Enantioselective conversion of certain derivatives of 6-hydroxyflavanone

Article abstract of DOI:10.1016/j.molcatb.2014.01.013

In the culture of Aspergillus niger MB, three racemic flavonoid derivatives (6-acetoxy-, 6-propionoxy-, and 6-butyryloxyflavanone) undergo microbial transformations resulting in optically pure (-)-(S)-6,4′- dihydroxyflavanone formation. In turn, biotransf

Full text of DOI:10.1016/j.molcatb.2014.01.013

Journal of Molecular Catalysis B: Enzymatic 102 (2014) 59–65
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Enantioselective conversion of certain derivatives of
6-hydroxyflavanone
Edyta Kostrzewa-Susłowa,∗, Monika Dymarskaa, Agata Białon´ skab, Tomasz Janeczkoa
a Department of Chemistry, Wrocław University of Environmental and Life Sciences, Norwida 25, 50-375 Wrocław, Poland
b Faculty of Chemistry, University of Wrocław, F. Joliot-Curie 14, 50-383 Wrocław, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
In the culture of Aspergillus niger MB, three racemic flavonoid derivatives (6-acetoxy-, 6-propionoxy-,
and 6-butyryloxyflavanone) undergo microbial transformations resulting in optically pure (−)-(S)-6,4ꢀ-
dihydroxyflavanone formation. In turn, biotransformation of ( )-6-methoxyflavanone by the same strain
led to optically pure (+)-(R)-6,4ꢀ-dihydroxyflavanone. Besides the optically pure flavanones, the product
of dehydration, 6-hydroxyflavone, was also formed.
Received 17 September 2013
Received in revised form 11 January 2014
Accepted 13 January 2014
Available online 2 February 2014
© 2014 Elsevier B.V. All rights reserved.
Keywords:
Biotransformation
6-Methoxyflavanone
6-Acetoxyflavanone
6-Propionoxyflavanone
Aspergillus niger
1. Introduction
6-acetoxy- and 6-butyryloxyflavanone. Additionally, it gives the
possibility to compare these transformations with transformations
The great therapeutic potential of flavonoid compounds of natu-
ral origin is well known and described [1–3]. In the case of aglycones
their potential application is limited by low solubility, low per-
meability through biological membranes and insufficient stability
in lipophilic media [4,5]. Therefore, the alternative may be to use
either synthetic flavonoids, such as their esters, or biotransfor-
mation products [6,7]. For example, 6-propionoxyflavanone and
6-hydroxyflavanone (the product of microbial hydrolysis of 6-
propionoxyflavanone) augment TRAIL-induced apoptosis in HeLa
cells and sensitize TRAIL-resistant cancer cells by engaging extrin-
sic and intrinsic apoptotic pathway via up-regulation of TRAIL-R2
expression and induction of ꢀꢁm loss [6].
However, from the pharmacological point of view, therapeutic
properties of a drug only weakly depend on activity of the admin-
istered compound itself, but they mainly depend on the products
of its metabolism. Therefore, it is important to determine possi-
ble transformations of flavonoid compounds in mammals. The first
step may be biomimetic study with the help of filamentous fungi
[8–10].
of 6-methoxyflavanone and 6-hydroxyflavanone.
In search for optically pure products of valuable biological
properties (including flavonoids) asymmetric synthesis, enzymatic
reactions or biotransformations have been used [11,12]. Interac-
tions between an enzymatic system and a substrate are often
stereospecific. In the case of flavanones stereochemistry at the C-
2 stereogenic center is of high importance. It has been proved that
(S)-naringenin was 2-fold more potent as an inhibitor of CYP19 and
CYP2C19 than (R)-naringenin, whereas (R)-naringenin was 2-fold
more potent for CYP2C9 and CYP3A [13]. Chiral flavanones (e.g.
naringenin) are difficult to separate into pure enantiomers [13].
Different biological properties of the opposite enantiomers are the
reasons for developing methods of obtaining pure enantiomeric
forms of chiral flavanones. In this paper we present biotransforma-
tion using the strain of Aspergillus niger MB as a useful method to
achieve both R and S enantiomers of 6,4ꢀ-dihydroxyflavanone.
2. Materials and methods
The study described in this paper allows to trace metabolic
2.1. Analysis
transformations of
a synthetic substrate of known biolog-
ical properties-6-propionoxyflavanone [6] and its analogs:
The course of microbial transformation was monitored by TLC
(SiO2, DC Alufolien Kieselgel 60 F254, Merck, Darmstadt, Germany).
Chromatograms were developed using the following develop-
ing systems: hexane:ethyl acetate (7:3), dichloromethane:ethyl
acetate (1:1), toluene:diethyl ether (4:1). Column chromatography
∗
Corresponding author. Tel.: +48 0713205252.
E-mail address: ekostrzew@gmail.com (E. Kostrzewa-Susłow).
