Biosynthesis of acaterin: Coupling of C5 unit with octanoate

Article abstract of DOI:10.1021/jo015614g

Acaterin (1), produced by Pseudomonas sp. A 92, is a secondary metabolite having a 2-penten-4-lide structure. Feeding experiments with ²H- and ¹³C-labeled decanoic acid, their 3-oxygenated congeners, and octanoic acid have suggested that 1 is biosynthesized via coupling of a C₅ unit with octanoate, rather than via introduction of a C₃ unit at the α position of a decanoate derivative. Further feeding study of [2,3-¹³C₂] decanoic acid concluded that the former route is operating in the biosynthesis of 1.

Full text of DOI:10.1021/jo015614g

VOLUME 66, NUMBER 17
AUGUST 24, 2001
© Copyright 2001 by the American Chemical Society
Articles
Biosyn th esis of Aca ter in : Cou p lin g of C5 Un it w ith Octa n oa te
Yasuyo Sekiyama,‡ Yoshinori Fujimoto,*,‡ Keiji Hasumi,§ and Akira Endo§
Department of Chemistry and Materials Science, Tokyo Institute of Technology,
Meguro, Tokyo 152-8551, J apan, and Department of Applied Biological Science, Tokyo Noko University,
Fuchu, Tokyo 183-8509, J apan
fujimoto@cms.titech.ac.jp
Received March 6, 2001
Acaterin (1), produced by Pseudomonas sp. A 92, is a secondary metabolite having a 2-penten-4-
olide structure. Feeding experiments with 2H- and 13C-labeled decanoic acid, their 3-oxygenated
congeners, and octanoic acid have suggested that 1 is biosynthesized via coupling of a C5 unit with
octanoate, rather than via introduction of a C3 unit at the R position of a decanoate derivative.
Further feeding study of [2,3-13C2]decanoic acid concluded that the former route is operating in the
biosynthesis of 1.
In tr od u ction
this group of compounds, was isolated from Pseudomonas
sp. A 92 as an inhibitor of acyl-CoA:cholesterol acyltrans-
ferase,17 and its absolute configuration was determined
as (4R,1′R).18 4-Dehydroacaterin (2), an immediate pre-
cursor of 1, was subsequently isolated from the same
strain when cultured in a medium supplemented with a
Natural compounds having a polyketide chain at the
C-2 position of a 2-penten-4-olide structure have been
found in a variety of organisms.1-16 Acaterin (1), one of
* To whom correspondence should be addressed.
‡ Tokyo Institute of Technology.
§ Tokyo Noko University.
(8) Bohlmann, F.; Grenz, M. Tetrahedron Lett. 1971, 3623-3626.
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198. (b) Zeng L.; Ye, Q.; Oberlies, N. H.; Shi, G.; Gu, Z.-M.; He, K.;
McLaughlin, J . L. Nat. Prod. Rep. 1996, 13, 275-306.
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1117-1122.
(11) Hadacek, F.; Greger, H.; