Glycosylation of methoxylated flavonoids in the cultures of isaria fumosorosea KCH J2

Article abstract of DOI:10.3390/molecules23102578

Flavonoids are widely described plant secondary metabolites with high and diverse pro-health properties. In nature, they occur mostly in the form of glycosides. Our research showed that an excellent way to obtain the sugar derivatives of flavonoids is thr

Full text of DOI:10.3390/molecules23102578

molecules
Article
Glycosylation of Methoxylated Flavonoids in the
Cultures of Isaria fumosorosea KCH J2
Monika Dymarska * , Tomasz Janeczko and Edyta Kostrzewa-Susłow
Department of Chemistry, Wrocław University of Environmental and Life Sciences, Norwida 25,
Wrocław 50-375, Poland; janeczko13@interia.pl (T.J.); ekostrzew@gmail.com (E.K.-S.)
*
Correspondence: monika.dymarska@gmail.com or monika.dymarska@upwr.edu.pl; Tel.: +48-71-320-5257
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 14 September 2018; Accepted: 6 October 2018; Published: 9 October 2018
Abstract: Flavonoids are widely described plant secondary metabolites with high and diverse
pro-health properties. In nature, they occur mostly in the form of glycosides. Our research showed
that an excellent way to obtain the sugar derivatives of flavonoids is through biotransformations
with the use of entomopathogenic filamentous fungi as biocatalysts. In the current paper,
0
we described the biotransformations of five methoxylated flavonoid compounds (2 -methoxyflavanone,
3
0
0
-methoxyflavanone, 4 -methoxyflavanone, 6-methoxyflavanone, and 6-methoxyflavone) in
cultures of Isaria fumosorosea KCH J2. As a result, we obtained twelve new flavonoid 4-O-
methylglucopyranosides. The products were purified with methods that enabled the reduction
of the consumption of organic solvents (preparative TLC and flash chromatography). The structures
1
13
of the products were confirmed with spectroscopic methods (NMR: H, C, HSQC, HMBC, COSY).
The compounds obtained by us expand the library of available flavonoid derivatives and can be used
in biological research.
Keywords:
flavonoids; biotransformations; Isaria; fungi; flavonoid glycosides; 4-O-
methylglucopyranoside; methoxyflavanone; methoxyflavone
1
. Introduction
Nowadays preventive medicine is increasing in importance. It is estimated that all over the
world, the cost of chronic diseases in 2015–2030 will reach $17 trillion (including health care costs
and reduced productivity). A sub-optimal diet is currently the leading risk factor for both disabilities
and death worldwide [1]. The prevention and control of non-communicable diseases (such as cancer,
cardiovascular diseases, and diabetes) will be one of the major challenges in healthcare in the next
5
0 years [2].
It is well known that vegetable and fruit consumption may be associated with lower mortality
and non-communicable diseases rates [ ]. Health professionals claim >50% of health problems can be
prevented by implementing an appropriate diet [ ]. The 2015–2020 Dietary Guidelines for Americans
strongly recommend the intake of more vegetables and fruits in our daily diet [ ]. Just consuming
–2 servings of fruit rich in anthocyanins (such as strawberries, raspberries or blueberries) every day
2
1
3
1
helps significantly reducing the risk of cardiovascular diseases [1]. In vitro and in vivo studies confirm
that flavonoid compounds are desirable from the pharmaceutical point of view activities: they are
anti-oxidants, anti-inflammatory, vasorelaxants, anticoagulants, cardioprotective, anti-obesity and
anti-diabetic, chemoprotective, neuroprotective, antidepressants, antimicrobials [3–8].
Many researchers argue that the presence of free hydroxyl groups is crucial for the biological
activity of flavonoid compounds [9–11]. However, some studies conclude that flavonoid derivatives
containing methoxy groups or sugar units in their structure also show desirable biological potential.
Molecules 2018, 23, 2578; doi:10.3390/molecules23102578
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Molecules 2018, 23, 2578
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Polymethoxyflavones are one of the types of flavonoid compounds found in significant amounts
in citrus. Those polyphenols may regulate metabolic disorders, prevent cardiovascular diseases, protect
neurons and have some other desirable bioactivities such as anti-atherosclerotic, anti-inflammatory,
neuroprotective, anti-cancer, anti-microbial, and anti-oxidative effects [12
A-ring polymethoxylated flavonoids possessing methoxyl groups in the B ring as wll) demonstrated
high anti-proliferative activity in cancer cells. In thecase of chrysin, methoxylation greatly enhanced the
anti-inflammatory properties. Akbar et al. demonstrated that 6-methoxyflavanone ( ) has the potential
,13]. Nobiletin and tangeretin
(
4
to easily cross the blood-brain barrier and possesses anti-allodynic and anti-vulvodynic activities in
the rat model of streptozotocin-induced diabetes mellitus. Therefore, this compound could be used to
treat neuropathic pain associated with diabetes mellitus [14]. In another study, Akbar et al. showed
that 6-methoxyflavanone (4) exerted an anxiolytic-like effect in several tests conducted with mice [15].
O-methylation has been proven to be also beneficial to the anti-viral, anti-bacterial, and anti-diabetic
properties of flavonoids [8].
Despite the extraordinary biological activities of flavonoids, these compounds are still relatively
rarely used as pharmaceuticals. This is due to their poor bioavailability [
a well-known method of improving the properties of flavonoid compounds, such as solubility
and stability [ 17]. Flavonoid glycosides often present a lower bioavailability than their parent
aglycones but not in the case of glucosides. The presence of a glucose unit attached to the
flavonoid skeleton is reported to improve its absorption [17 18]. In most cases, prior to absorption,
flavonoid glycosides need to be hydrolyzed into aglycones. Anthocyanins are a well-documented
exception: intact glycosides were detected in the circulation system [16 17 19]. Another relatively high
bioavailable group are methoxylated flavonoids. When compared to hydroxyl flavonoids, a 100-fold
higher plasma concentration has been obtained for methoxylated derivatives [ ]. The compounds
,7-dimethoxyflavone and 3 ,4 -dimethoxyflavone were reported to be more stable compared to
8,16]. Glycosylation is
3,6,
,
,
,
8
0
0
5
galangin (3,5,7-trihydroxyflavone) [16].
Bioavailability and biological activities of compounds strongly differ between distinct flavonoid
subclasses, and within subclasses, they depend on the substitution patterns. For this reason, it is
important to expand the flavonoid library with new derivatives, doing so gives us tools to further
understanding the metabolism of this complex group of plant secondary metabolites in humans and
may result in obtaining new pharmaceuticals of natural origin.
Our study is a continuation of research on the biotransformations of flavonoid aglycones
in cultures of entomopathogenic filamentous fungi [20
–
22]. In the current paper, we describe
0
the biotransformations of six methoxylated flavonoid compounds (2 -methoxyflavanone,
0
0
3
-methoxyflavanone, 4 -methoxyflavanone, 6-methoxyflavanone, and 6-methoxyflavone) in cultures
of Isaria fumosorosea KCH J2. As a result, we obtained twelve flavonoid 4-O-methylglucopyranosides
that were not previously described. Our current research provides an insight into the microbial
glycosylation of flavonoid compounds lacking free hydroxyl groups. The biotransformation products
expand the library of available flavonoid derivatives and can be used in biological research.
2
. Results and Discussion
In order to obtain new flavonoid 4-O-methylglucopyranosides, we used five flavonoid substrates
and a strain of the entomopathogenic filamentous fungus isolated from spider carcasses in Wrocław:
Isaria fumosorosea KCHJ2, which is so far the most efficient biocatalyst in the microbial glycosidation
of flavonoids in our collection. Detailed characteristics of the strain can be found in our previous
publication [20].
For the basis of the screening, we chose different durations of scale-up biotransformations for each
substrate. Each scale-up biotransformation was terminated on the day when, in the HPLC analysis of a
sample taken during the screening procedure, no peak was observed from the substrate, and the areas
under the peaks from the putative glycosylation products were the highest. Experiments conducted
on a larger scale allowed us to determine the chemical structures of products and their isolated