1381-1177/$ – see front matter © 2014 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2014.01.013

60
E. Kostrzewa-Susłow et al. / Journal of Molecular Catalysis B: Enzymatic 102 (2014) 59–65
(SiO2, Kieselgel 60, 230–400 mesh, 40–63 ␮m, Merck) was per-
formed using the same eluents. 1H NMR and 13C NMR spectra
were recorded with a Bruker Avance DRX 300 spectrometer. Mass
spectra were obtained using high resolution electrospray ioniza-
tion (ESI+-MS) (Waters LCT Premier XE mass spectrometer). Optical
rotation was measured on an Jasco P-2000-Na automatic polarime-
ter (ABL&E-JASCO, Kraków, Poland). CD spectrum was obtained on
Jasco J-715 CD/ORD spectropolarimeter in THF. Melting points were
determined with a Boetius apparatus (Kofler block). pH measure-
ments were carried out on a PH-100ATC pH-meter (Voltcraft).
HPLC analyses were performed with a Waters 2690 instru-
ment equipped with a Waters 996 photodiode array detector, using
ODS 2 column (4.6 mm × 250 mm, Waters) and a Guard-Pak Inserts
␮Bondapak C18 pre-column. Separation conditions were as fol-
lows: gradient 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.
(3H, m, H-3ꢀ, H-4ꢀ, H-5ꢀ), 7.35 (2H, m, H-2ꢀ, H-6ꢀ), and 7.51 (1H, d,
J5,7 = 2.7 Hz, H-5).
13C NMR (CDCl3) ı: 20.9 (CH3 ), 44.4 (C-3), 79.8 (C-2), 119.2
(C-5), 119.26 (C-8), 121.2 (C-10), 126.1 (C-7), 128.8 (C-2ꢀ, C-6ꢀ),
129.8 (C-3ꢀ, C-4ꢀ, C-5ꢀ), 138.4 (C-1ꢀ), 144.8 (C-6), 159.2 (C-9), 169.5
(>C = 0), and 191.1 (C-4). HRESI-MS [M+H+] (calculated/found) (m/z
283.1129/283.1124).
2.2.2. 6-Propionoxyflavanone (2)
C18H16O4; Melting point 112–115 ◦C; Rt 21.27 min (HPLC);
purity 99% (HPLC); [␣]58920 = 0, (c = 1.00, CH3CN).
1H NMR (THF-d8) ı: 1.19 (3H, t, J = 7.5 Hz, CH3CH2 ), 2.58 (2H,
q, J = 7,5 Hz, CH3CH2 ), 2.80 (1H, dd, J3eq,3ax = 16.8, J3eq,2 = 2.9 Hz,
H-3eq), 3.07 (1H, dd, J3ax,3eq = 16.7, J3ax,2 = 13.2 Hz, H-3ax), 5.58
(1H, dd, J2,3ax = 13.0, J2,3eq = 2.8 Hz, H-2), 6.80 (1H, dd, J7,8 = 8.8 Hz,
J7,5 = 3.0 Hz, H-7), 6.85 (1H, d, J5,7 = 2.9 Hz, H-5), 7.37 (3H, m, H-3ꢀ,
H-4ꢀ, H-5ꢀ), 7.52 (2H, m, H-2ꢀ, H-6ꢀ), and 7.87 (1H, d, J8,7 = 8.9 Hz,
H-8);
13C NMR (THF-d8) ı: 9.3 (CH3CH2 ), 28.2 (CH3CH2 ), 45.2 (C-
3), 81.2 (C-2), 111.9 (C-5), 116.4 (C-8), 119.9 (C-10), 127.3 (C-7),
128.8 (C-2ꢀ, C-6ꢀ), 129.4 (C-4ꢀ), 129.5 (C-3ꢀ, C-5ꢀ), 140.6 (C-1ꢀ), 157.9
(C-6), 163.5 (C-9), 172.3 (>C = 0), and 190.5 (C-4). HRESI-MS [M+H+]
(calculated/found) (m/z 297.1430/297.1426).
Enantiomeric excess values were determined using a Chi-
ralpak AD-H HPLC column, 4.6 mm × 250 mm (Diacel), with
hexane:isopropanol (9:1) as eluent (isocratic resolution).
Crystallographic measurements were performed at 100 K
using an Oxford Cryosystem device on a Kuma KM4CCD ꢂ-axis
diffractometer with a graphite-monochromated Mo K␣ radiation
Crystal data for (2): C18H16O4, Mw = 296.31, colorless plate, crys-
tal size 0.32 mm × 0.15 mm × 0.09 mm, monoclinic, space group
◦
˚
˚
˚
P21/n, a = 7.863(2) A, b = 17.876(4) A, c = 10.731(2) A, ˇ = 101.28(3) ,
˚
(ꢃ = 0.71073 A). The data were corrected for Lorentz and polariza-
3
V = 1479.2(6) A , Z = 4, Dx = 1.331 Mg m−3
, T = 100 K, R = 0.063,
˚
tion effects. No absorption correction was applied. Data reduction
and analysis were carried out with the CrysAlis CCD and CrysAlis
Red programs [14]. Structures were solved by direct methods
(program SHELXS97) and refined by the full matrix least-squares
method on all F2 data using the SHELXL97 programs [15] (for details
of refinement and the description of the crystal structure of 6-
propionoxyflavanone (2) and 6-hydroxyflavanone (5) see [16,17]).
wR = 0.140 (for 1906 with I > 2ꢄ(I)) for 263 variables [16].
