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S. Sang et al. / Bioorg. Med. Chem. 12 (2004) 459–467
eluted with 95% ethanol to give three catechins: epica-
techin gallate (ECG), epigallocatechin (EGC), and epi-
gallocatecin gallate (EGCG). Each catechin was further
purified by HPLCusing an RP C-18 column eluted with
a methanol–water solvent system.
6.00 1H d, J=2.4 Hz, 5.86 1H brs, 5.76 1H m, 5.67 1H
m, 5.21 1H s, 3.17 1H dd, J=4.8, 16.8 Hz, 3.09 1H dd,
J=4.8, 17.4, 2.91 2H m; 13CNMR (150 MHz, CD 3OD):
dC 185.7, 167.6, 167.1, 158.0, 157.9ꢃ3, 157.2, 156.5,
155.5, 151.3, 146.5, 146.22ꢃ2, 146.21ꢃ2, 139.8, 139.7,
134.2, 130.4, 128.9, 125.8, 122.8, 122.1, 121.1, 121.0,
117.5, 110.2ꢃ2, 110.1ꢃ2, 99.7, 99.3, 97.0ꢃ2, 96.1, 95.9,
80.1, 75.7, 69.4, 68.2, 27.4, 27.0 ppm; positive APCI-MS
m/z 869 [M+H]+.
4.3. Synthesis of theaflavin-type compounds
4.3.1. Theaflavin 9. EC(1.0 g) and EGC(1.0 g) were
dissolved in a mixture of acetone-pH 5.0 phosphate–
citrate buffer (1:10 v/v, 50 mL), which contained 4 mg
horseradish peroxidase. Four 2.0-mL aliquots of 3.13%
H2O2 were added during a period of 45 min while stir-
ring. The resulting reaction mixture was extracted with
ethyl acetate (3ꢃ50 mL). After concentration, the resi-
due was applied to a Sephadex LH 20 column and
eluted with acetone-water (2:3 v/v) to obtain 250 mg of
4.3.5. Neotheaflavin 13. Following the procedure for the
synthesis of 9, Catechin (1.0 g) and EGC (1.0 g) affor-
1
ded 120 mg of 13. H NMR (600 MHz, (CD3)2CO): dH
8.26 1H s, 7.46 1H s, 7.63 1H s, 6.06 1H d, J=2.4 Hz,
6.03 1H d, J=2.4 Hz, 5.96 1H d, J=2.4 Hz, 5.95 1H d,
J=2.4 Hz, 5.62 1H d, J=7.8 Hz, 5.01 1H s, 4.39 1H m,
4.15 1H m, 2.97 1H dd, J=5.4, 15.6 Hz, 2.91 1H dd,
J=4.2, 16.8, 2.84 1H dd, J=1.2, 16.8 Hz, 2.66 1H dd,
J=9.6, 15.6 Hz; 13CNMR ((CD 3)2CO, 150 MHz): dC
184.8, 157.6, 157.5, 157.4, 157.0, 156.7, 156.6, 154.4,
150.5, 146.2, 134.8, 132.2, 130.8, 128.6, 122.3, 121.6,
119.2, 100.7, 99.2, 96.4, 96.3, 95.6, 95.4, 81.5, 79.1, 69.5,
66.6, 30.0, 29.3 ppm; positive APCI-MS m/z 565
[M+H]+.
1
theaflavin. H NMR (600 MHz, CD3OD, TMS internal
reference): dH 7.97 1H s, 7.85 1H s, 7.34 1H s, 6.02 1H
d, J=2.4 Hz, 5.99 1H d, J=2.4 Hz, 5.97 1H d, J=2.4
Hz, 5.96 1H d, J=2.4 Hz, 5.64 1H brs, 4.91 1H brs, 4.45
1H m, 4.32 1H m, 2.98 1H dd, J=4.8, 16.8 Hz, 2.94 1H
dd, J=4.8, 16.8, 2.84 1H brd, J=16.8 Hz, 2.82 1H brd,
J=16.8 Hz; 13CNMR (150 MHz, CD 3OD): dC 185.1,
158.1, 158.0, 157.6, 157.5, 157.3, 156.6, 155.1, 150.9,
146.1, 134.4, 131.2, 129.0, 126.6, 123.7, 121.9, 118.3,
100.3, 99.8, 96.8, 96.7, 96.1, 96.0, 81.2, 77.1, 66.7, 65.6,
30.0, 29.4 ppm; positive APCI-MS m/z 565 [M+H]+.
4.3.6. Neotheaflavin 3-gallate 14. Following the proce-
dure for the synthesis of 9, Catechin (1.0 g) and EGCG
1
(1.0 g) were used to synthesize 170 mg of 14. H NMR
(600 MHz, CD3OD): dH 8.04 1H s, 7.59 1H s, 7.49 1H s,
6.92 2H s, 6.01 2H d, J=2.4 Hz, 5.98 1H d, J=2.4 Hz,
5.97 1H d, J=2.4 Hz, 5.67 1H brs, 5.56 1H m, 5.11 1H
s, 4.22 1H m, 3.03 1H dd, J=4.8, 17.4 Hz, 2.92 1H brd,
J=16.8 Hz, 2.83 1H dd, J=4.8, 16.8, 2.66 dd, J=8.4,
16.8; 13CNMR (150 MHz, CD 3OD): dC 185.8, 167.4,
158.0, 157.9, 157.8, 157.7, 156.7, 156.5, 155.3, 151.6,
146.9, 146.2ꢃ2, 139.9, 134.0, 132.0, 130.4, 127.7, 122.3,
121.3, 121.0, 117.6, 110.2ꢃ2, 100.6, 99.2, 96.9, 96.7,
95.9, 95.6, 80.5, 77.1, 69.9, 68.8, 28.5, 27.0 ppm; positive
APCI-MS m/z 717 [M+H]+.
