Molecules 2017, 22, 81
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3.4. Identification of the Products
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Purified products were identified by NMR spectra analysis. H-NMR, 13C-NMR, 1H-1H-NMR
(COSY) and 1H-13C-NMR (HSQC, HMBC) were recorded on a DRX Bruker Avance TM 600 (600 MHz)
instrument. Negative-ion HR-ESIMS spectra were measured on a Bruker ESI-TOF Mass Spectrometer
micrOTOF-Q. The direct infusion of ESI-MS parameters: The mass spectrometer was operated in
negative ion mode with the potential between the spray needle and the orifice 4, 5 kV, nebulizer
◦
pressure of 0.4 bar, and a drying gas flow rate of 4 L/min at 200 C. The sample flow rate was
180 µL/min. Ionization mass spectra were collected at the ranges m/z 150–3000. The instrument
was calibrated with an Agilent electrospray calibration solution (ESI-L low concentration Tuning
Mix-Agilent Technologies, Agilent Product Number: G1969-85000) that was diluted with acetonitrile.
3.5. Obtained Biotransformation Products
7-O-
β
-D-4”-O-Methyl-glucopyranosyl-40-hydroxyisoflavone (4”-O-methyldaidzin), (biocatalyst: Beauveria
1
bassiana AM 278, yield: 33.3%): H-NMR (600 MHz, DMSO-d6)
δ: 3.06 (1H, m, H-4”), 3.31 (1H, m,
H-2”), 3.45 (1H, m, H-3”), 3.47 (3H, s, 4”-OCH3) 3.50 (1H, m, Ha-6”), 3.52 (1H, m, H-5”), 3.65 (1H, m,
Hb-6”), 4.75 (1H, m, 6”-OH), 5.13 (1H, d, J = 7.8 Hz, H-1”), 5.32 (1H, d, J = 5.5 Hz, 3”-OH), 5.52
(1H, d, J = 5.3 Hz, 2”-OH), 6.82 (2H, m, H-30, H-50), 7.14 (1H, dd, J = 2.3; 8.9 Hz, H-6), 7.22 (1H, d,
J = 2.3 Hz, H-8), 7.40 (2H, m, H-20, H-60), 8.05 (1H, d, J = 8.9 Hz, H-5), 8.38 (1H, s, H-2), 9.56 (1H, s,
40-OH); 13C-NMR (150 MHz, DMSO-d6)
δ: 60.2 (C-6”), 73.3 (C-2”), 75.7 (C-5”), 59.7 (C-4”OCH3), 76.2
(C-3”), 78.9 (C-4”), 99.6 (C-1”), 103.3 (C-8), 115.0 (C-30, 50), 115.5 (C-6), 118.5 (C-10), 122.3 (C-10), 123.7
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(C-3), 127.0 (C-5), 130.1 (C-20, 60), 153.4 (C-2), 157.0 (C-9), 157.3 (C-40), 161.3 (C-7), 174.8 (C-4). H- and
13C-NMR data were found to be identical to that reported in the literature [25].
7-O-
β
-D-Glucopyranosyl-5,40-dihydroxyisoflavone (genistin), (biocatalysts: Absidia coerulea AM 93, yield:
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12.5%; Absidia glauca AM 177, yield: 21.9%): H-NMR (600 MHz, DMSO-d6)
δ: 3.17 (1H, m, H-4”), 3.26
(1H, m, H-2”), 3.30 (1H, m, H-3”), 3.44 (1H, m, H-5”), 3.47 (1H, m, Ha-6”), 3.71 (1H, m, Hb-6”), 4.61
(1H, m, 6”-OH), 5.06 (1H, d, J = 7.5 Hz, H-1”), 5.08 (1H, m, 4”-OH), 5.15 (1H, m, 3”-OH), 5.41 (1H, m,
0
0
2”-OH), 6.47 (1H, d, J = 2.2 Hz, H-6), 6.72 (1H, d, J = 2.2 Hz, H-8), 6.83 (2H, m, H-3 , H-5 ), 7.40 (2H, m,
H-20, H-60), 8.43 (1H, s, H-2), 9.63 (1H, s, 40-OH), 12.94 (1H, s, 5-OH); 13C-NMR (150 MHz, DMSO-d6)
δ
: 60.6 (C-6”), 73.1 (C-2”), 76.4 (C3”), 69.6 (C-4”), 77.2 (C-5”), 94.5 (C-8), 99.6 (C-6), 99.8 (C-1”), 106.1
(C-10), 115.1 (C-30, 50), 121.0 (C-10), 122.6 (C-3), 130.2 (C-20, 60), 154.6 (C-2), 157.2 (C-9), 157.5 (C-40),
161.6 (C-5), 163.0 (C-7), 180.5 (C-4). H- and 13C-NMR data were found to be identical to that reported
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in the literature [41,42].
