2260 J . Org. Chem., Vol. 67, No. 7, 2002
Schuhr et al.
to a pH of 2 by the addition of 1 M hydrochloric acid and
extracted with ethyl acetate. Evaporation of the solvent and
converted to the required thioester form as compared to
octanoate. Alternatively, the proffered hexyl malonate
could be enzymatically decarboxylated prior to utilization
for the biosynthesis of lipstatin. The incorporation data
are not sufficient to conclusively prove the proposed
hypothesis of R-carboxylation of octanoate.
distillation afforded 73 mmol of 3 (13.8 g, 94%, ee 87%, [R]25
D
+1.88° (c ) 0.1 g/mL in acetone)).
3: 1H NMR δ (ppm) 0.9 (t, J ) 7 Hz, 3H, 3′), 1.5-1.6 (m,
2H, 2′), 2.5/2.6 (2s, 4H, 2/4), 4.0 (t, J ) 7 Hz, 2H, 1′), 4.4 (m,
0.25H, 3); 13C NMR δ (ppm) 10.2 (3′), 21.8 (2′), 40.4/40.5 (2/4),
64.6 (3), 66.5 (1′), 171.9 (1), 176.1 (5).
We found that only 15N label but no 13C label is
incorporated into lipstatin from compound 10. Obviously,
incorporation can only take place after hydrolysis of the
formamide motif. The data also show that the assumed
formate moiety liberated by hydrolysis of the proffered
15N13C-labeled formyl leucine is not incorporated into
lipstatin to a significant extent.
All reported data are consistent with the biosynthetic
scheme shown in Figure 4. A fraction of the thioester of
3-hydroxytetradeca-5,8-dienoic acid is obviously con-
verted to hexyl malonic thioester via an octanoic acid
derivative. This hexyl malonic thioester undergoes a
condensation in conjunction with decarboxylation afford-
ing a C22-moiety with the main fraction of the hydroxy-
acyl thioester. Reduction of the keto group, ring closure
to the â-lactone, and introduction of the leucine residue
followed by its formylation afford lipstatin.
[3-2H ]-(3S)-3-ter t-Bu t yld ip h en ylsiloxyglu t a r ic Acid
Mon op r op yl Ester (4). tert-Butyldiphenylsilyl chloride (17.3
g, 62.9 mmol) was added dropwise to a solution containing 5
g (26.3 mmol) of 3 and 6.44 g (94.6 mmol) of imidazole in 50
mL of dry CH2Cl2 at 3 °C under a nitrogen atmosphere. The
solution was incubated at room temperature with shaking
overnight. An aqueous solution of 5% sodium carbonate (160
mL) was added. After several days, CH2Cl2 was evaporated,
and the remaining aqueous phase was extracted with diethyl
ether. It was adjusted to pH 2 and extracted again. The organic
phase was evaporated, affording a slightly yellow syrup (9.1
g, 21.3 mmol, 81%).
4: 1H NMR δ (ppm) 0.9 (t, J ) 7 Hz, 3H, 3′), 1.0 (s, 9H,
tBu-Me), 1.5-1.6 (m, 2H, 2′), 2.5-2.7 (br, 4H, 2/4), 3.9 (m, 2H,
1′), 4.5 (m, 0.25H, 3), 7.3-7.7 (m, 10H, Phe); 13C NMR δ (ppm)
10.4 (3′), 19.2 (tBu-C), 21.8 (2′), 26.8 (tBu-Me), 41.4/41.6 (2/4),
66.1 (1′), 67.0 (3), 127.7/129.8/133.2/135.9 (Phe), 170.8 (1), 176.9
(5).
[3-2H]-(3S)-5-Hyd r oxy-3-ter t-bu tyld ip h en ylsiloxyp en -
ta n oic Acid P r op yl Ester (5). A solution of borane-THF
complex in THF (11 mL, 1 M) was added to a solution of 4
(3.62 g, 8.4 mmol) in 13 mL of dry THF at -18 °C and under
an inert atmosphere. The mixture was allowed to warm to 0
°C. After 7 h, one volume of ice-water and sodium carbonate
solution were added slowly. The mixture was extracted with
diethyl ether. The organic phase was dried over sodium sulfate.
The solvent was evaporated under reduced pressure. The
residue was applied to a column of silica gel 60 (48 cm × 3
cm) that was developed with hexane/ethyl acetate (2:1) yielding
3.1 g (7.4 mmol, 88%) of 5.
5: 1H NMR δ (ppm) 0.9 (t, J ) 7 Hz, 3H, 3′), 1.0 (s, 9H,
tBu-Me), 1.5-1.6 (m, 2H, 2′), 1.7-1.8 (m, 2H, 4), 2.5 (s, 2H,
2), 3.6 (m, 2H, 5), 3.9 (t, J ) 7 Hz, 2H, 1′), 4.3 (m, 0.25H, 3),
7.3-7.7 (m, 10H, Phe); 13C NMR δ (ppm) 10.3 (3′), 19.2 (tBu-
C), 21.7 (2′), 26.8 (tBu-Me), 38.9 (4), 41.9 (2), 59.1 (5), 66.0/
68.6 (1′/3), 127.6/129.7/133.5/135.8 (Phe), 171.2 (1).
