Journal of Agricultural and Food Chemistry
Article
Figure 1. Biotransformation of 5,7-dimethoxyflavone (1) by I. farinosa KCh KW 1.1.
hydrolysis with sulfuric acid was carried out on the basis of the study
by Zhang et al.35 with solvent modification. In this case, the sample
was dissolved in dimethyl sulfoxide (DMSO). The reaction was
carried out in a round bottom flask on a magnetic stirrer. 15 mg of
compound (7) was dissolved in 800 μL of DMSO. 500 μL of
concentrated sulfuric acid (96% v/v) was added dropwise to the
sample; then 500 μL of water was added very carefully (Scheme 2).
The progress of the reaction was controlled by TLC plates every 30
min. After two hours of reaction, when the starting substrate was no
longer observed, 5 mL of H2O was added to the reaction. Then, the
reaction mixture was extracted three times with ethyl acetate. Organic
fractions were collected together, and the remains of water were
removed with anhydrous MgSO4. Then, the sample was evaporated
using a vacuum evaporator. The sample was then analyzed by NMR
and LC−MS spectroscopy to confirm the structure of the product
obtained.
2.3. Microorganisms. The microorganisms B. bassiana KCh J1.5,
KCh J2.1, KCh J1, KCh J3.2, and KCh BBT; B. caledonica KCh J3.3
and KCh J3.4; I. farinosa KCh KW 1.1; and I. fumosorosea KCh J2
were obtained from the public culture collection of the Department of
Chemistry, Wrocław University of Environmental and Life Sciences
(Wrocław, Poland). Isolation and identification procedures of all
strains were described in our previous papers.8,22
2.4. Screening Procedure. Erlenmeyer flasks (300 mL), each
containing 100 mL of the sterile cultivation medium (3% glucose, 1%
aminobac-bacteriological peptoneenzymatic hydrolysate of selected
animal tissue high in free amino acids and low molecular mass
peptides), were inoculated with a suspension of each entomopatho-
genic strain and then incubated for 3 days at 24 °C on a rotary shaker.
After this time, 10 mg of a substrate dissolved in 1 mL of dimethyl
sulfoxide (DMSO) was added. Samples were collected on the 1st, 3rd,
7th, and 10th day of the process. Then, all products were extracted
using ethyl acetate, and extracts were dried using MgSO4,
concentrated in vacuo, and analyzed using TLC and HPLC methods.
2.4.1. Scale-up Biotransformation. For the scale-up process,
Erlenmeyer flasks (2000 mL) were used, each containing 500 mL of
the same cultivation medium (3% glucose, 1% aminobac), which was
inoculated in the same way as described above. Three days after
inoculation, 100 mg of a substrate dissolved in 2 mL of DMSO was
added. Processes of substrate conversion were performed individually
depending on substrates and previously obtained HPLC results.
Products were extracted three times using ethyl acetate and then
analyzed using TLC, HPLC, and NMR spectroscopy (1H NMR, 13C
NMR, COSY, HMBC, and HSQC) analysis.
phase contained a mixture of chloroform and methanol in a 9:1 (v/v)
ratio. The products were isolated by scraping out successive bands
and extracted twice with ethyl acetate.
2.5.1. HPLC. 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 μBondapak
C18 pre-column, was used to perform HPLC analyses. The mobile
phase consisted of eluent A (80% acetonitrile in 4.5% acetic acid
solution) and eluent B (4.5% acetic acid) with gradient elution: 0−7
min, 10% A/90% B; 7−10 min, 50% A/50% B; 10−13 min. 60% A/
40% B; 15−20 min 80% A/20% B; 20−30 min, 90% A/10% B; 30−40
min, 100% A. The flow rate was 1.0 mL/min, injection volume was 10
μL, and detection wavelength was 323 nm.
2.5.2. NMR Spectroscopy. The NMR analysis was performed with
a DRX 600 MHz Bruker spectrometer (Bruker, Billerica, MA, USA)
with an UltraShield Plus magnet and measured in DMSO-d6 or
2.5.3. LC−MS. Molecular formulas of products were confirmed by
LC−MS 8045 SHIMADZU analysis. The mobile phase was a mixture
of 0.1% aqueous formic acid v/v (A) and acetonitrile (B). The
program was as follows: 80% B and 20% A in 5 min. The flow rate was
0.3 mL/min, and the injection volume was 2 μL. The column
(Kinetex 2.6 μm C18 100 Å, 100 mm × 3 mm, Phenomenex,
Torrance, CA, USA) was operated at 30 °C. The major operating
parameters were as follows: nebulizing gas flow: 3 L/min, heating gas
flow: 10 L/min, interface temperature: 300 °C, drying gas flow: 10 L/
min, data acquisition range m/z 100−1000 Da; and ionization mode-
positive. Data were collected with LabSolutions (Shimadzu, Kyoto,
Japan) software.
3. RESULTS AND DISCUSSION
The 5,7-methoxyflavones obtained by chemical synthesis were
biotransformed in the cultures of nine entomopathogenic
filamentous fungal strains. The cultures of five B. bassiana
strains (KCh J1.5, KCh J2.1, KCh J3.2, KCh J1, and KCh
BBT), two B. caledonica strains (KCh J3.3 and KCh J3.4), and
two Isaria strains (I. fumosorosea KCh J2 and I. farinosa KCh
KW 1.1.) were used as biocatalysts. All mentioned strains were
used in our recent work,31 in which we described the catalytic
abilities of these fungi toward flavones containing a methoxy
group/groups within the B ring.
All the used substrates, containing one to three methoxy
groups in the structure, were obtained by a two-step chemical
synthesis. Three chalcones were synthesized from 2′-hydroxy-
4,6-dimethoxyacetophenone and the appropriate benzaldehyde
in a basic medium in the first stage. Then, they were
transformed into the appropriate methoxyflavones by reaction
with I2 in DMSO. As a result of these reactions, 5,7-
2.5. Analysis. Basic analyses were carried out using TLC plates
(SiO2, DC Alufolien Kieselgel 60 F254 (0.2 mm thick), Merck,
Darmstadt, Germany). The mobile phase contained a mixture of
chloroform and methanol in a 9:1 (v/v) ratio. The plates were
observed using a UV lamp (254 and 365 nm). The scale-up
biotransformation products were separated using 1000 μm preparative
TLC silica gel plates (Anatech, Gehrden, Germany). The mobile
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J. Agric. Food Chem. 2021, 69, 3879−3886