1342
E. KITAMURA et al.
Table 1. Inhibitory Effects of the Converted Products on Lipid
Peroxidation in a Rat Brain Homogenate
of the epoxidation of 1-phenyl-4-butanol to synthesize
2-phenethyl-oxirane by CYP153A13a (data not
shown).19) The conversion of the flavones (flavone and
40-hydroxyflavone) was not apparent in this study, while
the flavanones were oxygenated. Our previous studies
have shown such a result in some cases.6,20,21) On the
other hand, such a characteristic of inserting oxygen at
C-3 (C ring) is likely to have been unique to P450 BM3
(F87V) among our enzymes that could oxygenate
flavonoids.6,20,21)
Among products 1–8, compounds 1, 3 and 4 have
been reported to be generated by conventional biotrans-
formation using microbial cells.11,13,14) The bioconver-
sion of naringenin to apigenin (5) has recently been
reported with E. coli cells expressing the cyanobacterial
P450 CYP110E1 gene.6) This is therefore the first report
of the preparation of compounds 2, 6, 7, and 8 by
microbial bioconversion achieved by using recombinant
E. coli cells that expressed the substrate-promiscuous
P450 gene. Moreover, the natural occurrence of com-
pounds 2, 6, 7, and 8 has never before been reported,
while many chemical reactions to prepare 6 and 7 have
been described.22,23) Compounds 2 and 8 were rare
flavonoids whose preparation by organic synthesis has
rarely been reported.12,18)
Compound
IC50 (mM)
flavanone
1
81
8.4
20-hydroxyflavanone
52
30-hydroxyflavanone
>100
0.78
0.77
>100
1.2
2
3
40,5,7-trihydroxyflavanone (naringenin)
4
5 (apigenin)
>100
>100
>100
>100
88
chalcone
6
7
isoflavanone
8
quercetin
15
0.78
from chalcone were identified as 2-hydroxy-1,3-diphenyl-
propane-1-one (compound 6)15) (Fig. 1) and 3-hydroxy-
1,3-diphenyl-propane-1-one (compound 7)16) (Fig. 1). The
product from isoflavanone was identified as 3-hydroxy-
3-phenyl-chroman-4-one (compound 8)17) (Fig. 1).
Since each substrate used in this study (excepting
chalcone) was a racemic mixture at C-2, we examined
the absolute configurations of the products by chiral
HPLC analyses {Daicel Chiralcel OD-H column, 10 mm
i.d. ꢁ 250 mm, Daicel Corporation, Tokyo, Japan; n-
hexane/2-propanol solvent [9:1 (for 1 and 7) or 3:1 (for
2)]; 3.0 mL/min flow rate; detection by monitoring the
maximum absorbance in the range of 200–500 nm},
using high-yield compounds 1, 2 (the product from 20-
hydroxyflavone), and 7 as representatives. All the tested
compounds gave two peaks derived from the two
enantiomers [1 (tR 15.6 min and tR 21.6 min), 2 (tR
10.5 min and tR 11.6 min), 7 (tR 20.8 min and tR
23.1 min)], the ratio of the two peaks being approx-
imately 1:1. We judged from these results that all the
products (1, 2, 3, 4, 6, 7, and 8) were racemic mixtures.
Since hydrogenated products may exhibit anti-oxida-
tive activity due to their structures, we evaluated their
in vitro inhibitory effects on free radical-induced lipid
peroxidation in a rat brain homogenate.18) The results
are shown in Table 1. Compounds 1, 2, 3, and 4 showed
superior antioxidative activity when compared to the
corresponding substrates. In particular, compounds 2, 3,
and 4, which possessed a para- or ortho-hydroquinone
structure in the B ring, showed potent antioxidative
activities, being almost identical to that of quercetin (a
natural potent antioxidative flavone).
We consider that our bioconversion method using
recombinant E. coli would be superior to conventional
biotransformation using microbial cells in respect of
product foreseeability. The products produced in this
study can be candidates for biologically active chem-
icals, including medicines, and further biological studies
on these products are in progress.
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Kitamura, Emi