Molecules 2018, 23, 2578
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yields. Percent yields are given in relation to the molar ratio of products to substrates. Enantiomeric
excesses of products and unreacted substrates were determined by HPLC using chiral column; we did
not observe any significant enantiomeric excesses. In the course of our study, we obtained twelve
biotransformation products, none of the products have been previously reported in the literature.
0
2
.1. Biotransformations of 2 -Methoxyflavanone (1)
0
As a result of the 11-day biotransformation of 2 -methoxyflavanone (
1
) in the culture of
0
0
I. fumosorosea KCH J2, we obtained two products: 2 -methoxyflavanone 5 -O-
glucopyranoside (1a) with an 18.6% yield and flavan-4-ol 2 -O-
β-D-(4”-O-methyl)-
0
β-D-(4”-O-methyl)-glucopyranoside
(1b) with a 24.9% yield (Scheme 1).
O
HO
CH3
H C
3
O
O
OH
O
3'
O
O
HO
H C
4'
5'
3
2'
8
5
O
7
6
1'
2
3
6'
O
1a)
4
Isaria fumosorosea KCH J2
(
11 days
OH
H C
3
O
O
HO
O
O
(
1)
OH
O
OH
(
1b)
0
Scheme 1. The microbial transformation of 2 -methoxyflavanone (
1) in the I. fumosorosea KCH J2 culture.
The presence of a glucose unit in molecules 1a and 1b is confirmed by five characteristic carbon
signals observed in the region from about = 80.0 ppm to about
= 62.0 ppm in the 13C-NMR
H ranging from about = 3.85 ppm to = 3.23 (3.28)
δ
δ
spectra, along with the proton signals of
ppm in the H-NMR spectra. Additionally, the attachment of a sugar unit to substrate 1 was
δ
δ
δ
1
1
confirmed by a one-proton doublet visible at
δ
= 4.90 ppm in the H-NMR spectrum of 1a and
1
δ = 4.85 ppm in the H-NMR spectrum of 1b, which are due to protons at the anomeric carbon atoms.
The
β
-configuration of the glucose unit was proved by the coupling constant (J = 7.8 Hz) for the
1
anomeric proton. A three-proton singlet at about
signal at about δ = 60.5 ppm in the 13C-NMR proved that one of the hydroxyl groups had been
δ
= 3.60 ppm in the H-NMR and the corresponding
methylated. O-methylation occurred in the C-4” hydroxyl group of the glucose moieties, which was
confirmed in the HMBC spectra, where the proton signals due to -OCH were coupled with the signal
3
of C-4 (δ = 80.2/79.8 ppm) in the glucose unit.
0
The sugar unit was attached to C-5 of 1a because, in the HMBC spectrum, the signal due
0
to the proton at the anomeric carbon atom (
δ = 152.8 ppm), which was shifted from
atom. In the COSY spectrum, it can be seen that the proton at C-6 became isolated. The C-NMR
δ
= 4.90 ppm) was coupled with the C-5 signal
(
δ = 121.6 ppm, indicating the attachment of an electronegative
0
13
0
0
signals from C-4 and C-6 were shifted towards higher field values, indicating the appearance of
an electron-withdrawing substituent in their vicinity. Chemical shifts of the other signals in the
H- and C-NMR spectra have only slightly changed, which indicates that the flavanone skeleton
remained intact.
In the case of compound 1b, the sugar unit was attached to C-2 , because in the HMBC spectrum
the signal due to the proton at the anomeric carbon atom (
signal ( = 155.4 ppm), which was only slightly shifted from
1
13
0
0
δ
= 4.85 ppm) was coupled with the C-2
= 157.0 ppm. The positions of the signals
δ
δ
from the remaining protons of the B-ring did not significantly change, indicating no changes in the
substitution pattern. In the C ring, the carbonyl group at C-4 was reduced. The proof is the lack of a

Molecules 2018, 23, 2578
4 of 18
characteristic signal at about
δ = 64.3 ppm along with one-proton singlet at δ = 4.83 ppm observed in the H-NMR spectrum of 1b.
δ
= 190 ppm on the 13C-NMR spectrum and the appearance of a signal at
1
0
2
.2. Biotransformations of 3 -Methoxyflavanone (2)
0
As a result of the 7-day biotransformation of 3 -methoxyflavanone (
2
) in the culture of I. fumosorosea
0
KCH J2, we obtained flavan-4-ol 3 -O-
β
-D-(4”-O-methyl)-glucopyranoside (2a) with a 15.4% yield and
-hydroxyflavanone 6-O-β-D-(4”-O-methyl)-glucopyranoside (2b) with a 16% yield (Scheme 2).
0
3
HO
CH3
O
O
OH
O
HO
H C
3
O
O
2'
3'
4'
5'
8
5
O
4
7
6
1'
2
6'
OH
2a)
3
Isaria fumosorosea KCH J2
(
7 days
OH
O
(
2)
OH
O
H C
3
O
HO
O
O
OH
O
(
2b)
0
Scheme 2. The microbial transformation of 3 -methoxyflavanone (2) in I. fumosorosea KCH J2 culture.
The presence of a glucose unit in molecules 2a and 2b was confirmed by five characteristic carbon
signals observed in the region from about
δ
= 80.0 ppm to
δ
= 62.0 ppm in the 13C-NMR spectra,
1
along with the proton signals of H ranging from about
δ
δ
= 3.90 ppm to
δ
= 3.30 ppm in the H-NMR
spectra. The attachment of a sugar unit to substrate 2 was also confirmed by a one-proton doublet
1
visible at δ = 5.03/4.93 ppm in the H-NMR spectrum of 2a
/
2b, which was due to the protons at the
anomeric carbon atoms. The
β
-configuration of the glucose unit was proved by the coupling constant
1
(
J = 7.8 Hz) for the anomeric proton. A three-proton singlet at
and the corresponding signal at
the hydroxyl group at C-4” of glucose, which was confirmed on the basis of the analysis of the HMBC
spectra of compound 2a 2b, where the proton signals due to -OCH3 ( = 3.60 ppm) were coupled with
δ
= 3.60 ppm in the H-NMR spectra
δ
= 60.5 ppm in the 13C-NMR spectra indicates the O-methylation of
/
δ
the signal of C-4” (δ = 80.1/79.9 ppm) in the glucose unit.
0
The sugar unit was attached to C-3 of 2a because in the HMBC spectrum the signal due to the
0
proton at the anomeric carbon atom (
which was only slightly shifted from
shifts of signals of the remaining protons in the B-ring indicating no changes in the substitution pattern.
In the 13C-NMR of 2a, there is no characteristic signal at about
= 190 ppm, suggesting a change
= 63.6 ppm along with one-proton signal at
δ
= 5.03 ppm) was coupled with the C-3 signal (
δ
= 158.9 ppm)
δ
= 160.9 ppm. There were no significant changes in the chemical
δ
in substitution at C-4. The appearance of a signal at
δ
1
δ = 4.81 ppm in the H-NMR of 2a prove that the carbonyl group at C-4 was reduced.
In the case of 2b, the glucose moiety was attached to C-6, which was proved on the basis of
the HMBC spectrum. The signal due to the proton at the anomeric carbon atom (
δ
= 4.93 ppm) was
coupled with the C-6 signal (
δ
= 153.1 ppm), which was shifted from
δ
= 122.3 ppm. There was no
signal from C-6 in the H-NMR spectrum of 2b. The 13C-NMR spectrum signals from C-5 and C-7 were
1
shifted towards higher field values (upfield), suggesting the appearance of a strongly electronegative
0
substituent in their vicinity. Demethylation at C-3 was confirmed on the basis of the HMBC spectrum
0
of 2b: the signal from C-3 (
δ
= 158.5 ppm) was coupled with a singlet signal from the hydroxyl group
proton (δ = 8.54 ppm) and there was also no signal from C-3 -OCH in the H-NMR.
0
1
3

