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2
+
performed in ‘selective’ ways. Among them, trans-d-viniferin
was obtained in 91% yield when catalyzed by [HRP–Mn ], and
M
incorporated HRP catalyzed oxidation of 2
1
1
The oxidation procedure was the same as HRP catalyzed
oxidation of 1.
C]C bond cleavage was found in presence of [HRP–Cu ] or
6
[
HRP–Fe ]. Initial mechanistic analysis of these ‘man-made’
3
HRP–metal complexes was also carried out using circular
dichroism (CD) and Atomic Absorption Spectrometry (AAS).
Product analysis and separation
Analysis of the product solution was carried out on HPLC with a
ꢀ
C-18 column (column temperature, 25 C; mobile phase,
methanol and water at the gradient: methanol, 0–8 min, 40–
Experimental section
5
3%, 8–15 min, 53%, 15–20 min, 53–90%; ow rate, 0.8 mL
General procedures
ꢂ1
min ; detection, 280 nm UV). HPLC yields were calculated by
peak area, assuming all the products share the same absorption
at 280 nm. Separation was carried out on a preparative HPLC
with a C-18 column, using gradient elution (methanol, 0–24
min, 40–53%; 24–40 min, 53%; 40–60 min, 53–90%) and
detected at 280 nm UV. Separations were only carried out in
some of the reactions ([HRP–Mn], [HRP–Fe], [HRP–Cu] and
native HRP). A Sephadex LH-20 gel column was occupied
together with a preparative HPLC to purify the products. The
yields of corresponding products in different reactions were
listed in Table S1, ESI.†
All reagents were purchased at the highest commercial quality
and used without further purication. Reactions were magnet-
ically stirred and monitored by thin-layer chromatography
(
TLC) or high performance liquid chromatography (HPLC).
HPLC analysis was carried out using a C18 column (250 mm ꢁ
.6 mm i.d., 5 mm). Purication of compounds was carried out
4
with silica gel (academic grade, 200–300) and a preparative
HPLC (C18 column, 250 ꢁ 20 mm i.d). NMR spectra were
recorded on a 500 MHz instrument. Mass spectroscopic data
were obtained using oa-TOF mass spectrometer.
The analysis data of products are listed and the NMR spectra
are in the end of ESI.†
2
+
Generation of M incorporated HRPs
trans-d-Viniferin (2) was obtained as a yellow amorphous
Aqueous solution of HRP was prepared in concentration of 1 mg
mL . All metal chloride solutions were prepared in concentration
of 10 mM. For a M incorporated HRP generation, 3 mL of HRP
solution was mixed with 3 mL metal chloride solution, well
shaken, and then incubated for 24 hours. Dialysis was then carried
out with dialysis tube (M
hours, the dialyzed solution was then lyophilized at ꢂ70 C.
1
powder. H NMR (500 MHz, acetone) d 8.48 (s, 1H), 8.24 (s, 2H),
ꢂ1
8
1
6
.20 (s, 2H), 7.44 (d, J ¼ 5.0 Hz, 1H), 7.26 (m, 3H), 7.06 (d, J ¼
2
+
5.0 Hz, 1H), 6.92 (d, J ¼ 15.0 Hz, 1H), 6.87 (m, 3H), 6.55 (d, 2H),
.28 (d, J ¼ 10.0 Hz, 2H), 6.20 (d, J ¼ 5.0 Hz, 2H), 5.46 (d, J ¼ 5.0
13
Hz, 1H), 4.48 (d, J ¼ 5.0 Hz, 1H); C NMR (125 MHz, acetone) d
W
¼ 3500) in deionized water. Aer 72
1
1
1
60.6, 159.7, 159.5, 158.4, 145.2, 140.8, 132.5, 132.1, 131.7,
29.1, 128.6, 127.2, 123.9, 116.2, 110.1, 107.4, 105.7, 102.7,
02.3, 94.0, 57.8, 55.4. HRMS(EI) calcd for C28
ꢀ
+
+
H
22
O
6
[M ]
2
+
454.1416, found 454.1414.
