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R. Duran-Patron et al. / Tetrahedron 59 (2003) 6267–6271
6270
could arise by an electrocyclic ring opening of a
cyclohexadiene reminiscent of the relatioship between
vitamin D and dehydrocholesterol. The dienes could also
be the precursors of the aromatic botryanes.2 Work is in
progress to establish the stage in the botryane pathway at
which these secobotryanes emerge.
containing 200 ml of Czapek–Dox medium.2 The pH of the
medium was adjusted to 7.0 and each flask was inoculated
with 3£106 fresh conidia. After 48 or 72 h of incubation (see
detailed experiments below) the mycelia was transferred
into the same number of 500 ml Erlenmeyer flasks, each
containing 200 ml of Czapek–Dox medium (without
glucose) and an aseptic solution of the labelled precursor
in H2O or EtOH. After 48 h from the administration of the
precursor, the culture medium and mycelia were separated
by filtration. The broth was saturated with NaCl, and
extracted with EtOAc (£4). The EtOAc extract was washed
with H2O (£3) and then dried over anhydrous Na2SO4.
Evaporation of the solvent at reduced pressure gave a
yellow oil that was separated by means of column
chromatography, with an increasing gradient of ethyl
acetate in petroleum ether. Final purification was carried
out with HPLC.
3. Experimental
3.1. General experimental procedures
Melting points were measured with a Reichert–Jung Kofler
block and are uncorrected. Optical rotations were deter-
mined with a Perkin–Elmer 241 polarimeter. IR spectra
were recorded on a Mattson Genesis spectrophotometer,
1
series FTIRe. H and 13C NMR spectra were recorded on
Varian Gemini 200 MHz (1H at 199.975 MHz, 13C at
50.289 MHz) and Varian Unity 400 MHz (1H at
399.952 MHz, 13C at 100.570 MHz) spectrometers. 2H
NMR Spectra were recorded on a Varian Unity 400 MHz
(2H at 61.395 MHz) spectrometer. Chemical shifts are
quoted relative to TMS (Me4Si) in CDCl3. Mass spectra
were recorded on GC-MS Thermoquest, model Voyager,
and VG-Autospec spectrometers. HPLC was performed
with a Hitachi/Merck L-6270 apparatus equipped with an
UV-VIS detector (L 4250) and a differential refractometer
detector (RI-71). TLC was performed on Merck Kiesegel 60
F254, 0.25 mm thick. Silica gel (Merck) was used for column
chromatography. Purification by means of HPLC was carried
out with a Si gel column (LiChrospher Si-60, 10 mm, 1 cm
wide, 25 cm long or 5 mm, 0.4 cm wide, 25 cm long).
3.5. Feeding of sodium [1-13C]-acetate to B. cinerea
Eleven cultured flasks with B. cinerea were fed with 360 ml
of an aseptic aqueous solution of sodium [1-13C]-acetate to
give a final concentration of 450 ppm per flask on day 2.
Extraction of the broth yielded a crude extract (61 mg),
which was purified as described in the general method to
afford secobotrytrienediol2 (1, 2.3 mg). The isotopic
composition of 1 was determined by HREIMS and is
shown in the Table 2.
3.6. Feeding of sodium [1,2-13C2]-acetate to B. cinerea
Six cultured flasks with B. cinerea were fed with 400 ml of
an aseptic aqueous solution of sodium [1,2-13C2]-acetate:
sodium acetate (1:2) to give a final concentration of
450 ppm per flask on day 2. Extraction of the broth yielded
a crude extract (51 mg), which was purified as described in
the general method to afford 12 (2.1 mg). The isotopic
composition of 1 was determined by HREIMS and is shown
in the Table 2.
3.2. Labelled precursors
Sodium [1-13C]-acetate (99 at.% 13C) and sodium
[1,2-13C2]-acetate (99 at.% 13C) were obtained from the
Aldrich Chemical Company. [10-2H]-Botrydiol (3)6 was
synthesised by treatment of dihydrobotrydial (4, 116 mg,
0.37 mmol) in MeOH (6 ml) with NaBD4 (3 mg,
0.07 mmol) ($99 at.% D, purchased from the Fluka
Chemical Company). After stirring for 2 h at room
temperature, the reaction mixture was poured onto ice,
acidified with 2N HCl (10 ml) and stirred for 10 min. The
solution was then diluted with H2O (45 ml) and extracted
with CHCl3 (30 ml, £3). The solvent was evaporated and
the crude extract subjected to column chromatography to
afford [10-2H]-botrydiol (3, 62 mg, 0.20 mmol, 53%, mp
170–1718C). The isotopic composition, calculated by
3.7. Feeding of [10-2H]-botrydiol to B. cinerea
Eleven cultured flasks with B. cinerea were fed with 375 ml
of an aseptic solution of [10-2H]-botrydiol (3) in EtOH to
give a final concentration of 150 ppm per flask on day 3.
Extraction of the broth yielded a crude extract (590 mg),
which was purified as described in the general method to
afford secobotrytrienediol2 (1, 2.8 mg), secobotrydiene-
3,10,15-triol (6, 1.2 mg), secobotrydiene-3,4,10,15-tetraol
(7, 0.5 mg), secobotrytriene-10,12,15-triol (8, 0.7 mg) and
[10-2H]-dehydroxydihydrobotrydialone7 (5, 14.5 mg). The
isotopic composition of 5, calculated by integration of the
1
integration of the resultant H NMR spectrum, was 98.9%.
3.3. Organism
1
resultant H NMR spectrum, was 88.1%.
´
B. cinerea 2100 was obtained from the Coleccion Espan˜ola
3.7.1. Secobotrydiene-3,10,15-triol (6). Colourless oil;
[a]2D5¼þ57 (c 0.1, CHCl3); IR (KBr) nmax 3354, 2925,
2862, 1739, 1451, 1373, 1261, 1033, 801 cm21; EIMS m/z
(%) 236 (1) [M2H2O]þ, 223 (10) [M2CH2OH]þ, 206 (90)
[M2CH2OH–OH]þ, 175 (82) [M22£CH2OH–OH]þ, 161
(58) [M23£CH2OH]þ, 145 (71), 133 (88), 119 (100);
HREIMS calcd. for C15H26O3 [M]þ 254.1882, found
254.1904. See Tables 3 and 4 for 1H and 13C NMR
spectroscopic data.
´
de Cultivos Tipo (CECT), Facultad de Biologıa, Universi-
dad de Valencia, Spain. Conidial stock suspensions of this
strain were maintained viable in 80% glycerol at 2208C.
3.4. Feeding experiments of labelled precursors:
extraction and isolation of metabolites
General method. B. cinerea 2100 was grown at 24–268C on
shake culture at 250 rpm in 500 ml Erlenmeyer flasks, each