2.2.3. 6-Butyryloxyflavanone (3)
C19H18O4; oily liquid; Rt 23.30 min (HPLC); purity 99% (HPLC);
[␣]58920 = 0, (c = 1.10, THF).
1H NMR (CDCl3) ı: 0.93 (3H, t, J = 7.4 Hz, CH3 ), 1.67 (2H,
m, CH3CH2 ), 2.42 (2H, t, J = 7.3 Hz, CH2 CH2 ), 2.80 (1H, dd,
J3eq,3ax = 16.9, J3eq,2 = 3.0 Hz, H-3eq), 2.93 (1H, dd, J3ax,3eq = 16.9,
J3ax,2 = 13.2 Hz, H-3ax), 5.36 (1H, dd, J2,3ax = 13.2, J2,3eq = 3.0 Hz, H-2),
6.95 (1H, d, J8,7 = 8.9 Hz, H-8), 7.12 (1H, dd, J7,8 = 8.9 Hz, J7,5 = 2.9 Hz,
H-7), 7.32 (5H, m, H-2ꢀ, H-6ꢀ,H-3ꢀ, H-4ꢀ, H-5ꢀ), and 7.50 (1H, d,
J5,7 = 2.9 Hz, H-5); 13C NMR (CDCl3) ı: 13.7 (CH3 ), 18.5 (CH3CH2 ),
36.1 (CH2CO ), 44.4 (C-3), 79.8 (C-2), 119.2 (C-5), 119.23 (C-8),
121.2 (C-10), 126.2 (C-7), 128.9 (C-2ꢀ, C-6ꢀ), 130.0 (C-4ꢀ, C-3ꢀ, C-5ꢀ),
138.5 (C-1ꢀ), 144.9 (C-6), 159.2 (C-9), 172.3 (>C = 0), and 191.3 (C-4).
HRESI-MS [M + H+] (calculated/found) (m/z 311.1627/311.1621).
2.2. Materials
Racemic substrates for biotransformation-6-methoxyflavanone
(4) and 6-hydroxyflavanone (5) were purchased from Sigma Chem-
ical Company (St. Louis, MO, USA).
6-Acetoxy- (1), 6-propionoxy- (2) and 6-butyryloxyflavanone
(3) were synthesized from 6-hydroxyflavanone (5) according to the
following procedure: to 0.25 mmol of racemic 6-hydroxyflavanone
(5) dissolved in 5 ml of tetrahydrofuran (THF) 0.62 mmol of pyri-
dine and 0.58 mmol of acetyl or propionyl or butyryl chloride were
added. The reaction mixture was stirred with a magnetic stirrer at
room temperature for 30 min (progress of the reaction was mon-
itored by TLC). When the substrate was fully consumed, 5 ml of
ethyl acetate was added and the reaction mixture was washed
with 0.5 M HCl until the solution became slightly acidic (pH = 5).
Then the organic layer was separated and the aqueous layer was
additionally extracted with ethyl acetate (3× 5 ml). The combined
organic layers were washed with brine until neutral and dried over
anhydrous MgSO4. After evaporation of the solvent and purifica-
tion by column chromatography (SiO2), ( )-6-acetoxyflavanone
(1), ( )-6-propionoxyflavanone (2), ( )-butyryloxyflavanone (3)
were obtained in 92%, 94% and 93% yields, respectively.
2.2.4. 6-Methoxyflavanone (4)
C16H14O3; melting point 140–143 ◦C; Rt 20.47 min (HPLC);
purity 98% (HPLC); 1H NMR (THF-d8) ı: 2.79 (1H, dd, J3eq,3ax = 16.8,
J3eq,2 = 2.8 Hz, H-3eq), 3.03 (1H, dd, J3ax,3eq = 16.8, J3ax,2 = 13.0 Hz, H-
3ax), 3.78 (3H, s, CH3O ), 5.48 (1H, dd, J2,3ax = 13.0, J2,3eq = 2.8 Hz,
H-2), 6.98 (1H, d, J8,7 = 8.9 Hz, H-8), 7.11 (1H, dd, J7,8 = 8.9 Hz,
J7,5 = 3.1 Hz, H-7), 7.32 (1H, d, J5,7 = 3.1 Hz, H-5), 7.36 (3H, m, H-3ꢀ,
H-4ꢀ, H-5ꢀ), and 7.51 (2H, m, H-2ꢀ, H-6ꢀ); 13C NMR (THF-d8) ı: 56.0
(OCH3), 45.5 (C-3), 80.8 (C-2), 108.5 (C-5), 120.1 (C-8), 122.1 (C-10),
125.2 (C-7), 127.3 (C-2ꢀ, C-6ꢀ), 129.2 (C-4ꢀ), 129.5 (C-3ꢀ, C-5ꢀ), 140.9
(C-1ꢀ), 155.5 (C-6), 157.2 (C-9) and 191.5 (C-4). HRESI-MS [M+H+]
(calculated/found) (m/z 255.1022/255.1013).
