N. Hirai et al. / Phytochemistry 80 (2012) 89–98
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carried out by B3LYP/6-31G(d) method with electric charge ꢀ1,
4.11. Electrolysis of ABA (1) in a D2O buffer, and isolation of
compounds 6 and 7-d2
spin multiplicity 2, and open shell. The method and basis set for
calculation of Mulliken charge and spin density was B3LYP/6-
31 + G(d,p) method with electric charge ꢀ1, spin multiplicity 2,
and open shell (Gaussian 98, 1998).
A buffer solution (15 ml) of pH 10 was prepared using D2O, and
ABA (21 mg) was electrolyzed in the buffer at ꢀ2.0 V for 4.5 h. The
electrolysis solution (13 ml) in the cathode compartment was acid-
ified with 1 M HCl to pH 2 and extracted with EtOAc (10 ml ꢁ 4).
The combined organic solubles were washed with H2O, dried
(Na2SO4), and concentrated to give crude products (19 mg). The
products were chromatographed on silica gel (Wakogel C-200,
30 g) column eluted with a mixture of toluene–EtOAc–HOAc
(90:10:0.1–40:60:0.1). The colorless oil (2.0 mg) eluted with 20%
and 30% EtOAc was purified by preparative HPLC using an ODS col-
umn (YMC AQ-311, 6 ꢁ 100 mm) with MeOH–H2O–HOAc
(45:55:0.1) and detection at 254 nm. The substances eluted at
12.9 min was collected, and concentrated to give colorless oil
(1.2 mg). The latter was purified by preparative HPLC using a silica
gel column (YMC PackSIL A-003) with a mixture of CHCl3–HOAc
(100:0.1) and detection at 254 nm. A material eluted at13.7 min
was collected, and concentrated to give 7-d2 (0.2 mg). The sub-
stance eluted from the silica gel column with 50% and 60% EtOAc
was concentrated to give 6 (3.0 mg).
4.9. Electrolysis of ABA (1), and isolation of compound 6
ABA (1, 21 mg) in 15 ml of the buffer solution of pH 7 was elec-
trolyzed at ꢀ2.0 V for 4.5 h. The electrolysis solution (13 ml) in the
cathode compartment was acidified with 1 M HCl to pH 2 and ex-
tracted with EtOAc (10 ml ꢁ 4). The combined organic solubles
were washed with H2O, dried (Na2SO4), and concentrated to give
colorless oil (21 mg). The latter applied to a silica gel (Wakogel
C-200, 14 g) column eluted with a mixture of toluene–EtOAc–
HCO2H (70:30:0.1–40:60:0.1). The compounds eluted with 50–
60% EtOAc in toluene were combined, and purified by preparative
HPLC using an ODS column (YMC AQ-311, 6 ꢁ 100 mm) with
MeOH–H2O–HCO2H (50:50:0.1–65:35:0.1, 12 min linear gradient).
A compound eluted at 5.0 min was collected, and concentrated to
give 6 (1.0 mg).
4.9.1. 5-[10-(4-carboxy-3-methyl-buta-1,3-dienyl)-3,10-dihydroxy-
2,4,4,9,11,11-hexamethyl-13-oxa-dispiro[5.0.5.1]trideca-1,8-dien-3-
yl]-3-methyl-penta-2,4-dienoic acid (6)
4.11.1. 10-Deoxy-ABA-d2 (7-d2)
Colorless oil; 1HNMR (500 MHz, CDCl3): d 0.99 (3H, s, H-90), 1.05
(3H, s, H-80), 1.95 (3H, s, H-70), 1.99 (3H, s, H-6), 2.08 (1H, d,
J = 16.9 Hz, H-50), 2.47 (1H, d, J = 16.8 Hz, H-50), 2.81 (0.35H, d,
J = 9.6 Hz, H-10), 5.74 (1H, s, H-30), 5.91 (0.11H, s, H-2), 6.00 (1H,
d, J = 15.7 Hz, H-5), 7.66 (1H, d, J = 15.7 Hz, H-4). Compound 7-d2
was treated with diazomethane to give a monomethyl ester 7-
Me-d2. EIMS (probe) 70 eV, m/z (rel. int.): 264 [Md2]+ (0.3), 263
[Md1]+ (0.2), 262 [Md0]+ (0.1), 125 (100); the total deuterium con-
tent at C-2 and -10 was 55.8%.
Colorless oil, yield 4.9%; ½a D22
ꢂ
+366° (c 0.10, MeOH); 1HNMR
(500 MHz, CD3OD, 325 K): d 0.87 (3H, s, H-90 or-80), 1.14 (3H, s,
H-80or -90), 1.33 (2H, m, H-50), 1.72 (3H, s, H-70), 194 (3H, s, H-6),
5.68 (2H, s, H-2 and H-30), 6.02 (1H, m, H-5), 7.76 (1H, d,
J = 16.1 Hz, H-4); UV k nm (loge): 262 (4.43), MeOHmax. Compound
6 was treated with diazomethane to give a dimethyl ester 6-Me.
