T. Tanaka et al. / Tetrahedron 59 (2003) 7939–7947
7945
dismutation of the dimer quinone 3a;13,14 however, his
greatly insightful proposal has long been ignored because no
chemical evidence could be demonstrated. However, our
result described in this report substantiates his
assumption.
with 80% MeOH to give 2 (7.0 mg, 0.02 mmol) and 4
(44.7 mg, 0.048 mmol).
4.1.1. Compound 2.
A
tan amorphous powder,
[a]2D0¼þ0.58 (c 0.1, MeOH); IR (KBr) nmax 3389, 1694,
1
1632 cm21; UV (EtOH) lmax (1 10600) 277 nm; H NMR
(500 MHz, d6-acetone) d 2.830 (1/3H, dd, J¼10.4, 15.6 Hz,
H-4), 2.838 (2/3H, dd, J¼2.0, 17.2 Hz, H-4), 2.949 (2/3H,
dd, J¼4.8, 17.2 Hz, H-4), 3.006 (1/3H, dd, J¼6.1, 15.6 Hz,
H-4), 5.184 (1/3H, ddd, J¼2.3, 6.1, 10.4 Hz, H-3), 5.216
(2/3H, ddd, J¼2.0, 2.8, 4.8 Hz, H-3), 5.520 (2/3H, dd,
J¼2.8, 5.0 Hz, H-2), 5.585 (1/3H, dd, J¼2.3, 5.3 Hz, H-2),
5.891 (1/3H, d, J¼2.3 Hz, H-8), 5.899 (2/3H, J¼2.3 Hz,
H-8), 6.051 (1/3H, d, J¼2.3 Hz, H-6), 6.056 (2/3H, d,
J¼2.3 Hz, H-6), 6.214 (1/3H, d, J¼5.3 Hz, C-2–OH), 6.316
(2/3H, d, J¼5.0 Hz, C-2–OH), 7.030 (4/3H, s, galloyl
H-2,6), 7.158 (2/3H, s, galloyl H-2,6);13C NMR (125 MHz,
d6-acetone) d 21.12, 21.83 (C-4), 67.73, 69.45 (C-3), 90.98,
91.12 (C-2), 96.15, 96.25, 96.40, 96.60 (C-6,8), 99.06, 99.62
(C-4a), 109.84, 109.99 (galloyl C-2,6), 121.51 (galloyl C-1),
138.90 (galloyl C-4), 145.93, 145.98 (galloyl C-3,5),
153.72, 153.86 (C-8a), 156.74, 156.92, 157.66, 158.01
(C-5,7), 166.16, 166.27 (galloyl C-7); FABMS m/z 351
[MþH]þ. Anal. calcd for C16H14O9 5/2H2O: C, 48.61; H,
4.84. Found: C, 48.54; H, 4.72.
4. Experimental
IR and UV spectra were obtained with JASCO FT/IR-410
and JASCO V-560 spectrophotometers. Optical rotations
were measured with a JASCO DIP-370 digital polarimeter.
CD spectra were measured with a JASCO J-720w apparatus.
1H and 13C NMR, 1H–1H COSY, NOESY, HSQC and
HMBC spectra were recorded with a Unity plus 500
spectrometer (Varian Inc, USA) operating at 500 MHz for
1H, and 125 MHz for 13C, respectively. FABMS were
recorded on a JMS DX-303 spectrometer (JEOL Ltd.,
Japan), and m-nitrobenzyl alcohol or glycerol used as a
matrix. Elemental analysis was obtained with a Perkin–
Elmer 2400 II analyzer (Perkin–Elmer, Inc.). Column
chromatography was done on MCI-gel CHP 20P
(Mitsubishi Chemical Co.), Chromatorex ODS (Fuji Silysia
Chemical Ltd., Japan), TSK gel Toyopearl HW-40F
(TOSOH Co.) and Sephadex LH-20 (Pharmacia Fine
Chemical Co.). Thin layer chromatography (TLC) was
performed on precoated Kieselgel 60 F254 plates, 0.2 mm
thick (Merck) with benzene-ethyl formate-formic acid
(1:7:1, v/v) or CHCl3–MeOH–H2O (14:6:1, v/v) and
spots were detected by UV illumination, sprayed with 2%
ethanolic FeCl3 or 10% sulfuric acid reagent, and followed
by heating. Analytical high pressure liquid chromotography
(HPLC) was performed on a Cosmosil 5C18-AR II,
250£4.6 mm i.d. column (Nacalai Tesque Inc., Japan)
with gradient elution from 10 to 30% (30 min) and 30–75%
(15 min) of CH3CN in 50 mM H3PO4 at a flow rate of
0.8 mL/min, and detected with a MD-910 photodiode
array detector (JASCO Co., Japan)]. (2)-Epigallocatechin
3-O-gallate (1) were isolated from commercial green
tea according to Nonaka et al. and recrystallized from
H2O.3 Japanese pear fruits were purchased in a local
market.