Molecules 2018, 23, 2578
5 of 18
0
.3. Biotransformations of 4 -Methoxyflavanone (3)
2
0
As a result of the 7-day biotransformation of 4 -methoxyflavanone (
3
) in the culture of
0
I. fumosorosea KCH J2, we obtained flavanone 4 -O-
β
-D-(4”-O-methyl)-glucopyranoside (3a) with
-D-(4”-O-methyl)-glucopyranoside (3b) with a 20.7% yield and
-D-(4”-O-methyl)-glucopyranoside (3c) with a 32.8% yield (Scheme 3).
0
a 11% yield, 4 -hydroxyflavanone 6-O-
β
0
0
3
,4 -dihydroxyflavanone 6-O-
β
HO
CH3
O
O
OH
O
HO
O
CH3
O
3'
O
OH
(
3a)
2'
4'
8
5
O
4
5'
OH
O
O
7
6
1'
H C
3
2
O
HO
O
6'
O
3
Isaria fumosorosea KCH J2
OH
7 days
O
(
3b)
(
3)
OH
OH
OH
O
O
3
H C
O
HO
O
O
OH
(
3c)
0
Scheme 3. The microbial transformation of 4 -methoxyflavanone (3) in I. fumosorosea KCH J2 culture.
The presence of a glucose unit in molecules 3a
carbon signals observed in the region from about
spectra and proton signals from about = 3.87 ppm to about
, 3b, and 3c was confirmed by five characteristic
13
δ
= 80.0 ppm to
δ
= 62.0 ppm in the C-NMR
1
δ
δ
= 3.27 ppm in the H-NMR spectra.
Additionally, the attachment of a sugar unit to substrate 3 was confirmed by a one-proton doublet
1
at
δ
= 5.02/4.93/4.92 ppm in the H-NMR spectrum of 3a
/3b/3c, which was due to the protons at
the anomeric carbon atoms. The
β-configuration of the glucose unit was proved by the coupling
constant (J = 7.8/7.7/6.3 Hz) for the anomeric proton. A three-proton singlet at
δ = 3.60 ppm in
1
= 60.5 ppm in the 13C-NMR prove that one of
the H-NMR and the corresponding signal at about
δ
the hydroxyl groups had been methylated. O-methylation occurred in the C-4” hydroxyl group of
the glucose moiety, which was confirmed in the HMBC spectra of the biotransformation products
3a/3b/3c, where the proton signals due to -OCH3 (δ = 3.60 ppm) were coupled with the signal of C-4”
(
δ = 80.1/79.9/79.9 ppm) in the glucose unit.
0
The sugar unit was attached to C-4 of compound 3a because in the HMBC spectrum the signal
0
due to the proton at the anomeric carbon atom (
δ
= 5.02 ppm) was coupled with the C-4 signal
(
δ = 158.9 ppm) which was only slightly shifted from
δ
= 160.9 ppm. There were no other changes in
1
the substitution pattern of the flavanone skeleton because chemical shifts of the other signals in the H
and 13C-NMR spectra had only slightly changed.
0
In the case of compound 3b and 3c, demethylation at C-4 occurred. In the HMBC spectrum of
0
the 3b signal from C-4 (δ = 158.6 ppm) was coupled with a singlet signal from the hydroxyl group
0
δ = 8.57 ppm); there was also no signal from C-4 -OCH in the H-NMR or C-NMR of
3
1
13
proton (
and 3c. The glucose unit was attached to C-6 because in the HMBC spectrum of 3b
signal due to the proton at the anomeric carbon atom ( = 4.93/4.92 ppm) was coupled with the C-6
= 122.1 ppm. The lack of a signal from C-6 in the
3
b
(3c) the
δ
signal (δ = 153.0 ppm), which was shifted from
δ
1
H-NMR spectrum of 3b and the shift of signals from C-5 and C-7 towards higher field values
in the 13C-NMR spectrum indicate the attachment of a sugar at C-6. In the case of compound
3
0
13
0
c
, an additional hydroxylation occurred in position C-3 , because the C-NMR signal from C-3

Molecules 2018, 23, 2578
6 of 18
was visible at δ = 146.0 ppm indicating the attachment of a strongly electronegative atom. In the
0
COSY spectrum, the signal from C-2 became isolated and was shifted towards higher field values in
the C-NMR.
13
2
.4. Biotransformations of 6-Methoxyflavanone (4)
As a result of the 8-day biotransformation of 6-methoxyflavanone (
4
) in the culture of I. fumosorosea
0
KCH J2, we obtained 6-methoxyflavanone 4 -O-
β-D-(4”-O-methyl)-glucopyranoside (4a) with a 6%
0
0
yield and 3 -hydroxy-6-methoxyflavanone 4 -O-
β-D-(4”-O-methyl)-glucopyranoside (4b) with a 7%
yield (Scheme 4).
HO
CH3
O
O
OH
O
HO
O
3'
2
'
4'
5'
8
5
O
4
O
7
1'
2
6'
CH3
O
6
3
Isaria fumosorosea KCH J2
(
4a)
O
8 days
OH
HO
CH3
CH3
O
O
O
OH
O
(
4)
HO
O
O
CH3
O
(
4b)
Scheme 4. The microbial transformation of 6-methoxyflavanone (4) in I. fumosorosea KCH J2 culture.
The presence of a glucose unit in molecule 4a
signals observed in the region from about = 80.0 ppm to about
H ranging from about = 3.90 ppm to
(
4b) was confirmed by five characteristic carbon
= 62.0 ppm in the 13C-NMR
= 3.26 ppm in the
δ
δ
spectra, along with proton signals of
δ
δ
δ
1
H-NMR spectra. Additionally, the attachment of a sugar unit to substrate 4 was confirmed by a
1
one-proton doublet at
anomeric carbon atoms. The
J = 7.8/7.9 Hz) for the anomeric proton. A three-proton singlet at
and the corresponding signal at
= 60.6 ppm in the 13C-NMR proved 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 HMBC spectra of compound 4a 4b), where the proton signals due to
δ
= 5.01 (4.81) ppm in the H-NMR spectrum of 4a
(
4b) from the protons at the
β
-configuration of the glucose unit was proved by the coupling constant
1
(
δ = 3.60 ppm in the H-NMR
δ
(
-
OCH (δ = 3.60 ppm) were coupled with the signal of C-4” (δ = 80.1/80.0 ppm) in the glucose unit.
3
0
In the case of 4a, a sugar unit was attached to C-4 , it was proved in the HMBC spectrum.
0
The signal due to the proton at the anomeric carbon atom (δ = 5.01 ppm) was coupled with the C-4
signal (
δ
= 158.9 ppm), which was shifted from
δ
= 129.3 ppm indicating a substitution with a strongly
0
electronegative atom. The substitution in position 4 was also evidenced by the characteristic two
1
0
0
0
doublets in the H-NMR spectrum from the protons at C-2 and C-6 (
δ
= 7.54 ppm) as well as C-3 and
0
C-5 (δ = 7.15 ppm).
0
In the case of 4b, 4-O-methylglucopyranose was also attached to C-4 . In the HMBC spectrum
of 4b, the signal due to the proton at the anomeric carbon atom (
δ
= 4.81 ppm) was coupled with
0
0
the C-4 signal (
δ
= 146.2 ppm). Additionally, hydroxylation into position C-3 occurred. In the
13
0
C-NMR spectrum, the signal from C-3 was shifted toward a lower field value (from δ = 129.5 ppm
0
0
to δ = 148.8 ppm) and the signals from C-2 and C-5 were shifted upfield by 12.3 ppm and 10.9 ppm,
respectively, indicating the appearance of strongly electronegative atoms in their vicinity. In the
1
0
0
H-NMR spectrum, there is no signal from the protons at C-3 and C-4 . The substitution pattern
0
was also confirmed on the basis of the COSY spectrum in which it can be seen that signals from C-2
became isolated.