2.2.1. 6-Acetoxyflavanone (1)
17H14O4; Melting point 105–107 ◦C; Rt 19.13 min (HPLC);
2.2.5. 6-Hydroxyflavanone (5)
C
C15H12O3; melting point 234–235 ◦C; Rt 16.07 min (HPLC);
purity 99% (HPLC). A full description of the 1H NMR, 13C NMR spec-
tra and crystal data of 6-hydroxyflavanone (5) can be found in our
previous paper [17,18]. HRESI-MS [M+H+] (calculated/found) (m/z
241.0862/241.0856).
purity 99% (HPLC); [␣]58920 = 0, (c = 1.00, THF).
1H NMR (CDCl3) ␦: 2.19 (3H, s, CH3 ), 2.80 (1H, dd, J3eq,3ax = 16.9,
J3eq,2 = 3.0 Hz, H-3eq), 2.93 (1H, dd, J3ax,3eq = 16.9, J3ax,2 = 13.3 Hz,
H-3ax), 5.36 (1H, dd, J2,3ax = 13.3, J2,3eq = 3.0 Hz, H-2), 6.95 (1H, d,
J8,7 = 8.9 Hz, H-8), 7.12 (1H, dd, J7,8 = 8.9 Hz, J7,5 = 2.7 Hz, H-7), 7.31

E. Kostrzewa-Susłow et al. / Journal of Molecular Catalysis B: Enzymatic 102 (2014) 59–65
61
2.3. Microorganism
3.2.2. 6-Hydroxyflavone (7)
C15H10O3; melting point 231–233 ◦C; Rt 14.0 min (HPLC); purity
99% (HPLC). Full description of the 1H NMR and 13C NMR spectra
of 6-hydroxyflavone (7) can be found in our previous paper [8,19].
HRESI-MS [M+H+] (calculated/found) (m/z 239.0765/239.0759).
A. niger MB was obtained from Wrocław University of Eco-
nomics (Poland). The microorganism was maintained on potato
slants (sterilized piece of potato) at 5 ◦C.
3. Biotransformations
4. Results and discussion
3.1. Initial biotransformations
Esterification of racemic 6-hydroxyflavanone (5) gave three
racemic derivatives: 6-acetoxy- (1), 6-propionoxy- (2) and 6-
butyryloxyflavanone (3), which were used as substrates for
biotransformations in the culture of the strain A. niger MB.
Biotransformations of racemic 6-methoxyflavanone (4) and 6-
hydroxyflavanone (5) with the help of the same species were also
carried out, for comparison.
In the first step we performed small-scale biotransformations,
using portions of 10 mg of the substrates. The results presented in
Figs. 1–4 show time–course of the reaction. After the first hour of
biotransformation of 6-acetoxyflavanone (1) 35.7% of the unreacted
substrate was observed in the reaction mixture (HPLC), along with
63.1% of the hydrolysis product – 6-hydroxyflavanone (5). After
3 h the substrate was fully consumed and 6-hydroxyflavanone (5)
was the only biotransformation product (95.7%). On the sixth day
two other products appeared: 6,4ꢀ-dihydroxyflavanone (6) and 6-
hydroxyflavone (7) with the yields of 14.8% and 10.1%, respectively.
With the progress of the reaction the amounts of 6 and 7 slightly
increased, along with a decrease in amount of 6-hydroxyflavanone
(5), to reach the yields of 25% and 17%, respectively, after 14 days
of biotransformation (Fig. 1).
In the case of biotransformation of 6-propionoxyflavanone (2)
catalyzed by enzymatic system of A. niger MB, after the first hour
49.8% of unreacted substrate was observed in the reaction mixture
and the only biotransformation product was 6-hydroxyflavanone
(5) (50.1%). The amount of the hydrolysis product increased with
time to rich the yield of 96.1% after the first day. On the third
day of the biotransformation two other products appeared: 6,4ꢀ-
dihydroxyflavanone (6) (24.7%) and 6-hydroxyflavone (7) (30.1%).
The amounts of 6 and 7 were slightly increasing with time, along
with a proportional decrease in amount of 6-hydroxyflavanone (5)
to reach the yields of 27.0% and 35.2%, respectively, after 14 days
of the reaction (Fig. 2).
For the next substrate – 6-butyryloxyflavanone (3) the percent
of conversion after 3 h was 95%. The only observed biotransfor-
mation product was the product of hydrolysis (5). Other products
appeared after one day of the reaction: 6,4ꢀ-dihydroxyflavanone (6)
(14.1%) and 6-hydroxyflavone (7) (17.3%). Their amounts increased
at a low rate during the progress of the biotransformation and after
14 days reached the yields of 15.4% and 18%, respectively (Fig. 3).