Generation of M incorporated apo-HRP
Leachianol F (3) and leachianol G (4) was obtained as a
17
apo-HRP was prepared following the reported procedure. To
brown amorphous powder. The product got was a 1 : 2 mixture.
2
+
generate M incorporated apo-HRP, the same procedure as
forementioned one was carried out, except that apo-HRP was
used instead of HRP.
1
Data of 3: H NMR (500 MHz, acetone) d 7.99 (br, 5H), 7.47
(
s, 1H), 6.84–6.88 (m, 4H), 6.70–6.73 (m, 2H), 6.67 (d, J ¼ 5.0 Hz,
2
1
1
H), 6.58 (d, 1H), 6.30 (d, J ¼ 5.0 Hz, 1H), 6.12 (m, 1H), 5.92 (d,
H), 4.48 (m, 1H), 4.22 (d, J ¼ 5.0 Hz, 1H), 4.08 (d, J ¼ 5.0 Hz,
1
3
H), 3.36 (m, 1H), 2.94 (t, J ¼ 5.0 Hz, 1H); C NMR (125 MHz,
Horseradish peroxidase catalyzed oxidation of 1
acetone) d 159.1, 158.8, 157.1, 156.3, 155.0, 150.6, 148.7, 137.4,
2
+
A mixture of 1 (0.4 mmol, 91.2 mg) and M incorporated HRP
160 mL, 1 mg mL aqueous solution) was added to a mixed
solvent consisted of 2 mL acetone and 2 mL water. This solution
was then stirred at 40 C for one hour. Then 60 mL fresh 30%
136.1, 129.3, 128.8, 122.5, 115.6, 115.4, 106.1, 106.0, 102.4,
ꢂ1
+
(
1
H
01.2, 76.6, 61.8, 59.4, 55.6. HRMS(EI) calcd for C28
H
24
O
7
[(M ꢂ
+
1
2
O) ] 454.1416, found 454.1419. Data of 4: H NMR (500 MHz,
ꢀ
acetone) d 8.10 (br, 5H), 7.44 (s, 1H), 7.07 (d, J ¼ 10.0, 2H), 6.85
H
2
O
2
was then added to the solution. Aer one hour, the reac- (m, 2H), 6.67 (m, 4H), 6.22 (d, 2H), 6.16 (d, 2H), 6.14 (d, J ¼ 5.0
tion was quenched by saturated Na
2
S
2
O
3
solution, evaporated, Hz, 2H), 5.74 (d, J ¼ 5.0 Hz, 1H), 4.48 (m, 1H), 4.26 (d, 1H), 4.14
13
and extracted with EtOAc and water. The organic layer was (d, 1H), 3.48 (t, J ¼ 5.0 Hz, 1H), 3.40 (m, 1H); C NMR (125 MHz,
washed by brine and water for three times, dried over anhy- acetone) d 159.2, 158.5, 157.2, 156.2, 154.8, 151.4, 147.3, 138.0,
drous sodium sulphate and concentrated.
1
5
35.7, 129.4, 129.3, 106.2, 105.6, 102.4, 101.0, 77.3, 62.5, 59.0,
+
+
6.0. HRMS(EI) calcd for C28
H
24
O
7
[(M ꢂ H
2
O) ] 454.1419,
found 454.1419.
Metal chlorides catalyzed oxidation of 1
1
Pallidol (5) was obtained as a brown amorphous powder. H
To make it as control, this oxidation was carried out following NMR (500 MHz, acetone) d 8.19 (s, 2H), 8.15 (s, 2H), 7.92 (s, 2H),
the procedures of HRP catalyzed oxidation, where HRP was 7.05 (d, J ¼ 10.0 Hz, 4H), 6.77 (d, J ¼ 10.0 Hz, 4H), 6.70 (s, 2H),
1
3
replaced by metal chlorides (FeCl
.04 mmol).
2
, CuCl
2
and MnCl
2
in 6.26 (s, 2H), 4.63 (s, 2H), 3.88 (s, 2H); C NMR (125 MHz,
0
acetone) d 159.0, 155.1, 150.2, 137.6, 129.0, 123.2, 115.8, 103.3,
This journal is ª The Royal Society of Chemistry 2013
RSC Adv., 2013, 3, 22976–22980 | 22979