
126.0
134.7
119.2
157.1
126.2
128.8
117.7
161.4
117.7
128.8
101.2
74.8
163.7
107.2
177.8
124.8
125.9
126.0
134.7
119.2
157.1
127.9
114.5
148.7
148.9
118.3
119.3
107.2
74.7
162.5
107.6
177.1
115.1
127.3
114.2
155.1
99.7
159.4
133.2
126.9
129.9
131.9
129.9
126.9
-
163.8
107.9
177.2
120.2
126.6
117.4
145.4
107.5
158.0
132.9
127.1
130.0
132.3
130.0
127.1
-
164.1
106.0
177.1
120.2
126.5
117.1
145.1
107.4
157.9
123.9
129.1
116.8
161.6
116.8
129.1
-
0
C-1
C-2
C-3
C-4
C-5
C-6
0
0
0
0
0
00
C-1
C-2
C-3
C-4
00
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
00
78.1
80.00
77.2
61.9
60.6
77.9
80.1
77.2
61.9
77.3
80.0
77.4
61.9
00
00
C-5
00
C-6
00
C-4 -OCH3
60.6
60.6
-COCH3
-COCH3
-
-
-
-
-
-
-
-
-
-
169.9
24.5
169.8
24.5

Molecules 2018, 23, 1356
10 of 15
Table 3. 1H-NMR shifts (
40,7-dihydroxyisoflavone (daidzein) (
δ
) of 5-hydroxyflavone (
2
), 6-hydroxyflavone (
3
), 7-hydroxyflavone (
4
) and
5) and products of their biotransformations (2a
,
3a,
4a, 5a) in
Acetone-d6, 600 MHz (Supplementary Materials).
Compound
Proton
2
-
2a
-
3
-
3a
-
4
4a
-
5
8.16 (s)
-
5a
H-2
H-3
-
8.24 (s)
-
6.96 (s)
6.87 (s)
6.85 (s)
6.88 (s)
6.79 (s)
6.84 (s)
7.53 (d)
J5,7 = 3.0
7.73 (d)
J5,7 = 1.9
8.02 (d)
J5,6 = 8.7
8.07 (d)
J5,6 = 8.8
8.09 (d)
J5,6 = 8.7
8.15 (d)
J5,6 = 8.7
H-5
-
-
7.03 (dd)
J6,5 = 8.7,
J6,8 = 2.2
6.84 (d)
J6,7 = 8.2
6.81 (d)
J6,7 = 8.4
7.19 (d)
J6,5 = 8.8
7.02 (d)
J6,5 = 8.8
7.18 (d)
J6,5 = 8.8
H-6
-
-
7.36 (dd)
J7,8 = 9.0, J7,8 = 8.8,
J7,5 = 3.0
7.57 (dd)
7.72 (t)
J = 8.3
7.69 (t)
J = 8.3
H-7
H-8
-
-
-
-
J7,5 = 2.0
7.21 (d)
J8,7 = 8.4
7.18 (d)
J8,7 = 8.4
7.68 (d)
J8,7 = 9.0
7.76 (d)
J8,7 = 9.0
7.11 (d)
J8,6 = 2.2
7.40 (s)
6.93 (m)
7.22 (s)
7.52 (d)
8.10 (d)
8.14 (d)
J = 7.1
8.10 (dd)
J = 6.5, 2.9
8.10 (d)
J = 7.3
7.51 (d)
J = 8.3
0
H-2
H-3
H-4
H-5
H-6
8.16 (m)
8.12 (m)
0
0
0
0
J2 ,3 = 8.6
J2 ,3 = 8.0
7.29 (d)
7.64 (d)
J = 5.9
6.93 (d)
J3 ,2 = 7.9
0
0
0
0
7.67 (m)
7.64 (m)
7.64 (m)
7.64 (m)
7.64 (m)
7.63 (m)
7.63 (m)
7.63 (m)
6.91 (m)
-
0
0
0
0
J3 ,2 = 8.6
7.64 (d)
J = 5.9
7.67 (m)
-
7.64 (m)
-
7.29 (d)
7.64 (d)
J = 5.9
6.93 (d)
J5 ,6 = 7.9
7.67 (m)
7.64 (m)
6.91 (m)
0
0
0
0
J5 ,6 = 8.6
8.10 (d)
8.14 (d)
J = 7.1
8.10 (dd)
J = 6.5, 2.9
8.10 (d)
J = 7.3
7.51 (d)
J = 8.3
7.52 (d)
J6 ,5 = 8.0
8.16 (m)
8.12 (m)
0
0
0
0
J6 ,5 = 8.6
5.15 (d)
J = 7.7
5.11 (d)