Molecules 2018, 23, 2578
7 of 18
2
.5. Biotransformations of 6-Methoxyflavone (5)
As a result of the 12-day biotransformation of 6-methoxyflavone (
5
) in the culture of
0
I. fumosorosea KCH J2 we obtained 6-methoxyflavone 3 -O-
β
-D-(4”-O-methyl)-glucopyranoside (5a
)
0
with a 2.6% yield, 6-methoxyflavone 4 -O-β-D-(4”-O-methyl)-glucopyranoside (5b) with a 7.8% yield
0
0
and 3 -hydroxy-6-methoxyflavone 4 -O-
Scheme 5).
β-D-(4”-O-methyl)-glucopyranoside (5c) with a 7.9% yield
(
HO
CH3
O
O
OH
O
HO
O
O
HO
HO
CH
CH
3
CH3
O
5a)
O
O
3
'
O
O
(
OH
2
'
4'
5'
HO
8
5
O
O
4
7
1'
2
6
'
6
3
Isaria fumosorosea KCH J2
O
O
13 days
OH
CH3
O
5b)
3
CH3
O
O
O
OH
(
(
5)
HO
O
O
CH3
O
(
5c)
Scheme 5. The microbial transformation of 6-methoxyflavone (5) in I. fumosorosea KCH J2 culture.
The presence of a glucose unit in molecule 5a
signals observed in the region from about = 80.0 ppm to about
along with proton signals of H ranging from about = 3.94 ppm (3.90 ppm, 3.92 ppm) to δ = 3.25 ppm
3.28 ppm, 3.29 ppm) in the H-NMR spectra. Additionally, the attachment of a sugar unit to substrate
(5b
,
5c) was confirmed by five characteristic carbon
δ
δ
= 62.0 ppm in the 13C-NMR spectra,
δ
δ
1
(
1
5
was confirmed by a one-proton doublet visible at
δ
= 5.14 ppm (5.14 ppm, 5.00 ppm) in the H-NMR
-configuration of the
spectrum of 5a
(
5b 5c) from the protons at the anomeric carbon atoms. The β
,
glucose unit was proved by the coupling constant (J = 7.8 Hz) for the anomeric protons. A three-proton
1
singlet at about
δ
= 3.60 ppm in the H-NMR and the corresponding signal at
δ
= 60.6 ppm in the
13
C-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 HMBC spectra of compound
5
a (5b, 5c), where the proton signals from-OCH were coupled with the signal of C-4” (δ = 80.3 ppm,
3
8
0.1 ppm, 80.0 ppm) in the glucose molecule.
0
The sugar unit was attached to C-3 of 5a because in the HMBC spectrum, the signal due to the
0
proton at the anomeric carbon atom (
The 13C-NMR signals from C-2 and C-4 were shifted upfield indicating the appearance of an
δ
= 5.14 ppm) was coupled with the C-3 signal (δ = 159.3 ppm)
.
0
0
electron-withdrawing substituent in their vicinity. In the COSY spectrum, it could be seen that
0
the signal from C-2 became isolated.
0
In the case of compounds 5b and 5c, the sugar unit was attached to C-4 . In the HMBC spectra,
signals due to the protons at the anomeric carbon atoms (
δ
= 5.14 ppm, 5.00 ppm) were coupled with
0
signals from C-4 (
δ
= 161.3 ppm,
δ
= 148.9 ppm) which were shifted downfield from
δ
= 132.4 ppm.
1
In the case of 5b, the characteristic two two-proton signals in the H-NMR spectrum from the protons
0
0
0
0
0
δ
at C-2 and C-6 (
δ
= 8.08 ppm) as well as C-3 and C-5 (
= 7.28 ppm) indicated the substitution
0
13
0
at C-4 . The compound 5c has also hydroxyl group at C-3 , in the C-NMR signal from C-3 was
0
shifted downfield by 18.8 ppm and the signal from C-2 was shifted upfield by 12.6 ppm indicating

Molecules 2018, 23, 2578
8 of 18
0
0
the attachment of strongly electronegative atom to C-3 . In the COSY spectrum, the signal from C-2
became isolated.
In the available literature, we have not found any reports on obtaining sugar derivatives of
substrates, whose biotransformations we describe in the present paper. The only two examples of
the microbial glycosylation of flavonoid compounds lacking hydroxyl groups are described by us
as the biotransformations of 6-methylflavone [20] and flavone [21] in cultures of entomopathogenic
filamentous fungi. In the case of 6-methylflavone, 4-O-methylglucopyranose was attached to C-8 in the
0
first product and to C-4 in the second product. The biotransformation of flavone yielded three products:
0
0
flavone 2 -O-
β
-D-(4”-O-methyl)-glucopyranoside, flavone 4 -O-
and 3 -hydroxyflavone 4 -O-β-D-(4”-O-methyl)-glucopyranoside.
β-D-(4”-O-methyl)-glucopyranoside
0
0
For microbial transformations of methoxy derivatives of flavonoids, the most frequently
described reaction is demethylation. Our team reported on the enantioselective conversion of four
derivatives of 6-hydroxyflavanone. Racemic 6-methoxyflavanone was hydrolyzed by filamentous
fungus Aspergillus niger MB to 6-hydroxyflavanone. The R-enantiomer of 6-hydroxyflavanone
0
was further hydroxylated at the C-4 position, while the S-enantiomer was biotransformed into
6
-hydroxyflavone [23]. In another publication, Kostrzewa-Susłow et al. described the biotransformations
of 6- and 7-methoxyflavones in cultures of A. niger and Penicillium chermesinum. In the
A. niger MB culture, 6-methoxyflavone underwent hydrolysis to 6-hydroxyflavone which was
0
further biotransformed into 4 ,6-dihydroxyflavone [24]. Cao et al. in an extensive review paper
described examples of various microbial transformations of bioactive flavonoids. In the case
0
of tangeretin (4 ,5,6,7,8-pentamethoxyflavone), A. niger ATCC 9142 selectively O-demethylated
0
the methoxy group at the C-4 position. A. alliaceus UI 315 performed the hydrolysis of
,3 -dimethoxyflavanone to 3 -hydroxy-2 -methoxyflavanone. Cuninghamella elegans NRRL
392 performed the O-demethylation of 7-O-methylnaringenin (4 ,5-dihydroxy-7-methoxyflavanone)
and 4 ,5-dihydroxy-3 ,7-dimethoxyflavanone to yield naringenin (4 ,5,7-trihydroxyflavanone) and
homoeriodictyol (4 ,5,7-trihydroxy-3 -methoxyflavanone). On the basis of their review, Cao et al.
suggested that the demethylation of polymethoxy flavonoid derivatives occurs more frequently
for the C-3 and C-4 positions [25]. Our experience shows that strains of the genus Isaria
perform demethylation only in the case of methoxy substituents in ring B. To time we
performed biotransformations of over thirty variously substituted flavones and flavanones using
entomopathogenic filamentous fungi as biocatalysts. Only for flavanones with methoxyl groups
in ring B did we observe the glycosylation into the C-6 position for flavonoid derivatives lacking
the hydroxy group in this position. In the case of 6-methoxyflavanone and 6-methoxyflavone,
0
0
0
0
2
1
0
0
0
0
0
0
0
0
0 0
4
-O-methylglucopyranose was attached most frequently to C-4 , but also to C-3 for 6-methoxyflavone.
When we used the above-mentioned 6-methylflavone as a substrate, we also observed the formation
of 6-methylflavone 8-O-β-D-(4”-O-methyl)-glucopyranoside [20].
A reaction, which we did not observe previously for other flavonoid substrates biotransformed
in the cultures of entomopathogenic filamentous fungi was the reduction of carbonyl group at C-4.
0
0
It occurred in the case of 2 -methoxyflavanone and 3 -methoxyflavanone.
3
. Materials and Methods
3
.1. Chemicals
The compound 6-methoxyflavanone was purchased from the Sigma Chemical Company (St. Louis,
MO, USA). The compound 6-methoxyflavanone was purchased from TCI EUROPE N.V. (Zwijndrecht,
0
0
0
Belgium). The compounds 2 -methoxyflavanone, 3 -methoxyflavanone, and 4 -methoxyflavanone
were chemically synthesized.
0
3
.1.1. 2 -Methoxyflavanone (1)
C H O , t 20.85; H-NMR, see Table 1; 13C-NMR, see Table 2.
1
16
14
3
R