4.3.2. Theaflavin 3-gallate 10. Following the procedure
for the synthesis of 9, EC(1.0 g) and EGCG (1.0 g) were
used to synthesize 220 mg of 10. H NMR (600 MHz,
1
CD3OD): dH 7.91 1H s, 7.80 1H s, 7.38 1H s, 6.80 2H s,
6.02 1H d, J=1.8 Hz, 6.00 1H d, J=2.4 Hz, 5.99 2H s,
5.78 1H m, 5.55 1H brs, 5.11 1H s, 4.16 m, 3.07 dd,
J=4.8, 16.8 Hz, 2.99 dd, J=4.2, 16.8, 2.91 brd, J=16.8
Hz, 2.83 brd, J=16.8 Hz; 13CNMR (150 MHz,
CD3OD): dC 185.6, 167.4, 158.0, 157.9, 157.8, 157.3,
156.4, 156.3, 155.4, 151.2, 146.4, 146.3ꢃ2, 139.9, 133.5,
131.3, 128.7, 125.7, 123.8, 121.9, 121.0, 117.5, 110.1ꢃ2,
100.2, 99.3, 96.9, 96.8, 96.2, 95.8, 79.8, 77.0, 69.0, 65.7,
30.1, 27.1 ppm; positive APCI-MS m/z 717 [M+H]+.
4.3.7. Theaflavate A 15. Following the procedure for the
synthesis of 9, ECG (0.85 g) itself was used to synthesize
60 mg of 15, and 600 mg ECG was recovered. 1H NMR
(600 MHz, CD3OD): dH 8.33 1H s, 7.81 1H s, 7.65 1H s,
6.87 1H dd, J=1.8, 7.8 Hz, 6.85 1H d, J=1.8 Hz, 6.80
2H, s, 6.53 1H d, J=7.8 Hz, 6.15 1H d, J=2.4 Hz, 6.11
1H d, J=2.4, 6.09 1H d, J=2.4 Hz, 5.98 1H, d, J=2.4
Hz, 5.69 1H brs, 5.64 1H brs, 5.52 1H, m, 5.11 1H s,
3.32 1H dd, J=4.8, 18.0 Hz, 3.10 1H dd, J=4.8, 18.0
Hz, 3.05 dd, J=1.8, 16.8 Hz, 2.91 d, J=16.8 Hz; 13C
NMR (150 MHz, CD3OD): dC 186.8, 167.8, 167.3,
158.2, 158.1, 158.0, 157.9, 157.2, 157.1, 155.3, 149.5,
146.4ꢃ2, 146.3, 146.0, 140.0, 133.5, 131.6, 131.2, 126.6,
124.8, 122.9, 122.6, 121.0, 119.0, 116.4, 115.8, 114.2,
110.2ꢃ2, 100.0, 99.4, 97.3, 97.2, 96.5, 96.4, 78.0, 75.6,
72.1, 68.9, 27.3, 26.7 ppm; positive APCI-MS m/z 853
[M+H]+.
4.3.3. Theaflavin 30-gallate 11. Following the procedure
for the synthesis of 9, ECG (1.0 g) and EGC (1.0 g) were
1
used to synthesize 110 mg of 11. H NMR (600 MHz,
CD3OD): dH 7.88 1H s, 7.87 1H s, 7.37 1H s, 6.84 2H s,
6.06 d, J=2.4 Hz, 6.00 1H d, J=2.4 Hz, 5.98 1H d,
J=2.4 Hz, 5.97 1H d, J=2.4 Hz, 5.87 1H brs, 5.81 1H
m, 4.94 1H brs, 4.33 1H brs, 3.09 1H dd, J=4.8, 17.4
Hz, 2.96 1H dd, J=4.8, 16.8, 2.88 1H brd, J=17.4 Hz,
2.861H dd, J=2.4, 16.8 Hz; 13CNMR (150 MHz,
CD3OD): dC 185.6, 167.2, 158.0, 157.9, 157.8, 157.7,
157.0, 156.6, 156.0, 155.5, 151.1, 146.2ꢃ2, 139.7, 134.8,
130.3, 128.8, 125.9, 123.0, 121.9, 120.9, 118.3, 110.1ꢃ2,
99.8, 99.6, 96.8, 96.7, 95.8, 95.7, 81.2, 75.8, 68.3, 66.5,
29.3, 27.2 ppm; positive APCI-MS m/z 717 [M+H]+.
4.3.4. Theaflavin 3,30-digallate 12. Following the proce-
dure for the synthesis of 9, ECG (1.0 g) and EGCG (1.0
g) afforded 100 mg of 12. 1H NMR (600 MHz,
CD3OD): dH 7.79 1H s, 7.76 1H s, 7.47 1H s, 6.88 2H s,
6.80 2H s, 6.07 1H d, J=2.4 Hz, 6.03 2H d, J=2.4 Hz,
4.3.8. Theaflavate B 16. Following the procedure for the
synthesis of 9, Catechin (1.0 g) and EGCG (1.0 g) were
used to synthesize 200 mg of 16. H NMR (600 MHz,
CD3OD): dH 8.26 1H s, 7.88 1H s, 7.59 1H s, 6.87 1H
dd, J=1.8, 7.8 Hz, 6.86 1H d, J=1.8 Hz, 6.55 1H d,
1