Spectroscopic data of 6-Me: ½a D29
ꢂ
+437° (c 0.11, MeOH); 1HNMR
(500 MHz, CD3OD, 325 K): d 0.86 (3H, s, H-90 or H-80), 1.16 (3H, s,
H-80 or H-90), 1.36 (1H, d, J = 14.8 Hz, H-50), 1.58 (1H, d,
J = 14.8 Hz, H-50), 1.74 (3H, s, H-70), 1.95 (3H, s, H-6), 3.67 (3H, s,
1-O-Me), 5.68 (1H, s, H-2), 5.69 (1H, s, H-30), 6.08 (1H, d,
J = 16.1 Hz, H-5), 7.64 (1H, d, J = 16.1 Hz, H-4); 13CNMR (75 MHz,
CDCl3): d 18.3 (C-70), 21.0 (C-6), 24.2 (C-80 or C-90), 25.5 (C-90 or
C-80), 37.7 (C-60), 41.4 (C-50), 51.1 (1-O-Me), 74.4 (C-40), 79.3 (C-
10), 117.5 (C-2), 124.9 (C-30), 127.1 (C-4), 138.7 (C-5), 140.5 (C-
4.12. Electrolysis of phaseic acid (3), and isolation of compound 8
Phaseic acid (3, 9 mg) was electrolyzed in 15 ml of a pH 3 buffer
solution at ꢀ2.0 V for 4.5 h. The total electric charge was 9.9 C cor-
responding to 170% of electric charge to reduce all 7. The cathodic
solution (12 ml) was recovered, diluted with H2O, and partitioned
with EtOAc (20 ml ꢁ 4). The combined organic solubles were
washed with H2O, and dried (Na2SO4). After filtration the organic
layer was concentrated to give a solid (5.8 mg). The solid was ap-
plied to silica gel (Wakogel C-200, 1.2 g), and eluted with mixtures
of toluene and EtOAc. The material eluted with 80% EtOAc in tolu-
ene was injected to an HPLC column (YMC AQ-311, 6 ꢁ 100 mm),
and eluted with a mixture of MeOH–H2O–HOAc (5:95:0.1) at a
flow rate of 1.0 ml/min and detected at UV 220 nm. A material
eluted at 14.0 min was collected, and concentrated to give 8
(1 mg).
20), 149.9 (C-3), 166.3 (C-1); UV k nm (log
e): 267 (4.30), MeOHmax;
IR mmax (CHCl3) cmꢀ1: 3550, 2953, 1713, 1707, 1606, 1245, 1221,
1167; EIMS (probe) 70 eV, m/z (rel. int.): 540 [M]+ (2), 522 [M-
H2O]+ (26), 504 [M-2H2O]+ (38), 490 (18), 486 [M-3H2O]+ (35),
472 (22), 439 (22), 262 (100), 247 (33), 125 (75); HR–EIMS: m/z
540.3097 [M]+ (calcd for C32H44O7 540.3087).
4.10. Electrolysis of ABA (1), and isolation of 10-deoxy-ABA (7)
ABA (1, 23 mg) in 15 ml of the pH 10 buffer solution was elec-
trolyzed at ꢀ2.0 V for 4.5 h. The cathodic solution (13 ml) was acid-
ified with 1 M HCl to pH 2 and extracted with of EtOAc (10 ml ꢁ 4).
The combined organic solubles were washed with H2O, dried
(Na2SO4), and concentrated to give colorless oil (21 mg). The latter
subjected to silica gel (Wakogel C-200, 14 g) CC with toluene–
EtOAc–HCO2H (20:80:0.05–60:40:0.05). The substances eluted
with 20–30% EtOAc in toluene were purified by preparative HPLC
using an ODS column (YMC AQ-311, 6 ꢁ 100 mm) with a mixture
of MeOH–H2O–HCO2H (45:55:0.01–50:50:0.01, a linear gradient
by 12 min). The substances eluted at 11.7 min were further puri-
fied by preparative HPLC using an ODS column (YMC AQ-311,
6 ꢁ 100 mm) with a mixture of MeOH–H2O–HCO2H (45:55:0.01).
The compound eluted at 13.0 min was collected, and concentrated
to give 7 (1.3 mg, 5.7% yield). Compound 7 did not show any signif-
icant optical rotation.
4.12.1. (E)-4-(3a,5-dihydroxy-3,6a-dimethylhexahydro-2H-3,5-
methanocyclopenta[b]furan-4-yl)-3-methylbut-3-enoic acid (8)
Colorless solid; ½a D28
ꢂ
+13° (c 0.1, MeOH);1HNMR (500 MHz,
CD3OD): d 1.09 (3H, s, H-9), 1.30 (3H, s, H-70), 1.71 (1H, d,
J = 12.4 Hz, H-50), 1.78 (1H, d, J = 11.9 Hz, H-30), 1.79 (3H, s, H-6),
1.82 (1H, d, J = 12.4 Hz, H-50), 2.04 (1H, d, J = 11.9 Hz, H-30), 2.94
(1H, d, J = 9.6 Hz, H-5), 3.09 (2H, s, H-2), 3.65 (1H, d, J = 7.6 Hz, H-
80pro-S), 3.67 (1H, d, J = 7.6 Hz, H-80pro-R), 5.40 (1H, d, J = 9.6 Hz, H-
13
4); CNMR (125 MHz, CD3OD): d 15.6 (C-6 or C-90), 17.2 (C-70),
21.5 (C-90 or C-6), 45.5 (C-2), 46.5 (C-60), 49.0 (C-30), 50.8 (C-50),
53.1 (C-5), 74.1 (C-40), 77.0 (C-80), 87.5 (C-10), 87.7 (C-20), 121.8
(C-4), 135.3 (C-3), 175.6 (C-1). Compound 8 (1 mg) was treated
with diazomethane to give 8-Me (1 mg). Spectral data of 8-Me.
½
a 2D6 +14° (c 0.1, MeOH);1HNMR (500 MHz, CD3OD):d 1.09 (3H, s,
ꢂ
H-90), 1.28 (3H, s, H-70), 1.70 (1H, d, J = 11.9 Hz, H50), 1.77 (1H, d,