J = 7.8
5.24 (d)
J = 7.7
5.21 (d)
J = 7.7
00
00
00
00
H-1
H-2
H-3
H-4
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
3.58 (t)
J = 8.4
3.57 (t)
J = 8.2
3.56 (m)
3.70 (m)
3.27 (m)
3.54 (m)
3.73 (m)
3.72 (t)
J = 9.3
3.71 (m)
3.31 (t)
J = 9.4
3.30 (t)
J = 9.3
3.28 (t)
J = 9.3
3.60 (dd)
J = 9.9,
2.5
00
00
H-5
-
-
3.58 (m)
-
-
-
-
3.65 (m)
-
-
3.67 (m)
3.89 (t)
J = 10.7
3.67 (m)
3.92 (d)
J = 11.3
3.75 (m)
3.89 (m)
3.76 (m)
3.92 (m)
3.75 (m)
H-6
00
C-4 -O
-OH
CH3
-
3.61 (s)
-
-
3.62 (s)
-
-
3.62 (s)
-
-
-
3.62 (s)
-
12.74
9.14
9.86
Table 4. 13C-NMR shifts (
δ
) of 5-hydroxyflavone (
2
), 6-hydroxyflavone (
3
), 7-hydroxyflavone (
4
) and
40,7-dihydroxyisoflavone (daidzein) (
5) and products of their biotransformations (2a
,
3a 4a
,
, 5a) in
Acetone-d6, 600 MHz (Supplementary Materials).
Compound
Carbon
2
2a
3
3a
4
4a
5
5a
C-2
C-3
C-4
C-4a
C-5
C-6
C-7
C-8
C-8a
164.7
105.7
183.6
110.6
160.8
111.1
135.8
107.2
156.5
131.2
165.4
105.3
184.3
111.3
161.7
111.8
136.4
108.0
157.3
125.5
163.6
107.0
177.9
125.7
108.9
155.9
123.7
120.6
151.1
133.0
163.8
102.7
177.7
125.5
111.1
156.0
124.9
120.6
152.5
132.8
163.4
107.7
177.3
118.0
127.7
115.6
163.5
103.6
158.9
132.9
163.7
107.9
177.3
119.6
127.4
116.3
162.9
104.9
158.5
132.8
153.1
125.2
175.7
118.3
128.4
115.8
163.6
103.1
158.1
124.4
153.5
124.2
175.7
120.2
128.1
116.4
162.6
104.4
158.3
125.4
0
C-1

Molecules 2018, 23, 1356
11 of 15
Table 4. Cont.
Compound
Carbon
2
2a
3
3a
4
4a
5
5a
0
C-2
C-3
C-4
C-5
C-6
C-1
C-2
C-3
126.6
129.2
132.2
129.2
129.2
117.7
161.8
117.7
129.2
101.1
74.9
77.9
80.1
77.2
62.0
127.1
130.0
132.3
130.0
127.2
130.0
132.5
130.0
127.2
102.2
74.89
77.6
79.9
77.1
61.9
60.6
127.0
129.9
132.2
129.9
127.1
130.0
132.4
130.0
127.1
101.3
74.8
77.8
80.0
77.3
61.9
131.1
115.8
158.9
115.8
131.1
115.9
158.4
115.9
131.1
101.3
74.8
77.9
80.0
77.3
62.0
0
0
0
0
126.6
127.1
127.0
131.1
00
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
00
00
00
C-4
C-5
00
00
C-6
00
C-4 -OCH3
60.6
60.6
60.6
3.2. Microorganism
The description of the collection of the material, propagation of structures of the fungi, as well as
genetic identification were detailed described in our previous publications [1,30]. The microorganisms
were maintained on potato slants at 4 ◦C and freshly subcultured before use in the experiments.
