M. Lang, A. Mühlbauer, E. Jägers, W. Steglich
FULL PAPER
1
and the residue was distributed between EtOAc and water. The
organic phase was washed with water (3ϫ), dried (Na2SO4) and
concentrated under reduced pressure. The crude product was puri-
fied by chromatography on acetylated polyamide-6, followed by
HSCCC (see isolation of 2). Yield 27 mg (13%), orange solid.
1H NMR (300 MHz, CDCl3, only data differing from those of 2
10:1). H NMR (300 MHz, CDCl3): δ = 1.58, 1.59, 1.60 (each s, 3
H, 18Ј-H, 19Ј-H, 20Ј-H), 1.68 (s, 3 H, 16Ј-H), 1.78 (s, 3 H, 17Ј-H),
1
3
1.91–2.12 (m, 12 H, 6ϫCH2), 3.44 (dd, JC,H = 128, JH,H
=
6.6 Hz, 2 H, 1Ј-H), 3.46, 3.50, 3.59 (each s, 3 H, 3ϫOCH2OCH3),
5.05–5.16 (m, 3 H, 4Ј-H, 8Ј-H, 12Ј-H), 5.10 (s, 2 H, OCH2OCH3),
5.12 (br. s, 4 H, 2ϫOCH2OCH3), 5.17–5.26 (m, 1 H, 2Ј-H), 6.78,
6.92 (2ϫd, 3J = 9.1 Hz, 2ϫ1 H, 5-H, 6-H) ppm. 13C NMR
(75 MHz, CDCl3): δ = 16.1 (s, 2ϫCH3, C-18Ј and C-19Ј), 16.3 (d,
3
1
given): δ = 1.74 (d, JC,H = 5.1 Hz, 3 H, 17Ј-H), 3.15 (ddd, JC,H
2
3
= 130 Hz, JC,H ≈ JH,H ≈ 7.2 Hz, 2 H, 1Ј-H), 5.02–5.13 (m, 3 H,
3ϫCHvinyl), 5.13 (dm, JC,H ≈ 156 Hz, 1 H, 2Ј-H) ppm. 13C NMR 3JC,C = 3.8 Hz, C-17Ј), 17.7 (C-20Ј), 23.7 (13C-1Ј), 25.8 (C-16Ј),
1
(75 MHz, CDCl3, only data differing from those for 2 given): δ = 26.72, 26.75, 26.84 (C-5Ј, C-9Ј, C-13Ј), 39.7–40.0 (m, 3ϫCH2, C-
1
16.18–16.28 (m, C-17Ј), 21.7 (d, JC,C = 43.0 Hz, 13C-1Ј), 26.5 (d,
4Ј, C-8Ј, C-12Ј), 55.9, 56.2, 57.6 (3ϫOCH2OCH3), 94.9, 95.9, 99.3
1
2
1
3JC,C = 3.0 Hz, C-5Ј), 116.1 (dd, JC,C = 44.1 Hz, JC,C = 4.0 Hz, (3ϫOCH2OCH3), 110.1 (C-5), 114.7 (C-6), 122.9 (d, JC,C
=
1
1
1
C-3), 119.1 (d, JC,C = 43.0 Hz, C-2Ј), 137.6 (d, JC,C = 74.3 Hz,
C-3Ј) ppm.
42.9 Hz, C-2Ј), 124.31, 124.33, 124.5 (3ϫCHvinyl), 126.2 (d, JC,C
= 44.0 Hz, C-3), 131.3 (C-15Ј), 134.9 (2ϫCvinyl), 135.0 (Cvinyl),
145.0 (C-1), 145.8, 150.97 (C-2 and C-4) ppm. IR (KBr):[28] ν =
˜
1,2,4-Tris(methoxymethoxy)benzene (7): A solution of 1,2,4-triacet-
oxybenzene (6, 5.04 g, 20 mmol) in MeOH (50 mL) was saturated
with argon. Hydrazine monohydrate (3.9 mL, 80 mmol) was then
added, and the mixture was stirred for 15 min at room temperature
under argon. After removal of the volatiles under reduced pressure,
the resulting residue was treated with HCl (2 , 50 mL, saturated
with argon), and the benzene-1,2,4-triol was extracted with EtOAc
(3ϫ50 mL). The combined organic phases were washed with brine,
dried (Na2SO4) and concentrated. The crude benzene-1,2,4-triol
(2.45 g, yield 97%) was suspended in dry CH2Cl2 (100 mL) under
argon and treated with chloro(methoxy)methane (6 in
EtOAc,[13b] 19 mL, 114 mmol) and N,N-diisopropylethylamine
(26 mL, 152 mmol). After the mixture had been stirred overnight,
NaOH (2 , 100 mL) was added, and the phases were separated.
