8260
M. Daoubi et al. / Tetrahedron 62 (2006) 8256–8261
(
Na SO and concentrated in vacuo, and the crude extract
3ꢁ5 mL). The combined organic layers were dried with
adjusted to 7.0 with aqueous NaOH, and the flasks were in-
oculated with a suspension (2.3 mL) of B. cinerea conidia
2
4
6
ꢂ
was subjected to column chromatography to afford botrydial
(0.30 g, 60%), which was identified by its NMR spectro-
scopic data.
(8ꢁ10 conidia/mL). The flasks were incubated at 25 C
for three days and stirred at 250 rpm; the mycelium was
then filtered and transferred into 10 flasks (500 mL) contain-
ing 200 mL of medium, which consisted of equal volumes of
sterile phosphate buffer (pH 7 with phosphate buffer 0.4 M)
and Czapek–Dox (as above but without glucose) and 90 ppm
1
3
.3.2. Botrydienal 9. A solution of 1 (100 mg, 0.32 mmol) in
AcOEt (10 mL) was treated with 6% aqueous oxalic acid so-
lution (15 mL) and refluxed for 12 h. The mixture was neu-
tralized with a saturated aqueous solution of NaHCO and
2
of the substrate botry-4,9-dien-[15,10- H]-ol (12) per flask.
The remaining two flasks were used as control. The broth
and the mycelia were separated further by filtration after
three days. The pH was measured (control: 6.58 pH units;
inoculated medium broth: 6.51 pH units) and the broth was
saturated with NaCl, and extracted four times with ethyl ace-
tate. The extract was dried over anhydrous sodium sulfate,
and the solvent was then evaporated under vacuum. Frac-
tionation of the extract (210 mg) was carried out by means
of column chromatography on silica gel (SiCC), eluting
with hexane/ethyl acetate (80:20). Final purification was
carried out by means of semi-preparative HPLC to afford
deuteriated known compounds dehydrobotrydienol (13;
3 mg), 11-hydroxydehydrobotrydienol (14; 130 mg) and
12-hydroxydehydrobotrydienol (15; 1.1 mg), unknown
3
extracted with AcOEt (ꢁ3). The organic layers were washed
withbrineand driedoveranhydrous Na SO . Thesolventwas
2
4
removed, and the mixture obtained was purified by means of
normal-phase HPLC to afford 8 (14 mg, 0.048 mmol,
2
,5
2
,9
15%), botrydienal 9 (48.3 mg, 0.21 mmol, 65%) and aro-
matic derivative 11 (7.4 mg, 0.032 mmol, 10%).
2,9
2
2
3
.3.3. [10- H,15- H]-Botry-1(9)-4-diendiol 12. NaBD
4
(
36.1 mg, 0.86 mmol) (ꢃ99 at. % D, purchased from the
Fluka Chemical Company) was added to a solution of 9
50 mg, 0.22 mmol) in methanol (10 mL), and the resulting
(
solution was stirred for 15 min at room temperature. The
mixture was poured onto ice, acidified with 2 N HCl
2
2
(
15 mL), and stirred for 10 min. The solution was diluted
deuteriated compounds, 1-hydroxy-4-oxo-[10- H, 15- H]-
botry-5(9)-endiol (16; 0.6 mg) and botry-[1(10),5(9)- H]-
dien-[15- H]-ol (17; 1.5 mg) and a new, undeuteriated,
2
with H O (45 mL) and extracted with CHCl (30 mL, ꢁ3).
2
3
2
The solvent was evaporated and the crude extract chromato-
graphed to yield 12 (50.3 mg, 0.21 mmol, 96%, HREIMS
metabolite compound 1b,4b-dihydroxy-5(9)-ene dihydrobo-
trydial (18; 0.7 mg). Their structures were established on the
basis of their one-dimensional and two-dimensional NMR
(
m/z): 238.1908 (calcd C H O D , 238.1901)).
15 22 2 2
1
2
13
3
.4. Biotransformation by B. cinerea
analyses ( H, H, C, HSQC and HMBC).
