R.W. King et al. / Phytochemistry 62 (2003) 77–82
81
MeOH with 30 pg of [17,17-2H2]-GAs added as internal
standards to give a deutero:protio ratio close to 1.0. The
GAs were purified by partitioning and solid phase
extraction. The samples were methylated, further pur-
ified, and then trimethylsilylated before injection into a
Hewlett-Packard 5890 GC equipped with a fused silica
capillary column with a chemically bonded DB-5 MS
stationary phase (J&W Scientific, Folsom, CA, USA).
The column effluent was introduced into the ion source
of a Jeol JMS-SX/SX102A four-sector tandem mass
spectrometer of B1E1–B2E2 geometry (Jeol, Tokyo,
Japan). The interface and the ion source temperatures
3.3.2. ent-3ꢁ-Acetoxy-2ꢁ-chloro-13-hydroxy-20-norgibb-
erell-16-en-19-oic acid 19,10-lactone 7-methoxymethyl
ester (3)
A solution of diol 2 (165 mg, 0.40 mmol) in MeCN
(2.2 ml) was heated to 40 ꢄC and treated with 2-acetoxy-
isobutyrylchloride (90 ml, 0.61 mmol). The mixture was
heated for a further 5 min, then diluted with EtOAc and
washed repeatedly with water until the pH was neutral.
The organic phase was dried (Na2SO4), filtered and
concentrated under reduced pressure. Chromatography
on silica gel (1, 2-dichloroethane/EtOAc 1:3) afforded
ester 3 (60 mg, 32%) in addition to a lower Rf material.
This was re-chromatographed (AcOH/MeOH/1,2-di-
chloroethane/EtOAc 0.05:0.5:1:3) to yield acid 4 (56 mg,
ꢄ
were 270 and 250 C, respectively. Ions were generated
with 70 eV at an ionization current of 600 mA. Mea-
surements were performed by GC/MS-selected reaction
monitoring (SRM).
1
33%). Ester 3: H NMR (CDCl3) ꢂ 1.08 (3H, s, 4-Me),
2.13 (3H, s, OAc), 2.49 (1H, d, J=15.4 Hz, H-14), 2.73
(1H, d, J=10.4 Hz, H-6), 3.17 (1H, d, J=10.4 Hz, H-5),
3.44 (3H, s, OCH2OMe), 4.18 (1H, d, J=6.0 Hz, H-2),
4.94 (1H, s, H0-17), 5.09 (1H, s, H-3), 5.24 (3 ꢃ H, s, H-
3.3. Synthesis of GA6
1
3.3.1. ent-2ꢁ,3ꢁ,13-Trihydroxy-20-norgibberell-16-en-19-
oicacid 19,10-lactone 7-methoxymethyl ester ( 2)
17, OCH2OMe). Acid 4: H NMR (CDCl3) ꢂ 1.10 (3H,
s, 4-Me), 2.14 (3H, s, OAc), 2.33 (1H, d, J=15.3 Hz, H-
15), 2.47 (1H, d, J=15.3 Hz, H-14), 2.71 (1H, d, J=10.5
Hz, H-6), 3.12 (1H, d, J=10.5 Hz, H-5), 4.19 (1H, d,
J=6.5 Hz, H-2), 4.95 (1H, s, H0-17), 5.09 (1H, s, H-3),
5.28 (1H, s, H-17).
A mixture of GA5 methyl ester (Murofushi et al.,
1974) (265 mg, 0.77 mmol) in MeOH (12 ml) and 2 M
NaOH (60 ml) was heated overnight under gentle reflux.
After cooling to room temp, most of the MeOH was
removed under reduced pressure and the residue acid-
ified with 10 MHCl and extracted with EtOAc. The
aqueous phase was concentrated and then extracted
with EtOAc. The combined organic extracts were
washed with water (ꢃ4) and brine and concentrated to
dryness. Without further purification the residue (237
3.3.3. ent-3ꢁ-Acetoxy-2ꢁ-chloro-13-hydroxy-20-norgibb-
erell-16-en-19-oic acid 19,10-lactone (4)
Ester 3 (60 mg, 0.13 mmol) was dissolved in THF (1.2
ml) and treated with MeOH (90 ml) and Me3SiCl (278
ml, 2.18 mmol). The mixture was stirred at room temp
for 3 h, then diluted with EtOAc, washed with water
(until the pH was neutral) and brine, dried (Na2SO4),
filtered and concentrated to dryness. Chromatography
on silica gel (AcOH/MeOH/1,2-dichloroethane/ EtOAc
0.05:0.5:1:3) gave the title compound 4 (42 mg, 78%).
ꢄ
mg) was dissolved in CH2Cl2 (5 ml) and cooled to 0 C
under an atmosphere of nitrogen. Hunig’s base (iPr2NEt)
(119 mg, 0.92 mmol) and methoxymethyl chloride (74
mg, 0.92 mmol) were then added and the mixture stirred
ꢄ
at 0 C for 2 h, after which EtOAc (30 ml) was added
and the mixture washed with 1 MHCl, sat. NaHCO 3 and
dried (Na2SO4). A portion of the resulting methoxy-
methyl ester (115 mg, 0.31 mmol), water (150 ml),
N-methylmorpholine N-oxide (40 mg, 0.34 mmol),
t-butanol (300 ml) and p-toluenesulfonic acid (29 mg,
0.15 mmol) in acetone (1.2 ml) was cooled in an ice bath
and treated with osmium tetroxide (36 mg, 0.14 mmol).
The solution was stirred at 0 ꢄC for 40 min, then diluted
with butan-2-ol/EtOAc (10 ml, 1:4) and washed with
saturated NaHCO3 (ꢃ3) and brine. The organic phase
was dried (Na2SO4), filtered and concentrated under
reduced pressure. Chromatography on silica gel (1,
2-dichloroethane/EtOAc 1:3) gave the starting alkene
(27 mg, 24% recovery) and triol 2 (49 mg, 51% at 76%
conversion). 1H NMR (CDCl3/d4-MeOH) ꢂ 1.04 (3H, s,
4-Me), 2.52 (1H, d, J=10.2 Hz, H-6), 3.14 (1H, d,
J=10.2 Hz, H-5), 3.33 (3H, s, OCH2OMe), 3.50 (1H, d,
J=4.2 Hz, H-3), 3.63 (1H, m, H-2), 3.92 (1H, br s, OH),
4.80 (1H, s, H0-17), 5.08 (1H, s, H-17), 5.07, 5.15 (2ꢃH,
ABd, J=6.0 Hz, OCH2OMe).
3.3.4. ent-2ꢁ,3ꢁ-Epoxy-13-hydroxy-20-norgibberell-16-
en-19-oicacid 19,10-lactone (GA 6) (5)
Acid 4 (98 mg, 0.23 mmol) was dissolved in MeOH
(2.8 ml) and treated with K2CO3/KOH solution (1.4 ml
of a stock solution prepared from 1.38 g of K2CO3 and
150 mg of KOH in 10 ml of water). The mixture was
stirred overnight at room temp. and then most of the
MeOH removed under reduced pressure. The residue
was acidified with 1 N HCl and extracted with EtOAc.
The combined organic layers were washed with water
until neutral, then dried (Na2SO4), filtered and con-
centrated under reduced pressure to give GA6 (5) (65
1
mg, 81%). H NMR (CDCl3) ꢂ 1.33 (3H, s, 4-Me), 2.64
(1H, d, J=10.0 Hz, H-6), 3.02 (1H, d, J=10.0 Hz, H-5),
3.13–3.18 (2H, m, H-2, H-3), 4.94 (1H, s, H0-17), 5.22
(1H, s, H-17), 6.40 (1H, br s, CO2H). 13C NM R
(CDCl3) ꢂ 14.6 (C-18), 17.5 (C-11), 31.8 (C-1), 38.0 (C-
12), 42.6 (C 14), 45.1 (C_15), 48.2 (C-8), 48.4, 49.6, 49.7
(C-2, C-3, C-6), 50.2 (C-4), 53.3 (C-9), 56.7 (C-5), 78.2