The aqueous phase was extracted with CH2Cl2 (2 ϫ 50 mL). The
combined organic phases were washed with water and brine and
dried (Na2SO4). The product was purified by flash chromatography
(hexanes/EtOAc, 3:1). Yield 3.21 g (62%), yellowish oil. Rf (TLC)
= 0.35 (hexanes/EtOAc, 3:1). 1H NMR (300 MHz, CDCl3): δ =
3.46, 3.50, 3.51 (each s, 3 H, 3ϫOCH2OCH3), 5.10, 5.14, 5.20
2956 (s, sh), 2925 (s, br), 2849 (s), 2826 (s, sh), 1666 (w), 1593 (w),
1485 (s), 1440 (s), 1399 (m), 1382 (m), 1250 (s), 1206 (m), 1154 (s),
1089 (s), 1046 (ss, br), 944 (s), 926 (s), 805 (m), 717 (w), 679
(w) cm–1. EI-MS: m/z (%) = 531 (6) [M]+, 486 (2) [M – C2H5O]+,
455 (5), 454 (5), 354 (3), 286 (8), 236 (9), 218 (22), 192 (15), 190
(19), 152 (27), 140 (15), 135 (12), 121 (15), 109 (11), 107 (13), 95
(13), 93 (11), 81 (25) [C6H9]+, 69 (56) [C5H9]+, 45 (100) [C2H5O]+,
41 (16) [C3H5]+. HR EI-MS:[28] m/z = 530.3617 [M]+ (calcd. for
C32H50O6: 530.3607).
3-(E,E,E)-[1Ј-13C](Geranylgeranyl)benzene-1,2,4-triol ([1Ј-13C]De-
acetylsuillin, 9#): Acetyl chloride (23 µL, 0.32 mmol) was added un-
der argon to a solution of 8# (85 mg, 0.16 mmol) in MeOH (2 mL).
After completion of the reaction (3 h, TLC monitoring), H2O
(25 mL) was added, and the crude product was extracted with
EtOAc (3 ϫ 15 mL). The combined organic phases were washed
with water (2 ϫ 15 mL) and concentrated under reduced pressure.
Flash chromatography (hexanes/acetone, 3:1) afforded 9# (43.5 mg,
68%) as a yellowish waxy solid. Rf (TLC) = 0.19 (hexanes/acetone,
1
2:1). H NMR (300 MHz, CDCl3): δ = 1.60 (s, 9 H, 18Ј-H, 19Ј-H,
3
4
(each s, 2 H, 3ϫOCH2OCH3), 6.64 (dd, J = 8.9, J = 2.9 Hz, 1
H, 5-H), 6.88 (d, 4J = 2.9 Hz, 1 H, 3-H), 7.05 (d, 3J = 8.9 Hz, 1
H, 6-H) ppm. 13C NMR (75 MHz, CDCl3): δ = 56.0, 56.1, 56.2
(3ϫOCH2OCH3), 95.1, 95.5, 96.3 (3ϫOCH2OCH3), 106.6, 109.4
(C-3 and C-5), 118.3 (C-6), 142.3 (C-1), 148.3 (C-2), 153.0 (C-
20Ј-H), 1.68 (s, 3 H, 16Ј-H), 1.82 (s, 3 H, 17Ј-H), 1.91–2.17 (m, 12
1
3
H, 6ϫCH2), 3.44 (dd, JC,H = 128, JH,H = 7.0 Hz, 2 H, 1Ј-H),
5.04–5.16 (m, 3 H, 3ϫCHvinyl), 5.24–5.33 (m, 1 H, 2Ј-H), 5.60 (br.