Microbial transformation of 6-methoxyflavanone (4) in the cul-
ture of A. niger MB proceeded slower than for esters 1–3. The
hydrolysis product 6-hydroxyflavanone (5) appeared after one day
in 57.2% yield. Products 6 and 7 were observed after 3 days in
the yields of 16.7% and 16.1%, respectively, which after 14 days
increased to 27.3% and 29.5%. On the 14th day there was still
16.1% of unreacted substrate (3) and 14.2% of hydrolysis product
(5) in the reaction mixture (Fig. 4). Also in this case when mon-
itoring the reaction course between 1st and 3rd day one can see
that compounds 6 and 7 are the products of transformation of
6-hydroxyflavanone (5), which is formed first.
Cultivation media consisted of 3% glucose (The Industrial and
Trading Enterprise “Stanlab” Co. Ltd., Poland) and 1% peptobac (BTL
sp. z o.o., Poland) in water. The microorganism was transferred from
the slants to 500 ml Erlenmayer flasks, each containing 200 ml of
the medium. Pre-incubation was performed at 25 ◦C for 24–48 h.
Then portions of 1 ml of the culture solution were transferred to
inoculate 500 ml flasks, each containing 200 ml of the medium.
After cultivation at 25 ◦C for 24 hours on a rotary shaker, 10 mg
of a substrate, dissolved in 0.5 ml of THF, was added to the grown
culture. Control cultivation with no substrate was also performed.
After 1, 3, 6, 9 h and 1, 3, 6, 7, 9, 10, 14 days of incubation under
the above conditions, portions of 5 ml of the transformation mix-
ture were withdrawn and extracted with ethyl acetate (3× 3 ml).
The extracts were dried over MgSO4 (5 min), concentrated in vacuo
and analyzed by TLC. Quantitative analyses of the mixtures were
performed by means of HPLC. Calibration curves for quantitative
analyses were prepared using isolated and purified biotransforma-
tion products as standards.
Additionally, stability tests for substrates were carried out, using
water solutions of citric acid of the same pH as in biotransformation
mixtures.
3.2. Preparative biotransformations
Portions of 1 ml of the pre-incubation culture solution were used
to inoculate three 2000 ml flasks, each containing 500 ml of the cul-
tivation medium. The cultures were incubated at 25 ◦C for 48 h on
a rotary shaker. Then 50 mg of the substrate dissolved in 2.5 ml of
THF was added to each flask (100 mg of the substrate per 1 l of
the cultivation mixture). After certain time of incubation the mix-
tures were extracted with ethyl acetate (3× 200 ml), dried (MgSO4)
and concentrated in vacuo. The transformation products were sep-
arated by column chromatography. Pure products were identified
by means of spectral analyses (TLC, 1H NMR, 13C NMR).
Physical and spectral data of the products obtained are pre-
sented below.
3.2.1. 6,4ꢀ-Dihydroxyflavanone (6)
C
15H12O4; melting point 223–224 ◦C; Rt
13.10 min
(HPLC); purity 99% (HPLC); 1H NMR (CD3CN) ı: 2.73 (1H,
dd, J3eq,3ax = 16.9 Hz, J3eq,2 = 2.9 Hz, H-3eq), 3.06 (1H, dd,
J3ax,3eq = 16.9 Hz, J3ax,2 = 13.1 Hz, H-3ax), 5.37 (1H, dd, J2,3ax = 13.1 Hz,
J2,3eq = 2.8 Hz, H-2), 6.84 (2H, d, J = 8.5 Hz, H-3ꢀ, H-5ꢀ), 6.89 (1H, d,
J8,7 = 8.9 Hz, H-8), 7.03 (1H, dd, J7,8 = 8.9, J7,5 = 3.0 Hz, H-7), 7.17 (1H,
d, J5,7 = 2.9 Hz, H-5), and 7.32 (2H, d, J = 8.4 Hz, H-2ꢀ, H-6ꢀ); 1H NMR
(THF-d8) ı: 2,71 (1H, dd, J3eq,3ax = 16.9 Hz, J3eq,2 = 2.8 Hz, H-3eq),
3.02 (1H, dd, J3ax,3eq = 16.9 Hz, J3ax,2 = 13.5 Hz, H-3ax), 5.34 (1H, dd,
J2,3ax = 13.5 Hz, J2,3eq = 2.8 Hz, H-2), 6.80 (2H, d, J = 8.4 Hz, H-3ꢀ, H-5ꢀ),
6.88 (1H, d, J8,7 = 8.9 Hz, H-8), 6.98 (1H, dd, J7,8 = 8.9, J7,5 = 3.0 Hz,
H-7), 7.21 (1H, d, J5,7 = 3.0 Hz, H-5), 7.33 (2H, d, J = 8.4 Hz, H-2ꢀ,
H-6ꢀ), 8.38 (1H, s, 4ꢀ-OH), and 8.52 (1H, s, 6-OH); 13C NMR (CD3CN)
ı: 44.7 (C-3), 79.1 (C-2), 111.2 (C-5), 116.2 (C-8), 122.1 (C-10),
125.3 (C-7), 129.2 (C-2ꢀ, C-6ꢀ), 131.5 (C-3ꢀ, C-5ꢀ), 139.2 (C-1ꢀ), 152.1
(C-6), 156.4 (C-9), 158.2 (C-4ꢀ), and 193.1 (C-4); HRESI-MS [M+H+]
(calculated/found) (m/z 257.0853/257.0848).