3.3. Analysis
The course of the biotransformation was evaluated by chromatographic methods (TLC, HPLC).
TLC analysis was carried out using TLC Silica gel 60/Kieselguhr F254 plates (Merck, Darmstadt,
Germany). The developing system was a mixture of chloroform and methanol in the ratio of 9:1.
Compounds were visualized using 5% aluminum chloride solution in ethanol. The plates were
observed at two wavelengths: 254 and 365 nm.
HPLC analyses were performed using a Waters 2690 instrument equipped with a Waters 996
photodiode array detector, using an ODS 2 column (4.6
×
250 mm, Waters, Milford, MA, USA) and
a Guard-Pak Inserts Bondapak C18 pre-column. Separation conditions were as follows: Gradient
µ
elution, using 80% of acetonitrile in 4.5% formic acid solution (eluent A) and 4.5% formic acid (eluent B);
flow, 1 mL/min; detection wavelength 280 nm; program: 0–7 min, 10% A 90% B; 7–10 min, 50% A
50% B; 10–13 min, 60% A 40% B; 13–15 min, 70% A 30% B; 15–20 min 80% A 20% B; 20–30 min 90% A
10% B; 30–40 min, 100% A.
Separation of the products obtained by the scaled-up biotransformation was achieved using
1000 µm preparative TLC silica gel plates (Analtech, Gehrden, Germany). After development of
the chromatograms in chloroform:methanol 9:1, compounds were extracted from the selected gel
fragments using ethyl acetate (twice) and tetrahydrofuran (once). The extracts were combined and the
solvents were removed under reduced pressure.
NMR analysis was carried out using a Bruker Avance 600 MHz NMR spectrometer (Billerica,
MA, USA) with an UltraShield Plusmagnet.
Optical rotation was measured using digital polarimeter P-2000-Na (ABL&E-JASCO,
Kraków, Poland).
Molecular formulas of products were confirmed using high-pressure liquid chromatography
HPLC 1200 Agilent Technologies with DAD, FLD, and Mass detectors Triple Quad LC/MS (Agilent
Technologies, Santa Clara, CA, USA).
3.4. Screening Procedure
Experiments were carried out using Sabouraud medium (1% peptone, 3% glucose).
The microorganism was transferred to a 300 mL flask containing 100 mL of the medium. Pre-incubation
was carried out on a rotary shaker (140 rpm) at 25 ◦C for 72 h. Screening was carried out in 100 mL
Erlenmeyer flasks containing 30 mL of Sabouraud liquid medium. The pre-grown culture (0.5 mL)

Molecules 2018, 23, 1356
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was transferred to a flask and after 72-h incubation, 3 mg of the substrate dissolved in 0.5 mL of
tetrahydrofuran was added. The molar concentrations of the substrates were: 0.45 M (flavone), 0.42 M
(5-hydroxyflavone, 6-hydroxyflavone, 7-hydroxyflavone, 7-aminoflavone), and 0.39 M (daidzein).
We used a separate flask for culture for each sample collection. The biotransformation was carried out
under the same conditions as pre-incubation. After 4, 7, and 12 days of biotransformation the mixtures
were extracted with 30 mL of ethyl acetate. The extracts were dried over MgSO4 (5 min), concentrated
in vacuo and analyzed by TLC and HPLC.
Stability of the substrate was evaluated under analogous conditions, without using a biocatalyst.
3.5. Scaled-Up Biotransformations
Scaled-up biotransformations were carried out in 2 L flasks containing 500 mL of the medium.
The pre-incubation culture (1 mL) was transferred to the flask. After 72 h of incubation, 50 mg of
the substrate dissolved in 1 mL of tetrahydrofuran was added. The molar concentrations of the
substrates were: 0.45 M (flavone), 0.42 M (5-hydroxyflavone, 6-hydroxyflavone, 7-hydroxyflavone,
7-aminoflavone), and 0.39 M (daidzein). The scale-up biotransformation was carried out under the
◦
same conditions as the screening (140 rpm, 25 C). After complete consumption of the substrate,
different for each substrate used, metabolites were extracted three times using each time 200 mL of
ethyl acetate. The extracts were dried out using MgSO4 and concentrated on a rotary evaporator.