Molecules 2018, 23, 2578
9 of 18
)
1
0
Table 1. The H-NMR shifts (
δ
) (ppm) and coupling constants (JH,H) (Hz) of 2 -methoxyflavanone (
1
0
and 3 -methoxyflavanone (
2
) and the products of their biotransformations (1a 1b 2a 2b) in Acetone-d6,
,
,
,
6
00 MHz (Supplementary Materials).
Compound
1b
Proton
1
1a
2
2a
5.31 (dd)
J2,3a = 11.7, J2,3a = 12.7,
2b
H-2
H-3a
H-3e
5.89 (dd)
J2,3a = 13.3,
J2,3e = 2.8
5.85 (dd)
J2,3a = 13.0,
J2,3e = 2.8
5.84 (d)
J = 11.4
5.65 (dd)
J2,3a = 13.0,
J2,3e = 2.9
5.57 (dd)
J2,3e = 2.1
J2,3e = 3.0
3.05 (dd)
J3a,2 = 13.3,
J3a,3e = 16.7
3.01 (m)
2.88 (m)
-
2.49 (d)
J = 14.0
3.19 (dd)
J3a,2 = 13.1,
J3a,3e = 16.8
2.26 (m)
3.12 (dd)
J3a,2 = 12.7,
J3a,3e = 16.8
2.88 (m)
2.01 (m)
2.90 (dd)
J3e,2 = 2.9,
J3e,3a = 16.8
2.15 (m)
4.81 (m)
2.87 (m)
H-4
H-5
-
4.83 (m)
-
-
7.90 (dd)
J5,6 = 8.2,
J5,7 = 1.8
7.89 (dd)
J5,6 = 7.8,
J5,7 = 1.6
7.60 (d)
J5,6 = 7.0
7.88 (dd)
J5,6 = 8.0,
J5,7 = 1.8
7.39 (dd)
J5,6 = 7.6,
J5,7 = 1.6
7.49 (t)
J = 3.1
H-6
H-7
7.12 (m)
7.13 (m)
7.19 (t)
J = 7.4
7.13 (m)
6.96(dt)
J = 7.4,
J = 1.1
-
7.62 (m)
7.63 (m)
7.33 (m)
7.33 (m)
7.63 (ddd)
J7,5 = 1.7,
J7,6 = 7.2,
J7,8 = 8.3
7.25 (m)
7.37 (dd)
J7,5 = 3.1,
J7,8 = 9.0
H-8
7.12 (m)
7.13 (m)
7.03 (d)
7.13 (m)
6.91 (dd)
J8,6 = 0.9,
J8,7 = 8.1
7.07 (m)
0
H-2
7.22 (m)
-
7.23 (m)
-
7.07 (m)
-
0
H-3
7.12 (m)
7.41 (m)
6.92 (d)
J3
0
0
= 8.9
J
0
0
= 8.2
,4
3 ,4
0
H-4
7.11 (dd)
4.25 (m)
7.00 (ddd)
7.08 (ddd)
6.89 (ddd)
J4
0
0
= 9.1,
= 2.9
J
J
0
0
= 0.8,
= 8.3,
= 2.6
J
J
J
0
0
= 0.7,
= 8.5,
= 2.6
J
J
J
0
0
= 0.8,
= 8.1,
= 2.3
,3
4 ,2
4 ,2
4 ,2
J4
0
0
0
0
0
0
0
4 ,5
0
,6
4 ,5
4 ,5
J4
0
0
0
0
0
0
,6
4 ,6
4 ,6
0
H-5
7.12 (m)
7.68 (dd)
-
6.96 (t)
J = 7.4
7.40 (m)
7.36 (t)
J = 8.0
7.29 (t)
J = 7.9
0
H-6
7.41 (d)
= 2.8
7.42 (d)
J = 6.5
7.19 (m)
7.17 (d)
7.07 (m)
J6
0
0
= 1.5,
0
= 7.6
,5
J
0
0
J
0
0
= 7.6
,4
6 ,4
6 ,5
J6
0
H-1”
-
4.90 (d)
J = 7.8
4.85 (d)
J = 7.8
-
5.03 (d)
J = 7.8
4.93 (d)
J = 7.8
H-2”
H-3”
-
-
3.52 (m)
3.65 (m)
3.53 (m)
-
-
3.51 (m)
3.66 (m)
3.49 (m)
3.63 (t)
J = 8.5
3.67 (dt)
J = 9.0,
J = 3.7
H-4”
H-5”
-
-
3.23 (m)
3.48 (m)
3.28 (t)
J = 9.3
-
-
3.26 (m)
3.51 (m)
3.27 (t)
J = 9.3
3.42 (ddd)
J = 9.4,
3.49 (m)
J = 4.0,
J = 2.0

Molecules 2018, 23, 2578
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Table 1. Cont.
Compound
Proton
H-6”
1
1a
1b
2
2a
2b
-
3.72 (m)
.85 (m)
3.84 (dd)
J = 5.2,
J = 2.4
-
3.85 (m)
3.72 (m)
3.87 (m)
3.76 (m)
3
3
.74 (dd)
J = 11.0,
J = 3.8
C-4”-OCH3
-
3.59 (s)
3.59 (s)
-
3.60 (s)
3.60 (s)
0
C-2 -OCH3
3.92 (s)
3.89 (s)
-
-
-
-
-
-
-
-
0
C-3 -OCH3
-
-
-
-
3.88 (s)
-
-
0
C-3 -OH
8.54 (s)
Table 2. The 13C-NMR shifts (
δ) (ppm) of 2 -methoxyflavanone (1) and 3 -methoxyflavanone (2) and the
0 0
products of their biotransformations (1a
,
1b,
2a,
2b) in Acetone-d , 151 MHz (Supplementary Materials).
6
Compound
Carbon
1
1a
1b
2
2a
2b
C-2
C-3
C-4
C-4a
C-5
C-6
C-7
C-8
C-8a
75.5
44.1
192.1
121.9
127.4
122.2
136.8
118.9
162.8
128.4
157.0
111.8
130.4
121.6
127.4
-
75.0
44.0
68.9
38.4
64.3
133.2
127.2
124.1
129.5
118.5
156.1
125.7
155.4
117.6
130.0
121.1
131.7
104.3
75.1
77.6
79.8
76.9
61.8
60.5
-
80.3
45.0
191.8
122.0
127.4
122.3
136.8
118.9
162.4
141.9
112.9
160.9
114.7
130.6
119.3
-
-
-
-
-
-
-
-
73.8
39.6
63.6
126.1
131.5
121.1
129.9
117.5
155.6
144.2
115.4
158.9
116.5
130.3
120.7
101.5
75.0
78.0
80.1
77.0
62.1
60.5
-
80.3
44.9
192.1
121.9
127.4
122.3
136.8
119.0
162.7
129.2
152.0
112.7
117.8
152.8
116.3
102.4
75.3
78.0
80.2
77.0
62.2
60.5
56.4
-
191.7
122.1
113.7
153.1
126.8
118.2
157.8
141.9
114.2
158.5
116.2
130.6
119.9
102.6
75.0
77.8
79.9
77.0
62.0
60.5
-
-
0
C-1
C-2
C-3
C-4
C-5
C-6
0
0
0
0
0
C-1”
C-2”
C-3”
C-4”
C-5”
-
-
-
-
-
-
C-6”
C-4”-OCH3
0
C-2 -OCH3
56.0
-
0
C-3 -OCH3
-
55.6
-
0
3
.1.2. 3 -Methoxyflavanone (2)
C H O , t 19.30; H-NMR, see Table 1; 13C-NMR, see Table 2.
1
16
14
3
R
0
3
.1.3. 4 -Methoxyflavanone (3)
C H O , t 19.01; H-NMR, see Table 3; 13C-NMR, see Table 4.
1
16
14
3
R