So as to isolate the products and to establish their struc-
tures preparative-scale biotransformations were carried out. In a
6-day biotransformation of 6-acetoxyflavanone (1) we obtained
6-hydroxyflavanone (5) in 39.28% yield, 6,4ꢀ-dihydroxyflavanone
(6) in 12.28% yield and 6-hydroxyflavone (7) in 10.7% yield. A
nine-day biotransformation of 6-propionoxyflavanone (2) gave

62
E. Kostrzewa-Susłow et al. / Journal of Molecular Catalysis B: Enzymatic 102 (2014) 59–65
Fig. 1. Biotransformation of 6-acetoxyflavanone (1) in A. niger MB culture – yield (%) of products determined by HPLC.
Fig. 2. Biotransformation of 6-propionoxyflavanone (2) in A. niger MB culture – yield (%) of products determined by HPLC.
Fig. 3. Biotransformation of 6-butyryloxyflavanone (3) in A. niger MB culture – yield (%) of products determined by HPLC.
Fig. 4. Biotransformation of 6-methoxyflavanone (4) in A. niger MB culture – yield (%) of products determined by HPLC.

E. Kostrzewa-Susłow et al. / Journal of Molecular Catalysis B: Enzymatic 102 (2014) 59–65
63
OH
O
O
HO
4'
O
O
2
A. niger MB
(6) (-)-(S)-6,4'-dihydroxyflavanone
6
3
RO
4
HO
5
O
O
O
(1) R1 -C(O)CH3 ( )-6-acetoxyflavanone
(5) ( )-6-hydroxyflavanone
(2) R2 -C(O)CH2CH3 ( )-6-propionoxyflavanone
(3) R3 -C(O)CH2CH2CH3 ( )-6-butyryloxyflavanone
HO
O
(7) 6-hydroxyflavone
Scheme 1. Microbial transformations of esters of 6-hydroxyflavanone (1–3) in the culture of A. niger MB.
6-hydroxyflavanone (5) in 33.69% yield, 6,4ꢀ-dihydroxyflavanone
(6) in 9.88% yield and 6-hydroxyflavone (7) in 14.18% yield. A
13-day biotransformation of 6-butyryloxyflavanone (3) led to 6-
hydroxyflavanone (5) in 36.1% yield, 6,4ꢀ-dihydroxyflavanone (6)
in 16.7% yield and 6-hydroxyflavone (7) in 17.3% yield. In all the
cases the substrates were fully consumed. Preparative-scale bio-
transformation of 6-methoxyflavanone (4) was carried out for 7
days. We obtained 24.7% of 6-hydroxyflavanone (5), 18.3% of 6,4ꢀ-
dihydroxyflavanone (6) and 5.34% of 6-hydroxyflavone (7). 17.7% of
unreacted substrate 4 ([␣]58920 = 0, c = 1.20, THF) remained in the
reaction mixture. For comparison, we subjected to the biotrans-
formation racemic 6-hydroxyflavanone. The only reaction product
was 6-hydroxyflavone (7), which after 12 days reached the yield
of 70% and the substrate was fully consumed. Structures of the
products were determined by 1H NMR and 13C NMR.
Enantiomeric excesses of products 5 and 6 and unreacted
substrate 4 were determined by HPLC, using chiral column. In
all the experiments 6-hydroxyflavanone (5), obtained either by
hydrolysis or demethylation, was racemic, as well as unreacted 4.
Whereas, 6,4ꢀ-dihydroxyflavanone (6) was obtained with 100% of
enantiomeric excess in each of the experiments. Specific rotation
measurements and circular dichroism analysis revealed that the
optically pure product of biotransformation of esters: 6-acetoxy-
(1), 6-propionoxy- (2) and 6-butyryloxyflavanone (3) was found to
be (−)-(S)-6,4ꢀ-dihydroxyflavanone (6) (retention time 30.93 min,
HPLC chiral column) ([␣]58920 = −19.49, c = 0.70, SD = 0.469, THF)
(Scheme 1). The absolute configuration at C-2 was established
by CD analysis [20,21]. The CD spectrum showed a high ampli-
tude negative Cotton effect at the ␲→␲ absorption band in the
*
280–300 nm region ([ꢅ]289 = −13.26), which allowed an assignment
of S-configuration at the C-2 stereocenter. The structure of prod-
uct 6 obtained in biotransformation of 6-methoxyflavanone (4)
was found to be (+)-(R)-6,4ꢀ-dihydroxyflavanone (retention time
33.56 min, HPLC chiral column) ([␣]58920 = +4.65, c = 1.9, SD = 0.195,
THF) (Scheme 2). The CD spectrum showed a high amplitude posi-
tive Cotton effect in the 280–300 nm region ([ꢅ]289 = 10.01), which
allowed an assignment of R-configuration at the C-2 stereocenter.