Product separation was carried out using preparative TLC plates. Product structure was determined
by spectroscopic methods (1H-NMR, 13C-NMR, COSY, HMBC, HSQC).
The physical and spectral data of the products obtained are presented below (Tables S1–S4)
(Supplementary Materials).
3.5.1. Flavone 20-O-β-D-(400-O-Methyl)-glucopyranoside (1a)
C22H22O8, Mw = 414.4; tR 11.44; [α]20 = −29.7◦; 1H-NMR, see Table S1; 13C-NMR, see Table S2.
D
3.5.2. Flavone 40-O-β-D-(400-O-Methyl)-glucopyranoside (1b)
C22H22O8, Mw = 414.4; tR 10.65; [α]20 = −48.0◦; 1H-NMR, see Table S1; 13C-NMR, see Table S2.
D
3.5.3. 30-Hydroxyflavone 40-O-β-D-(400-O-Methyl)-glucopyranoside (1c)
C22H22O9, Mw = 430.4; tR 10.50; [α]20 = −28.0◦; 1H-NMR, see Table S1; 13C-NMR, see Table S2.
D
3.5.4. 5-Hydroxyflavone 40-O-β-D-(400-O-Methyl)-glucopyranoside (2a)
C22H22O9, Mw = 430.4; tR 11.72; [α]20 = −76.5◦; 1H-NMR, see Table S3; 13C-NMR, see Table S4.
D
3.5.5. Flavone 6-O-β-D-(400-O-Methyl)-glucopyranoside (3a)
C22H22O8, Mw = 414.4; tR 11.10; [α]20 = −34.4◦; 1H-NMR, see Table S3; 13C-NMR, see Table S4.
D
3.5.6. Flavone 7-O-β-D-(400-O-Methyl)-glucopyranoside (4a)
C22H22O8, Mw = 414.4; tR 10.68; [α]20 = −40.3◦; 1H-NMR, see Table S3; 13C-NMR, see Table S4.
D
3.5.7. 40-Hydroxyisoflavone 7-O-β-D-(400-O-Methyl)-glucopyranoside (5a)
C22H22O9, Mw = 430.4; tR 10.00; [α]20 = −26.0◦; 1H-NMR, see Table S3; 13C-NMR, see Table S4.
D
3.5.8. 7-Acetamidoflavone (6a)
C17H13NO3, Mw = 279.3; tR 13.67; 1H-NMR, see Table S1; 13C-NMR, see Table S2.
3.5.9. 7-Acetamido-40-hydroxyflavone (6b)
C17H13NO4, Mw = 295.3; tR 11.22; 1H-NMR, see Table S1; 13C-NMR, see Table S2.

Molecules 2018, 23, 1356
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4. Conclusions
The catalytic capacity of four strains of entomopathogenic filamentous fungi of the genus Isaria
was evaluated. Only I. fumosorosea KCH J2 was able to effectively carry out biotransformations of all
substrates used. In the case of flavone, 5-hydroxyflavone, 6-hydroxyflavone, 7-hydroxyflavone and
daidzein we observed the formation of O-methylglucosides. 7-Aminoflavone was transformed into
two acetamido derivatives. Our study not only allowed us to find new biocatalysts for glycosylation of
flavones but also, as a result, we obtained seven new flavonoid derivatives. The substrates and products
obtained by us can be used to assess the biological activity and bioavailability of aglycone/glucoside
pairs of flavonoids, and in the future to become the basis for the creation of new pharmaceuticals or
food additives.
Supplementary Materials: The supplementary materials are available online.
Author Contributions: Formal analysis, M.D., T.J. and E.K.-S.; Funding acquisition, M.D. and E.K.-S.;
Investigation, M.D.; Methodology, M.D. and E.K.-S.; Project administration, M.D. and E.K.-S.; Resources, E.K.-S.;
Supervision, E.K.-S.; Visualization, M.D.; Writing-original draft, M.D.; Writing-review & editing, M.D. and E.K.-S.
Funding: This research received no external funding.
Acknowledgments: The costs of publishing in open access were financially supported by the Wroclaw Centre of
Biotechnology, Leading National Research Centre (KNOW) program for the years 2014 to 2018.
Conflicts of Interest: The authors declare no conflicts of interest.
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the authors.
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2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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