4878
K. Barkov aꢁ et al. / Tetrahedron 67 (2011) 4874e4878
2
ꢀ3
ꢀ1
5
.3. Product identification
ascorbic acid, 4ꢁ10ꢀ mL DMF and 5ꢁ10 e5ꢁ10 mM of the
2 2
flavonoid. The reactions were initiated with 1.8e2.2 mM H O and
ꢀ2
Reaction products were analyzed by HPLC using an Agilent HP
stopped by addition of 2ꢁ10 mL of 50% trichloroacetic acid after
20 s, at which time less than 5% of the apigenin had been consumed.
The resulting 6-hydroxyapigenin was quantified by HPLC as
described above, and the K
culated by nonlinear regression using the MicheliseMenten model
1200 liquid chromatograph (Agilent, Waldbronn, Germany)
equipped with diode array (DAD) and mass detectors (LC/MSD VL),
or a ProStar 210e510 liquid chromatograph (VARIAN, Walnut
Creek, USA) equipped with a diode array detector (DAD). Both
systems were used along with Synergi 4u Fusion RP 18 80A re-
m
and kcat values for apigenin were cal-
in the Sigma Plot 11.0 Enzyme Kinetics 1.3 program as apparent
3
5
versed phase columns (4.6 mm diameter by 250 mm length, 4
mm
constants.
particle size, Phenomenex Ltd., Aschaffenburg, Germany), which
ꢀ
1
ꢂ
was eluted at 1 mL min and 40 C with aqueous 0.01% vol/vol
ammonium formate (pH 3.5)/acetonitrile (95:5), followed by a 25-
min stepwise acetonitrile gradient (5 min to 50%, 15 min to 90%,
Acknowledgements
We thank M. Brandt and U. Schneider for technical assistance.
Financial support of the German Environmental Foundation (DBU,
project numbers 20008/959 and 13225e32) is gratefully
acknowledged.
2
min to 5%, 3 min at 5% acetonitrile). Products were identified
relative to authentic standards, based on their retention times,
þ
ꢀ
UV-absorption spectra, and [MþH] or [MꢀH] ions.
The reaction products 6-hydroxyapigenin and 6-hydrox-
1
3
yluteolin were analyzed via H and C NMR (Bruker, 400 MHz,
Bruker BioSpin GmbH, Rheinstetten, Germany). For these mea-
surements, the substances were dissolved in deuterated dimethyl
sulfoxide. Quantitative amounts (5 mg) of 6-hydroxyapigenin (yield
7.5%) and 6-hydroxyluteolin (yield 16.5%) were obtained from
semi-continuous enzymatic conversions.
References and notes
1
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3
1
2
4
For apigenin: the complete reaction mixture (65 mL) contained
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1
the peroxygenase (1.2 U mL ), potassium phosphate buffer
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5
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(
2
O
2
2 2
O
using a syringe pump (for 67 h). Apigenin and ascorbic acid (one
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1
the peroxygenase (1.3 U mL ), potassium phosphate buffer
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(
6
047e6053.
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2
O
2
2 2
O
1
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1
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ꢂ
NX12C18 column (110 A, 21.2 mmꢁ150 mm, 10
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2
2
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5
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ꢀ
1
ꢂ
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2
2
2
2
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2
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6
,7-dihydro-6,7-epoxyflavone) was analyzed by reversed phase
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ꢂ
1
10 A column (2 mm diameter by 150 mm length, 3
size, Phenomenex Ltd., Aschaffenburg, Germany). A mixture of
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mm particle
5
ꢂ
ꢀ1
as solvent at constant 40 C at a flow rate of 0.2 mL min under the
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3
3
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5
.4. Enzyme kinetics
The kinetics of apigenin hydroxylation were analyzed in stirred
ꢂ
ꢀ4
reaction mixtures (0.20 mL, 23 C) that contained 10
mM of
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