In the case of esters 1–3 of 6-hydroxyflavanone, the first step
of biotransformation in the culture of A. niger MB is hydroly-
sis, then enantioselective hydroxylation at C-4ꢀ in ring B occurs,
leading to (−)-(S)-6,4ꢀ-dihydroxyflavanone. Additionally, in the
second step of the transformation, along with hydroxylation
of 6-hydroxyflavanone (5) to (−)-(S)-6,4ꢀ-dihydroxyflavanone,
dehydration product is obtained: 6-hydroxyflavone (7). Biotrans-
formation of 6-methoxyflavanone (4) proceeds in an analogous
way, with this difference that in the second step the enan-
tioselective hydroxylation leads to the opposite enantiomer:
(+)-(R)-6,4ꢀ-dihydroxyflavanone.
The absolute configuration of the obtained product – either
(−)-(S)- or (+)-(R)-6,4ꢀ-dihydroxyflavanone – depends on the
kind of a substrate used for the biotransformation. When 6-
hydroxyflavanone was used as a substrate – no optically active
product of biotransformation was found. Optically pure prod-
ucts were formed only in the case when 6-hydroxyflavanone was
OH
O
HO
O
O
O
O
A. niger MB
(6) (+)-(R)-6,4'-dihydroxyflavanone
CH3O
HO
O
O
(4) ( )-6-methoxyflavanone
(5) ( )-6-hydroxyflavanone
HO
O
(7) 6-hydroxyflavone
Scheme 2. Microbial transformations of 6-methoxyflavanone (4) in the culture of A. niger MB.

64
E. Kostrzewa-Susłow et al. / Journal of Molecular Catalysis B: Enzymatic 102 (2014) 59–65
OH
O
O
O
dehydratase
2-hydroxylase
HO
HO
HO
H2O
O
O
O
6-hydroxyflavone
(R)-6-hydroxyflavanone
Scheme 3. Probable course of dehydration of 6-hydroxyflavanone.
by Britsch and co-workers, we assume that at first hydroxylation
at C-2 in 6-hydroxyflavanone (5) takes place, which is followed
by dehydration leading to a double bond between C-2 and C-3
(Scheme 3) [22].
5. Conclusions
In the case of biotransformation of 6-acetoxy-, 6-propionoxy-
and 6-butyryloxyflavanone enzymatic system of A. niger MB in the
first step catalyzes hydrolysis of the substrates, which is followed
by enantioselective hydroxylation at C-4ꢀ in the B-ring, lead-
ing to (−)-(S)-6,4ꢀ-dihydroxyflavanone. In the second step of the
transformation, along with (−)-(S)-6,4ꢀ-dihydroxyflavanone, from
6-hydroxyflavanone the product of dehydration is also formed:
6-hydroxyflavone. Biotransformation of 6-methoxyflavanone pro-
ceeds in an analogous way, only with this difference that
in the second step the enantioselective hydroxylation at C-
4ꢀ leads to (+)-(R)-6,4ꢀ-dihydroxyflavanone. Thus, depending on
the substrate used (either the ester of 6-hydroxyflavanone
or 6-methoxyflavanone) we obtain enantiopure either (−)-
(S)-6,4ꢀ-dihydroxyflavanone or (+)-(R)-6,4ꢀ-dihydroxyflavanone,
respectively.
Fig. 5. Molecular structure of the overlapping enantiomers of
propionoxyflavanone (2) [16].
( ) – 6-
an intermediate product in the biotransformation catalyzed by
the strain A. niger MB. In our earlier research concerning trans-
formations of racemic 6-hydroxyflavanone in the cultures of A.
niger strains we obtained only the products of dehydrogenation
or carbonyl group reduction [8]. Enzymatic system of A. niger
KB transformed the substrate into racemic 6-hydroxyflavan-4-ol,
whereas A. niger 13/5 catalyzed formation of 6-hydroxyflavone
[8].
Enantioselectivity of the enzymatic system of A. niger MB
may be dependent on molecular structure of a substrate and the
intermediate product 6-hydroxyflavanone (5). X-ray analysis of
(
)-6-propionoxyflavanone (2) (Fig. 5) shows that two enantiomers
are randomly and non-systematically arranged in the unit cell.