Molecules 2018, 23, 2578
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1
0
Table 3. The H-NMR shifts (
δ
) (ppm) and coupling constants (JH,H) (Hz) of 4 -methoxyflavanone
(
(
3
) and the products of its biotransformations (3a
,
3b
,
3c) in Acetone-d6, 600 MHz
Supplementary Materials).
Compound
Proton
H-2
3
3a
3b
3c
5.60 (dd)
J2,3a = 13.1,
J2,3e = 2.8
5.63 (dd)
J2,3a = 13.0,
J2,3e = 2.8
5.51 (ddd)
J2,3a = 13.1,
J2,3e = 2.8
J = 4.8
5.45 (ddd)
J2,3a = 12.9,
J2,3e = 2.9
J = 5.2
H-3a
3.22 (dd)
J3a,2 = 13.1,
J3a,3e = 16.7
3.21 (m)
3.17 (ddd)
J3a,2 = 13.2,
J3a,3e = 16.8, J3a,3e = 16.8,
3.13 (ddd)
J3a,2 = 12.9,
J = 0.9
J = 1.3
H-3e
H-5
2.85 (dd)
J3e,2 = 2.9,
J3e,3a = 16.8
2.86 (m)
2.81 (dt)
J = 16.8,
J = 3.0
2.81 (m)
7.88 (dd)
J5,6 = 7.7,
J5,7 = 1.8
7.88 (dd)
J5,6 = 7.8,
J5,7 = 1.7
5.49 (t)
J = 3.0
7.49 (t)
J = 3.0
H-6
H-7
7.12 (m)
7.61 (m)
7.11 (m)
7.61 (m)
-
-
7.35 (dd)
J7,5 = 3.1,
J7,8 = 9.0
7.35 (dd)
J7,5 = 3.1,
J7,8 = 9.0
H-8
7.09 (d)
J8,7 = 8.7
7.11 (m)
7.55 (m)
7.03 (dd)
J8,7 = 8.9,
J = 1.4
7.02 (dd)
J8,7 = 9.0,
J = 1.4
0
H-2
7.56 (m)
6.44 (m)
7.08 (t)
J = 1.9
0
H-3
7.04 (m)
7.04 (m)
7.56 (m)
-
7.16 (m)
7.16 (m)
7.55 (m)
6.93 (m)
6.93 (m)
6.44 (m)
-
0
H-5
6.91 (m)
6.91 (m)
0
H-6
H-1”
5.02 (dd)
J = 7.8,
J = 1.9
4.93 (d)
J = 7.7
4.92 (d)
J = 6.3
H-2”
H-3”
-
-
3.52 (m)
3.49 (m)
3.67 (m)
3.49 (m)
3.67 (dt)
J = 9.1,
J = 3.9
3.67 (t)
J = 9.0
H-4”
-
3.25 (m)
3.27 (t)
J = 9.3
3.27 (t)
J = 9.3
H-5”
H-6”
-
-
3.52 (m)
3.87 (m)
3.49 (m)
3.49 (m)
3.87 (m)
3.75 (m)
3.87 (d)
J = 11.4
3
.74 (m)
3
.75 (dd)
J = 10.9,
J = 5.5
C-4”-OCH3
-
3.60 (s)
3.60 (s)
-
3.60 (s)
0
C-4 -OCH3
3.87 (s)
-
-
-
-
-
0
C-4 -OH
8.57 (s)

Molecules 2018, 23, 2578
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Table 4. The 13C-NMR shifts (
δ) (ppm) of 4 -methoxyflavanone (3) and the products of its
0
biotransformations (3a, 3b, 3c) in Acetone-d , 600 MHz (Supplementary Materials).
6
Compound
Carbon
3
3a
3b
3c
C-2
C-3
C-4
C-4a
C-5
C-6
C-7
C-8
C-8a
80.2
44.8
192.1
121.9
127.4
122.1
136.8
118.9
162.5
132.2
128.9
114.8
160.9
114.8
128.9
-
80.1
44.8
80.4
44.8
80.4
44.8
192.0
121.9
127.4
122.2
136.8
118.9
162.5
133.8
128.7
117.4
158.9
117.4
128.7
101.6
74.0
192.1
122.0
113.7
153.0
126.8
119.9
158.0
131.1
129.0
116.2
158.6
116.2
129.0
102.7
75.0
192.1
122.0
113.7
153.0
126.8
119.9
157.9
131.9
114.7
146.0
146.3
119.2
116.0
102.6
75.0
0
C-1
C-2
C-3
C-4
C-5
C-6
0
0
0
0
0
C-1”
C-2”
-
C-3”
C-4”
-
-
78.0
80.1
77.9
79.9
77.8
79.9
C-5”
C-6”
-
-
77.1
62.1
77.0
62.0
76.9
62.0
C-4”-OCH3
C-4 -OCH3
-
60.6
-
60.5
-
60.5
-
0
55.6
3
3
.1.4. 6-Methoxyflavanone (4)
C H O , t 21.36; H-NMR, see Table 5; 13C-NMR, see Table 6.
1
16
14
3
R
.1.5. 6-Methoxyflavone (5)
C H O , t 19.63; H-NMR, see Table 5; 13C-NMR, see Table 6.
1
16
12
3
R
0
0
3
4
.1.6. Chemical Synthesis of 2 -Methoxyflavanone (1), 3 -Methoxyflavanone (2),
-Methoxyflavanone (3)
0
0
0 0
1), 3 -methoxyflavanone (2), and 4 -methoxyflavanone
The compounds 2 -methoxyflavanone (
3) were prepared according to the method we described earlier [26]. A total of 10 g of
(
0
0
the 2 -hydroxychalcone with a metoxy substituent (obtained from 2 -hydroxyacetophenone and
methoxy-substituted benzaldehyde) was dissolved in ethanol (75 mL) with the addition of sodium
acetate (8 g), and the reaction mixture was refluxed for 48 h. Crystallization from ethanol afforded
pure methoxyflavanone.