[3-2H]-(3S)-5-Oxo-3-ter t-b u t yld ip h en ylsiloxyp en t a n o-
ic Acid P r op yl Ester (6). A 3.65 g (8.8 mmol) portion of 5
was added to 3.75 g (17.4 mmol) of pyridinium chlorochromate
in 30 mL of dry CH2Cl2. The solution was incubated for 3 h
with shaking under a dry atmosphere. The mixture was
extracted with ether. The organic phase was concentrated
under reduced pressure and applied to a column of silica gel
(43 cm × 3 cm), which was developed with hexane/ethyl acetate
(2:1), yielding 2.34 g (5.7 mmol, 64%) of 6.
6: 1H NMR δ (ppm) 0.9 (t, J ) 7 Hz, 3H, 3′), 1.0 (s, 9H,
tBu-Me), 1.5-1.7 (q, J ) 7 Hz, 2H, 2′), 2.5-2.8 (br, 4H, 2/4),
3.9 (t, J ) 7 Hz, 2H, 1′), 4.6 (m, 0.25H, 3), 7.3-7.8 (m, 10H,
Phe), 9.6 (s, 1H, 5); 13C NMR δ (ppm) 10.3 (3′), 19.1 (tBu-C),
21.8 (2′), 26.8 (tBu-Me), 41.9 (2), 50.3 (4), 65.9/66.1 (1′/3), 127.7/
129.9/133.2/135.7 (Phe), 170.5 (1), 200.7 (5)
[3,10,11,12-2H ]-(3S,5Z,8Z)-3-ter t-Bu t yld ip h en ylsiloxy-
tetr a d eca -5,8-d ien oic Acid P r op yl Ester (8). The phos-
phonium salt of labeled bromononene (7) (4.31 g, 9.2 mmol)
was added to 9.0 mL of a 1 M solution of sodium bis-silyl amide
at -17 °C in a dry THF/toluene solution (5:1) under nitrogen.
The orange-colored solution was allowed to warm to room
temperature and was then cooled to -90 °C. A 3.1 g portion
of 6 (7.5 mmol) in 5 mL of THF was added dropwise, and the
mixture was shaken overnight with warming to room tem-
perature. A saturated aqueous solution of ammonium chloride
was added, and the mixture was extracted with diethyl ether.
The extract was concentrated under reduced pressure. The
residue was applied to a column of silica gel (37 cm × 2.5 cm),
which was developed with pentane/diethyl ether (97:3), yield-
ing pure 8 (2.42 g, 4.6 mmol, 62%).
Exp er im en ta l Section
Ma ter ia ls. Commercially available reagents were used
without further purification. Pig liver esterase (EC 3.1.1.1) was
purchased from Fluka (Buchs, Switzerland). The phosphonium
salt 7 was synthesized as published and is a complex mixture
of isotopomers.6 The reactions were done in dry solvents at
room temperature unless stated otherwise.
Syn th esis of 2H-La beled In ter m ed ia tes. (3S,5Z,8Z)3-
Hyd r oxytetr a d eca -5,8-d ien oic Acid (9). The synthesis of
isotope labeled 9 was achieved by methods modified from ref
8.
[3-2H]-3-Hydr oxyglu tar ic Acid Dipr opyl Ester (2). 3-Ox-
oglutaric acid (1) (21 g, 144 mmol) was dissolved in 50 mL of
dry propanol (40 g, 0.66 mol) containing 8.75 g (0.24 mmol) of
HCl. The mixture was incubated with shaking at 35 °C for 10
min and was then kept at room temperature overnight. The
solution was extracted with diethyl ether. The organic phase
was washed with aqueous sodium carbonate, dried over
sodium sulfate, and evaporated to dryness under reduced
pressure, yielding 30.9 g (134 mmol, 93%) of the liquid dipropyl
ester of 1.
Without further purification, the ester was dissolved in 160
mL of propanol containing 1% (v/v) of triethylamine. Catalytic
deuteration was performed with D2 at atmospheric pressure
over Raney nickel (7.2 g) as catalyst. The catalyst was removed
by filtration after 18 h, and the solution was evaporated under
reduced pressure. Vacuum distillation of the residue (bp 100-
112 °C, ∼0.1 mbar) afforded 2 (118 mmol, 27.5 g, 88%). The
deuterium abundance at position 3 was about 75%, as the
catalyst was produced in nondeuterated solvents.
2: 1H NMR δ (ppm) 0.9 (t, J ) 8 Hz, 6H, 3′/3′′), 1.6-1.7 (m,
4H, 2′/2′′), 2.6 (s, 4H, 2/4), 3.5 (s, 1H, OH), 4.1 (t, J ) 7 Hz,
4H, 1′/1′′), 4.5 (m, 0.25H, 3); 13C NMR δ (ppm) 10.2 (3′, 3′′),
21.8 (2′, 2′′), 40.6 (2, 4), 64.6 (3), 66.2 (1′, 1′′), 171.7 (1, 5).
[3-2H]-(3S)-3-Hyd r oxyglu ta r ic Acid Mon op r op yl Ester
(3). Pig liver esterase (3620 U) in 270 mL of 30 mM phosphate
buffer (pH ) 7) was added to 18 g (77.5 mmol) of 2. The
mixture was incubated with stirring at 4 °C. The pH was
continually adjusted to 7.0 by the addition of 1 M NaOH. The
reaction came to an end after the consumption of 1 equiv of
the base (44 h). The solution was brought to pH 9 and
extracted with diethyl ether. The aqueous phase was adjusted
(8) Baader, E.; Bartmann, W.; Beck, G.; Bergmann, A.; Fehlhaber,
H.-W.; J endralla, H.; Kesseler, K.; Saric, R.; Schu¨ssler, H.; Teetz, V.;
Weber, M.; Wess, G. Tetrahedron Lett. 1988, 29, 2563-2566.