3
4
s, 1 H, OH), 6.27 (dd, JH,H = 8.6, JC,H = 1.0 Hz, 1 H, 5-H),
6.61 (d, J = 8.6 Hz, 1 H, 6-H) ppm; 2ϫOH obscured. 13C NMR
3
4) ppm. IR (KBr): ν = 2994 (m), 2956 (s), 2901 (s), 2827 (m), 2787
˜
(75 MHz, CDCl3): δ = 16.08, 16.14 (C-18Ј and C-19Ј), 16.3 (d,
3JC,C = 3.9 Hz, C-17Ј), 17.8 (C-20Ј), 23.0 (13C-1Ј), 25.8 (C-16Ј),
26.4, 26.7, 26.9 (C-5Ј, C-9Ј, C-13Ј), 39.7–39.9 (m, C-4Ј, C-8Ј, C-
12Ј), 106.9 (C-5), 113.1 (C-6), 114.6 (d, 1JC,C = 43.4 Hz, C-3), 121.3
(w), 1609 (m), 1596 (m), 1505 (s), 1469 (m), 1442 (m), 1402 (m),
1261 (m), 1223 (s), 1194 (s), 1153 (s), 1126 (m), 1080 (s), 1008 (s,
br), 923 (s), 850 (m), 811 (m), 767 (w), 708 (w), 666 (w), 631
(w) cm–1. EI-MS: m/z (%) = 259 (4) [M + 1]+, 258 (35) [M]+, 213
(2) [M – C2H5O]+, 195 (2), 182 (28), 152 (52), 137 (7), 123 (2), 121
(2), 79 (2), 45 (100) [C2H5O]+. C12H18O6 (258.27): calcd. C 55.81,
H 7.02; found C 55.82, H 7.26.
1
(d, JC,C = 42.0 Hz, C-2Ј), 123.6, 124.3, 124.5 (3ϫCHvinyl), 131.4
(C-15Ј), 135.1, 135.8, 137.5 (3ϫCvinyl), 139.5 (C-1), 143.1, 148.5
(C-2 and C-4) ppm. EI-MS: m/z (%) = 400 (18) [M + 1]+, 399 (60)
[M]+, 263 (10), 259 (11), 207 (10), 195 (14), 191 (11), 180 (36), 178
(30), 140 (100), 139 (45), 123 (21), 121 (15), 109 (18), 107 (13), 95
(17), 93 (12), 81 (30) [C6H9]+, 69 (39) [C5H9]+, 41 (20) [C3H5]+. HR
EI-MS:[28] m/z = 398.2828 [M]+ (calcd. for C26H38O3: 398.2821).
3-(E,E,E)-[1Ј-13C]Geranylgeranyl-1,2,4-tris(methoxymethoxy)-
benzene (8#): A solution of 7 (65 mg, 0.25 mmol) in dry THF
(2.5 mL) in a Schlenk tube was treated under argon at 0 °C with
nBuLi[27] (1.7 in hexanes, 0.176 mL, 0.30 mmol), and the mixture
was stirred at room temperature for 0.5 h. After the system had
been cooled to 0 °C, CuCl (99.995+%, Aldrich, 30 mg, 0.30 mmol)
was added, and the stirring was continued at 0 °C for 0.5 h. The
mixture was then cooled to –20 °C and treated over 0.5 h with a
Feeding Experiments
Feeding Experiments with Fruit Bodies (Table 2 and Table 4):
DMSO solutions and cyclodextrin preparations of the potential
solution of [1-13C]geranylgeranyl bromide[20] (5#, 89 mg, precursors were injected by syringe deep into the stalks of young
0.25 mmol) in THF (2.5 mL). The mixture was stirred for an ad-
ditional 0.5 h at –20 °C and subsequently warmed to room tem-
perature. Saturated aq. NH4Cl was added, and the product was
extracted with Et2O. The combined organic phases were dried
(Na2SO4) and concentrated under reduced pressure. Compound 8#
was purified by flash chromatography (hexanes/EtOAc, 10:1). Yield
97 mg (73%), colourless oil. Rf (TLC) = 0.23 (hexanes/EtOAc,
specimens (cap diameter: approx. 2 cm) of S. bovinus. For the feed-
ing of the liposome preparations, a deep hole was gouged into a
young fruit body; if necessary, the reservoir volume was extended
by insertion of an appropriate short glass tube. The resulting cavity
was soaked with the liposome preparation (1 mL). The toadstools
were harvested after 4–7 d, frozen in liquid nitrogen and kept at
–20 °C until workup and isolation of the metabolites.
3550
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Eur. J. Org. Chem. 2008, 3544–3551