Molecules 2018, 23, 2578
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1
Table 5. The H-NMR shifts (
δ
) (ppm) and coupling constants (JH,H) (Hz) of 6-methoxyflavanone (
) and the products of their biotransformations (4a 4b 5a 5b 5c) in Acetone-d6,
00 MHz (Supplementary Materials).
4)
and 6-methoxyflavone (
5
,
,
,
,
6
Compound
Proton
H-2
4
4a
4b
5
5a
5b
5c
5.62 (dd)
7.56 (dd)
5.52 (dd)
-
-
-
-
J2,3a = 13.1, J2,3a = 13.0, J2,3a = 12.8,
J2,3e = 2.9
J2,3e = 2.8
J2,3e = 2.9
H-3a
3.15 (dd)
J3a,2 = 13.1, J3a,2 = 13.0,
3.17 (dd)
3.14 (m)
-
-
-
-
J3a,3e
6.8
=
J3a,3e =
16.8
1
H-3e
2.88 (m)
-
2.87 (m)
-
2.85 (m)
-
H-3
6.88 (s)
6.88 (s)
6.80 (s)
6.79 (s)
(flavone)
H-5
H-7
7.33 (d)
J5,7 = 3.2
7.32 (d)
J5,7 = 3.1
7.31 (m)
7.21 (m)
7.54 (d)
J5,7 = 3.1
7.54 (m)
7.54 (d)
J5,7 = 3.1
7.54 (d)
J5,7 = 3.1
7.23 (dd)
J7,5 = 3.2,
J7,8 = 9.0
7.22 (dd)
J7,5 = 3.2,
J7,8 = 9.0
7.43 (dd)
J7,5 = 3.1,
J7,8 = 9.1
7.43 (dd)
J7,5 = 3.2,
J7,8 = 9.1
7.41 (dd)
J7,5 = 3.2,
J7,8 = 9.1
7.42 (dd)
J7,5 = 3.2,
J7,8 = 9.1
H-8
7.08 (d)
J8,7 = 9.0
7.05 (d)
J8,7 = 9.0
7.05 (m)
7.14 (d)
7.73 (d)
J8,7 = 9.1
7.78 (d)
J8,7 = 9.1
7.72 (d)
J8,7 = 9.1
7.74 (d)
J8,7 = 9.1
0
H-2
7.62 (d)
7.54 (d)
8.13 (m)
7.64 (m)
7.81 (m)
-
8.08 (m)
7.28 (m)
7.61 (m)
-
J2
0
0
= 7.4
J
0
0
= 8.6
J
0
0
= 2.2
,3
2 ,3
2 ,5
0
H-3
7.49 (t)
J = 7.5
7.15 (d)
J3 = 8.6
-
0
0
,2
0
H-4
7.43 (m)
-
-
7.64 (m)
7.64 (m)
7.31 (m)
7.54 (m)
-
-
0
H-5
7.49 (t)
J = 7.5
7.15 (d)
J5 = 8.6
7.00 (dd)
= 2.1,
0
= 8.4
,6
7.28 (m)
7.37 (d)
0
0
J
0
0
J
0
0
= 8.5
,6
5 ,2
5 ,6
J5
0
0
H-6
7.62 (d)
7.54 (d)
= 8.6
7.24 (d)
= 8.3
8.13 (m)
-
7.77 (m)
8.08 (m)
7.58 (dd)
J6
0
0
= 7.4
J
0
0
J
0
0
J
0 0
= 2.3,
0
= 8.5
,5
,5
6 ,5
6 ,5
6 ,2
J6
0
H-1”
-
5.01 (d)
4.81 (d)
5.14 (d)
5.14 (d)
5.00 (d)
J = 7.8
J = 7.9
J = 7.8
J = 7.8
J = 7.8
H-2”
H-3”
-
-
3.51 (m)
3.53 (m)
-
-
3.57 (m)
3.71 (m)
3.55 (m)
3.69 (m)
3.57 (m)
3.72 (m)
3.67 (t)
J = 8.9
3.67 (t)
J = 9.0
H-4”
H-5”
-
-
3.26 (t)
J = 9.3
3.26 (m)
-
-
3.25 (dd)
J = 9.5,
J = 9.1
3.28 (m)
3.59 (m)
3.29 (m)
3.57 (m)
3.53 (m)
3.50 (m)
3.65 (ddd)
J = 9.7,
J = 5.5,
J = 1.9
H-6”
-
3.86 (m)
3.88 (m)
3.73 (m)
-
3.94 (m)
3.76 (m)
3.90 (m)
3.75 (m)
3.92 (m)
3.77 (m)
3
.72 (dd)
J = 12.0,
J = 4.8
C-4”-OCH3
C-6-OCH3
-
3.60 (s)
3.85 (s)
3.60 (s)
3.85 (s)
-
3.62 (s)
3.97 (s)
3.61 (s)
3.96 (s)
3.61 (s)
3.96 (s)
3.86 (s)
3.96 (s)

Molecules 2018, 23, 2578
14 of 18
Table 6. The 13C-NMR shifts (
and the products of their biotransformations (4a
δ
) (ppm) of 6-methoxyflavanone (
4
) and 6-methoxyflavone (
5)
,
4b
,
5a
,
5b, 5c) in Acetone-d , 151 MHz
6
(Supplementary Materials).
Compound
5
Carbon
4
4a
4b
5a
5b
5c
C-2
C-3
C-4
C-4a
C-5
C-6
C-7
C-8
C-8a
80.4
45.0
191.8
121.9
108.3
155.2
125.3
120.2
156.9
140.5
127.3
129.5
129.3
129.5
127.3
-
80.1
44.8
80.1
44.9
163.6
107.2
177.7
125.5
105.8
158.0
124.0
120.8
151.8
132.9
127.1
130.0
132.4
130.0
127.1
-
163.2
107.4
177.7
125.4
105.7
158.1
124.0
120.8
151.8
134.2
115.0
159.3
120.6
131.0
120.9
101.7
75.0
163.5
106.1
177.6
125.5
105.8
158.0
123.8
120.7
151.7
126.3
128.7
117.6
161.3
117.6
128.7
101.2
74.9
163.4
106.4
177.6
125.5
105.8
158.0
123.8
120.7
151.7
128.0
114.5
148.8
148.9
118.3
119.2
103.2
74.9
192.0
121.9
108.3
155.1
125.2
120.2
157.0
133.9
128.7
117.4
158.9
117.4
128.7
101.6
74.8
191.9
121.9
108.3
155.1
125.2
120.2
156.9
136.3
115.0
148.8
146.2
118.6
119.4
104.3
74.8
0
C-1
C-2
C-3
C-4
C-5
C-6
0
0
0
0
0
C-1”
C-2”
-
-
C-3”
C-4”
-
-
77.9
80.1
78.7
80.0
-
-
78.1
80.3
78.0
80.1
77.4
80.0
C-5”
C-6”
-
-
77.1
62.0
77.3
61.9
-
-
77.3
62.2
77.2
62.1
77.4
62.0
C-4”-OCH3
C-6-OCH3
-
60.6
56.1
60.6
56.0
-
60.6
56.2
60.6
56.2
60.6
56.2
56.0
56.2
3
.2. Microorganism
The molecular identification of the strain I. fumosorosea KCH J2 was described in our previous
◦
publication [20]. The microorganism was stored at 4 C on potato slants and freshly subcultured
before use.
3
.3. Analysis
The course of the biotransformation was determined with the use of chromatographic methods
TLC, HPLC). The TLC analysis was carried out on TLC Silica gel 60/Kieselguhr F254 plates (Merck,
(
Darmstadt, Germany) with a mixture of chloroform and methanol in the ratio of 9:1 as developing
system. The solution of 5% aluminum chloride in ethanol was used as the staining solution. The plates
were observed at two wavelengths: 254 and 365 nm.
HPLC analyses were performed with a Waters 2690 instrument equipped with a Waters
9
96 photodiode array detector, using an ODS 2 column (4.6
and a Guard-Pak Inserts Bondapak C18 pre-column. The following separation conditions were used:
gradient elution, using 80% of acetonitrile in 4.5% acetic acid solution (eluent A) and 4.5% acetic acid
eluent B); flow: 1 mL/min; detection wavelength 280 nm; program: 0–7 min, 10% A 90% B; 7–10 min,
×
250 mm, Waters, Milford, MA, USA)
µ
(
5
9
0% 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
0% A 10% B; 30–40 min, 100% A.
Enantiomeric excess values were determined using a Chiralpak AD-H HPLC column,
.6 mm × 250 mm (Diacel), with hexane:isopropanol (9:1) as the eluent (isocratic resolution).
4
0
Separation of the products obtained by the scale-up biotransformation of 3 -methoxyflavanone,
0
4
-methoxyflavanone, 6-methoxyflavanone, and 6-methoxyflavone was accomplished using a 1000 µm
preparative TLC silica gel plates (Analtech, Gehrden, Germany). The developing system was a mixture
of chloroform and methanol (9:1). After the separation, selected fractions were scraped out and