7-O-
β
-D-4”-O-Methyl-glucopyranosyl-5,40-dihydroxyisoflavone (4”-O-methylgenistin) (biocatalyst:
1
Beauveria bassiana AM 278, yield: 27.2%): H-NMR (600 MHz, DMSO-d6)
δ: 3.0 (1H, m, H-4”), 3.27 (1H,
m, H-2”), 3.44 (1H, m, H-3”), 3.46 (3H, s, 4”-OCH3) 3.49 (1H, m, Ha-6”), 3.52 (1H, m, H-5”), 3.63 (1H,
m, Hb-6”), 4.73 (1H, m, 6”-OH), 5.09 (1H, d, J = 7.8 Hz, H-1”), 5.30 (1H, d, J = 5.5 Hz, 3”-OH), 5.48 (1H,
0
0
d, J = 5.2 Hz, 2”-OH), 6.47 (1H, d, J = 2.2 Hz, H-6), 6.71 (1H, d, J = 2.2 Hz, H-8), 6.83 (2H, m, H-3 , H-5 ),
7.40 (2H, m, H-2 , H-6 ), 8.43 (1H, s, H-2), 9.62 (1H, s, 4 -OH), 12.94 (1H, s, 5-OH); 13C-NMR (150 MHz,
0
0
0
DMSO-d6) δ: 59.7 (C-4”-OCH3), 60.2 (C-6”), 73.2 (C-2”), 75.7 (C-5”), 76.1 (C3”), 78.9 (C-4”), 94.5 (C-8),
99.4 (C-1”), 99.5 (C-6), 106.1 (C-10), 115.1 (C-30, 50), 121.0 (C-10), 122.6 (C-3), 130.2 (C-20, 60), 154.6 (C-2),
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157.2 (C-9), 157.5 (C-40), 161.7 (C-5), 162.9 (C-7), 180.5 (C-4). H- and 13C-NMR data were found to be
identical to that reported in the literature [25,30].
7-O-
β
-D-Glucopyranosyl-5-hydroxy-40-methoxyisoflavone (sissotrin), (biocatalysts: Absidia coerulea AM
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93, yield: 4.6%; Absidia glauca AM 177, yield: 40.3%): H-NMR (600 MHz, CD3OD)
δ: 3.44–3.48 (1H,
m, H-4”), 3.47–3.51 (1H, m, H-2”), 3.71–3.74 (1H, m, Ha-6”), 3.83 (3H, s, 40-OCH3), 3.90–3.93 (1H, m,
Hb-6”), 5.05 (1H, m, H-1”), 6.51 (1H, d, J = 2.2 Hz, H-6), 6.70 (1H, d, J = 2.2 Hz, H-8), 6.98 (2H, pd,
J = 8.7 Hz, H-30, H-50), 7.48 (2H, pd, J = 8.7 Hz, H-20, H-60), 8.16 (1H, s, H-2); 13C-NMR (150 MHz,
CD3OD) δ
: 55.8 (C-40OCH3), 62.4 (C-6”), 71.2 (C-4”), 74.7 (C-2”), 77.8 (C-3”), 78.4 (C5”), 95.9 (C-8),