4.1.2. Treatment of 4 with o-phenylenediamine. To a
solution of 4 (10 mg) in EtOH (1.0 mL) was added
o-phenylenediamine (5 mg) and AcOH (0.3 mL), and the
mixture was heated at 808C for 2.5 h. The mixture was
applied to a Sephadex LH-20 column (1.0 cm£15 cm) with
EtOH to give 1 (2.7 mg, 27.6%) and phenazine derivative
1b (3.3 mg, 29.2%):
a
red amorphous powder,
[a]2D0¼2240.48 (c 0.2, MeOH); IR (KBr) nmax 3325,
1692, 1608 cm21; UV (EtOH) lmax 370 (1 8920), 269
(61400) nm; 1H NMR (500 MHz, d6-acetone) d 3.058 (1H,
dd, J¼1.7, 17.5 Hz, H-4), 3.218 (1H, dd, J¼2.0, 17.5 Hz,
H-4), 5.593 (1H, br s, H-2), 5.904 (1H, br t, J¼1.3 Hz, H-3),
6.143, 6.206 (each 1H, d, J¼2.3 Hz, H-6 and H-8), 7.008
(2H, s, galloyl-H-2,6), 7.527 (1H, d, J¼1.6 Hz, H-20), 7.913
(2H, m, H-100, H-110), 8.016 (1H, br s, H-6), 8.195 (2H, m,
H-90, H-120); 13C NMR (125 MHz, d6-acetone) d 26.63
(C-4), 68.88 (C-3), 78.04 (C-2), 95.84, 96.78 (C-6,8), 98.87
(C-4a), 109.26 (C-20), 109.77 (galloyl C-2,6), 117.74 (C-60),
121.29 (galloyl C-1), 130.01,0 130.29, 131.29, 131.73
(C-90,100,110,120), 135.63 (C-4 ), 138.87 (galloyl C-4),
142.15 (C-10), 144.01, 144.39, 144.82 (C-50,70,80), 145.88
(galloyl C-3,5), 153.36 (C-30), 156.53, 157.54, 157.92
(C-5,7,8a), 165.90 (galloyl C-7); FABMS m/z 529
[MþH]þ. Anal. calcd for C28H20O9N2 5/4H2O: C, 61.04;
H, 4.12; N, 5.08. Found: C, 61.07; H, 4.38; N, 4.69.
4.1. Oxidation of 1 with a Japanese pear homogenate
Japanese pear fruits (100 g) were homogenized with H2O
(100 mL) and filtered through four layers of gauze. The
homogenate (120 mL) was added to a solution of 1 (0.5 g,
1.092 mmol) in H2O (50 mL) and vigorously stirred at room
temperature for 2 h. Vigorous stirring is necessary to supply
sufficient oxygen molecules, which are indispensable to
progress the reaction. The mixture was filtered through filter
paper (No. 101) and the filtrate was directly applied to an
MCI gel CHP20P column (2 cm£15 cm) without concen-
tration. After washing the column with 0.1% TFA in H2O,
elution was undertaken with 0.1% TFA containing increas-
ing proportions of MeOH (20–30%) to give 3 (103.5 mg,
0.11 mmol). The fractions containing 3 were concentrated
by rotary evaporator below 358C until MeOH was removed
and then lyophilized. Elution of the column with 30–40%
MeOH in 0.1% TFA afforded a fraction containing 4, which
was subjected to Sephadex LH-20 column chromatography
4.2. Decomposition of 4
A solution of 4 (11.5 mg) in H2O (1.15 mL) was heated at
1008C for 15 min and the mixture was subjected to column
chromatography over Sephadex LH-20 (1.0 cm£15 cm)
with 80% MeOH to give 1 (5.3 mg) and 2 (0.6 mg).
4.3. Isolation of 2 from black tea
Commercial black tea (600 g), a blended tea produced in
India and Sri Lanka, was extracted six times with boiling