Molecules 2018, 23, 2578
15 of 18
compounds were extracted using ethyl acetate (twice) and tetrahydrofuran (once). The extracts were
combined and the solvents were removed using a rotary evaporator.
0
Products obtained by the scale-up biotransformation of 2 -methoxyflavanone were separated
®
with the use of Reveleris PREP Purification System (BUCHI Corporation, Flawil, Switzerland) using
Reveleris Silica 4 g column. The separation conditions were as follows: gradient elution, using
®
chloroform (eluent A) and methanol (eluent B); flow: 20 mL/min; detection wavelengths: 254 nm,
2
90 nm, and 360 nm; mode: Flash Dry.
NMR spectra were obtained using a Bruker Avance600 MHz NMR spectrometer (Bruker, Billerica,
MA, USA) with an UltraShield Plusmagnet.
Molecular formulas of products were confirmed with the use of high-pressure liquid
chromatography HPLC 1200 Agilent Technologies with DAD, FLD, and Mass detectors Triple Quad
LC/MS (Agilent Technologies, Santa Clara, CA, USA).
Optical rotation was measured by means of the digital polaimeter P-2000-Na (ABL&E-JASCO,
Kraków, Poland).
3
.4. Screening Procedure
Sabouraud medium (1% peptone, 3% glucose) was used in all the experiments. The fungus
was transferred to a 300 mL Erlenmeyer flask containing 100 mL of the medium. Pre-incubation
◦
was conducted at 25 C for 72 h using a rotary shaker (140 rpm). The screening was performed
in 100 mL Erlenmeyer flasks containing 30 mL of the medium. The medium was inoculated wit
0.5 mL of pre-grown culture and after 72-h incubation, 3 mg of the substrate dissolved in 0.5 mL of
tetrahydrofuran was added. A separate flask of microbial culture was used for each sample collection.
The biotransformation was carried out under the same conditions as pre-incubation. After 2, 5,
and 12 days of biotransformation, the metabolites were extracted adding 30 mL of ethyl acetate to
the fungal culture. The mixture was then shaken and the organic phase (extract) was separated.
The extracts were dried over MgSO (5 min), concentrated on a rotary evaporator and analyzed using
4
TLC and HPLC.
The stability of the substrate was determined under the same conditions, without the microorganism.
3
.5. Scale-Up Biotransformations
Scale-up biotransformations were conducted in 2 L flasks containing 500 mL of the Sabouraud
medium. A duration of 72 h of incubation followed the inoculation after which 50 mg of the substrate
dissolved in 1 mL of tetrahydrofuran was added. The scale-up biotransformation was conducted
◦
under analogous conditions to the screening (140 rpm, 25 C). After the time was determined on the
base of the screening experiments (different for each substrate used), the metabolites were extracted
using 200 mL of ethyl acetate (repeated three times, the procedure was as in the case of screening).
The extracts were dried over MgSO and concentrated in vacuo. Product separation was carried
4
0
out using preparative TLC plates or flash chromatography (in the case of 2 -methoxyflavanone (
The products structures were confirmed by means of spectroscopic methods ( H-NMR, C-NMR,
COSY, HMBC, HSQC).
1)).
1
13
The physical and spectral data of the products obtained are presented below (Tables 1–6)
(Supplementary Materials).
0
0
3
.5.1. 2 -Methoxyflavanone 5 -O-β-D-(4”-O-methyl)-glucopyranoside (1a)
C H O , Mw = 446.4, t 11.87, [α] = −5.4 , H-NMR, see Table 1; 13C-NMR, see Table 2.
2
0
◦
1
23
26
9
R
D
0
3
.5.2. Flavan-4-ol 2 -O-β-D-(4”-O-methyl)-glucopyranoside (1b)
C H O , Mw = 418.4, t 12.16, [α] = −25.7 , H-NMR, see Table 1; 13C-NMR, see Table 2.
2
0
◦
1
22
26
8
R
D

Molecules 2018, 23, 2578
16 of 18
0
.5.3. Flavan-4-ol 3 -O-β-D-(4”-O-methyl)-glucopyranoside (2a)
3
3
3
3
3
3
3
3
3
3
4
C H O , Mw = 418.4, t 10.90, [α] = −6.5 , H-NMR, see Table 1; 13C-NMR, see Table 2.
0
2
0
◦
1
22
26
8
R
D
.5.4. 3 -Hydroxyflavanone 6-O-β-D-(4”-O-methyl)-glucopyranoside (2b)
C H O , Mw = 432.4, t 10.54, [α] = −24.0 , H-NMR, see Table 1; 13C-NMR, see Table 2.
2
0
◦
1
22
24
9
R
D
0
.5.5. Flavanone 4 -O-β-D-(4”-O-methyl)-glucopyranoside (3a)
C H O , Mw = 416.4, t 11.49, [α] = −19.1 , H-NMR, see Table 3; 13C-NMR, see Table 4.
2
0
◦
1
22
24
8
R
D
0
.5.6. 4 -Hydroxyflavanone 6-O-β-D-(4”-O-methyl)-glucopyranoside (3b)
C H O , Mw = 432.4, t 10.34, [α] = −36.0◦ 1H-NMR, see Table 3; 13C-NMR, see Table 4.
20
22
24
9
R
D
0
0
.5.7. 3 ,4 -Dihydroxyflavanone 6-O-β-D-(4”-O-methyl)-glucopyranoside (3c)
C H O , Mw = 448.4, t 10.01, [α] = −25.7 , H-NMR, see Table 3; 13C-NMR, see Table 4.
2
D
0
◦
1
22
24 10
R
0
.5.8. 6-Methoxyflavanone 4 -O-β-D-(4”-O-methyl)-glucopyranoside (4a)
C H O , Mw = 446.4, t 11.68, [α] = −19.2 , H-NMR, see Table 5; 13C-NMR, see Table 6.
2
0
◦
1
23
26
9
R
D
0
0
.5.9. 3 -Hydroxy-6-methoxyflavanone 4 -O-β-D-(4”-O-methyl)-glucopyranoside (4b)
C H O , Mw = 462.4, t 10.45, [α] = −19.9 , H-NMR, see Table 5; 13C-NMR, see Table 6.
2
D
0
◦
1
23
26 10
R
0
.5.10. 6-Methoxyflavone 3 -O-β-D-(4”-O-methyl)-glucopyranoside (5a)
C H O , Mw = 444.4, t 11.23, [α] = −34.2 , H-NMR, see Table 5; 13C-NMR, see Table 6.
2
0
◦
1
23
24
9
R
D
0
.5.11. 6-Methoxyflavone 4 -O-β-D-(4”-O-methyl)-glucopyranoside (5b)
C H O , Mw = 444.4, t 10.95, [α] = −13.0 , H-NMR, see Table 5; 13C-NMR, see Table 6.
2
0
◦
1
23
24
9
R
D
0
0
.5.12. 3 -Hydroxy-6-methoxyflavone 4 -O-β-D-(4”-O-methyl)-glucopyranoside (5c)
C H O , Mw = 460.4, t 10.81, [α] = −25.1 , H-NMR, see Table 5; 13C-NMR, see Table 6.
2
D
0
◦
1
23
24 10
R
. Conclusions
Hereby, we reported on microbial glycosylation of methoxylated flavonoids. The strain
I. fumosorosea KCH J2 has an unprecedented ability to attach a sugar unit to flavonoid aglycones
lacking hydroxyl groups. In the case of flavanones with methoxy substituents in ring B,
4-O-methylglucopyranose was attached to ring B carbons but also attached to the C-6 position.
In the case of 6-methoxyflavanone and 6-methoxyflavone glycosylation occurred only in the B ring.
As a result, we obtained twelve flavonoid 4-O-methylglucopyranosides. None of the products has
been previously reported in the literature. The method presented by us is relatively easy, cheap,
and environmentally friendly and it allows us to obtain products in amounts that enable their further
use, for example, to assess their biological activities or to evaluate the bioavailability of flavonoid
sugar derivatives.
Supplementary Materials: Supplementary Materials can be found online.
Author Contributions: Conceptualization, M.D. and E.K.-S.; Formal analysis, M.D.; Funding acquisition, M.D.
and E.K.-S.; Investigation, M.D. and T.J.; Methodology, M.D. and E.K.-S.; Project administration, M.D. and E.K.-S.;
Resources, E.K.-S.; Supervision, E.K.-S.; Validation, M.D.; Visualization, M.D.; Writing—original draft, M.D.;
Writing—review & editing, M.D. and E.K.-S.
Funding: 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.

Molecules 2018, 23, 2578
17 of 18
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 1, 1a, 1b, 2, 2a, 2b, 3, 3a, 3b, 3c, 4, 4a, 4b, 5, 5a, 5b, 5c are
available from the authors.
©
2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
CC BY) license (http://creativecommons.org/licenses/by/4.0/).
(