In the presented analysis the atom at position 2 in the pyrone
ring [C2 and H2 (major component) and C2A and H2A (minor
component)] and phenyl ring [C1ꢀ-C6ꢀ (major component) and
C1Aꢀ–C6Aꢀ (minor component)] are clearly resolved. Thus, the
two enantiomers occupy equivalent sites in the unit cell, how-
ever in a non-systematic way. The ratio of two enantiomers (R:S)
in the asymmetric part of the unit cell is approximately equal
to 0.8:0.2, which gives a 4:1/1:4 ratio in the crystal structure
overall [16]. In the case of the intermediate product ( )-6-
hydroxyflavanone (5) the ratio of two enantiomers (R:S) in the
asymmetric part of the unit cell is approximately equal to 0.75:0.25,
which gives a 3:1/1:3 ratio in the crystal structure overall (Fig. 6)
[17].
In the second step of biotransformation of esters (1–3) the enzy-
matic system of A. niger MB transforms one of the enantiomers
of the intermediate product (5), e.g. the S enantiomer (Fig. 6)
into (−)-(S)-6,4ꢀ-dihydroxyflavanone (6) (Scheme 1), whereas the
R enantiomer undergoes dehydration to 6-hydroxyflavone (7).
According to the mechanism of dehydration of flavanones proposed
Acknowledgements
This work was financed by the project “Biotransformations
for pharmaceutical and cosmetics industry” No. POIG.01.03.01-00-
158/09, which is partly financed by the European Union within the
“European Regional Development Fund for the Innovative Econ-
omy”.
References
[1] G. di Carlo, N. Mascolo, A.A. Izzo, F. Capasso, Life Sci. 65 (1999) 337–353.
[2] E. Middleton, C. Kandaswami, T.C. Theoharides, Pharmacol. Rev. 52 (2000)
673–751.
[3] B.H. Havsteen, Pharmacol. Ther. 96 (2002) 67–202.
[4] A. Arora, M.G. Nair, G.M. Strasburg, Arch. Biochem. Biophys. 356 (1998)
133–141.
[5] E. Kostrzewa-Susłow, J. Dmochowska-Gładysz, J. Oszmian´ ski, J. Mol. Catal. B:
Enzym. 49 (2007) 113–117.
[6] E. Szliszka, E. Kostrzewa-Susłow, J. Bronikowska, D. Jaworska, T. Janeczko, Z.P.
Czuba, W. Król, Molecules 17 (2012) 11693–11711.
[7] E. Kostrzewa-Susłow, T. Janeczko, Molecules 17 (2012) 14810–14820.
[8] E. Kostrzewa-Susłow, J. Dmochowska-Gładysz, A. Białon´ ska, Z. Ciunik, W.
Rymowicz, J. Mol. Catal. B: Enzym. 39 (2006) 18–23.
[9] E. Kostrzewa-Susłow, J. Dmochowska-Gładysz, T. Janeczko, Z. Naturforsch. C J.
Biosci. 65c (2010) 55–60.
[10] A. Wang, F. Zhang, L. Huang, X. Yin, H. Li, Q. Wang, Z. Zeng, T. Xie, J. Med. Plant
Res. 4 (2010) 847–856.
[11] S. Zhou, Y. Zhou, Y. Xing, N. Wang, A. Cao, Chirality 23 (2011) 504–506.
[12] E. Kostrzewa-Susłow, T. Janeczko, Sci. World J. (2012), http://dx.doi.org/10.
1100/2012/254929.
[13] W. Lu, V. Ferlito, C. Xu, D. Flockhart, S. Caccamese, Chirality 23 (2011)
891–896.
[14] Oxford Diffraction, Poland Sp. z o.o., CrysAlis CCD and CrysAlis Red, V166,
2001.
[15] Bruker, SHELXTL-NT. Version 5. 1, Bruker AXS Inc., Madison, WI, USA,
1999.
[16] E. Kostrzewa-Susłow, A. Białon´ ska, T. Janeczko, Acta Crystalogr. E66 (2010)
o1401, sup-9.
Fig. 6. Molecular structure of the overlapping enantiomers of
hydroxyflavanone [17].
( ) – 6-

E. Kostrzewa-Susłow et al. / Journal of Molecular Catalysis B: Enzymatic 102 (2014) 59–65
[17] A. Białon´ ska, Z. Ciunik, E. Kostrzewa-Susłow, J. Dmochowska-Gładysz, Acta [20] W. Gaffield, Tetrahedron 26 (1970) 4093–4108.
65
Crystalogr. E63 (2007) 430–431.
[21] A. Bartman´ ska, E. Huszcza, T. Tronina, J. Mol. Catal. B: Enzym. 61 (2009)
[18] E. Kostrzewa-Susłow, J. Dmochowska-Gładysz, A. Białon´ ska, Z. Ciunik, J. Mol.
221–224.
Catal. B: Enzym. 52–53 (2008) 34–39.
[19] E. Kostrzewa-Susłow, J. Dmochowska-Gładysz, T. Janeczko, K. Sroda, K. Micha-
[22] L. Britsch, W. Heller, H. Grisebach, Z. Naturforsch. C J. Biosci. 36c (1981)
742–750.
´
lak, A. Palko, Z. Naturforsch. C J. Biosci. 67c (2012) 411–417.