6
2
2
A suspension (4.2 mL) of B. cinerea conidia (2.48ꢁ10 con-
3.5.1. 1-Hydroxy-4-oxo-[10- H,15- H]-botry-5(9)-endiol
16. Colorless oil, IR (film): n 3375, 2930, 1660, 1355,
1059, 758 cm ; H NMR (CDCl , 600 MHz) d (ppm):
ꢂ
cultures in 12 Erlenmeyer flasks (500 mL) containing
idia/mL) was inoculated at 25 C and 250 rpm in shake
max
ꢀ
1
1
3
2
tract (1 g), KH PO (5 g), NaNO (2 g), MgSO (0.5 g)
and FeSO (10 mg) per litre of distilled water. Before the in-
4
oculation, the pH medium was carefully adjusted to 7.0 with
aqueous NaOH. After 72 h, the mycelium was transferred,
into 10 flasks (500 mL) containing 200 mL of Czapek–
Dox medium (without glucose) and the substrate 12
00 ml of Czapek–Dox medium: glucose (50 g), yeast ex-
1.09 (3H, d, J11–2¼6.2 Hz, H-11), 1.27* (3H, s, H-13),
1.28* (3H, s, H-12), 1.38 (3H, s, H-14), 1.51 (1H, d, J
7a–7b
¼
2
4
3
4
12.9 Hz, H-7a), 1.76 (1H, d, J7b–7a¼12.9 Hz, H-7b), 2.39
0
3.53 (1H, s, H-15), 3.76 (1H, s, H-15 ). C NMR
(3H, m, H-2, H-3b, H-3a), 3.72–3.74 (2H, m, H10, H10 ),
0
(600 MHz, CDCl ) d (ppm): 14.6 (q, C-11), 26.1 (q, C-
13
3
14), 28.2* (q, C-13), 29.8* (q, C-12), 42.0 (d, C-2), 42.4
(t, C-6), 44.3 (s, C-3), 52.0 (s, C-7), 52.4 (t, C-8), 64.4
(dt , C-10), 68.4 (dt , C-15), 75.6 (s, C-1), 145.0 (s, C-5),
(
90 ppm). The additional two flasks were used as control.
#
#
After three days, the mycelium was filtered. The pH was
measured (control: 6.09 pH; inoculated mediums broth:
#
169.1 (s, C-9), 197 (s, C-4). *: Interchangeable; : coupling
C– H (d) and C– H (t). EIMS (m/z): 270 [M ] (3%), 252
1
3
1
13
2
+
6
.03 pH). The broth (2 L) was saturated with sodium chlo-
+
+
+
ride and extracted four times with ethyl acetate. Extracts
were dried over anhydrous sodium sulfate, and the solvent
was evaporated under reduced pressure. The residue
[M –H O] (0.2%), 238 [M –CDHOH] (55%), 220 [M –
2
H O–CDHOH] (100%). HREIMS (m/z): 270.1772 (calcd
2
C D H O , 270.1800). Deuterium percentage: 44%.
15 2 22 4
(
203 mg) was purified by chromatography on a Si gel
column and then by HPLC affording dehydrobotrydienol
13; 1.8 mg), 11-hydroxydehydrobotrydienol (14; 120 mg)
and 12-hydroxydehydrobotrydienol (15; 3.5 mg), novel
2
2
3.5.2. Botry-[1(10),5(9)- H]-dien-[15- H]-ol 17. Colorless
oil, [a]D +12.20 (c 1.5, ethyl acetate); IR (film): n
max
2
5
(
ꢀ
1
1
3386, 2950, 2933, 1459, 1375 cm ; H NMR (CDCl ,
3
2
2
deuteriated compounds 1-hydroxy-4-oxo-[10- H, 15- H]-
botry-5(9)-endiol (16; 0.3 mg) and botry-[1(10),5(9)- H]-
dien-[15- H]-ol (17; 4 mg). Their structures were established
on the basis of their NMR spectroscopy: H, H and
HSQC, HMBC.
400 MHz) d (ppm): 1.12 (3H, d, J11–2¼6.7 Hz, H-11),
1.25 (3H, s, H-12), 1.17 (3H, s, H-13), 1.29 (3H, s,
H-14), 1.54 (1H, d, J7a–7b¼13.0 Hz, H-7a), 2.09 (1H, d,
2
2
1
2
13
C
J7b–7a¼13.0 Hz, H-7b), 1.65 (1H, ddd, J
¼13.2 Hz,
3b–3a
3a–3b
J3a–2¼10 Hz, J
¼4.8 Hz, H-3a), 1.8 (1H, ddd, J
¼
3a–4
1
H-2), 3.41 (1H, br s, H-15), 3.8 (1H, br s, H-15 ), 4.39
3.2 Hz, J
¼4.8 Hz, J
¼3.9 Hz, H-3b), 2.6 (1H, m,
3
b–4
3b–4
0
3.5. Biotransformation by B. cinerea on
buffered medium
(1H, dd, J4–3a¼4.8 Hz, J
¼4.8 Hz, H-4), 4.9 (1H, s,
4–3b
1
3
H-10), 5.11 (1H, s, 10). C NMR (100 MHz, CDCl )
3
Twelve Erlenmeyer flasks (500 mL) were filled with 200 mL
of Czapek–Dox medium as cited above. The pH was
d (ppm): 24.2 (q, C-11), 19.3 (q, C-14), 31.15 (q, C-12),
28.39 (q, C-13), 33.12 (d, C-2), 41.62 (t, C-3), 50.67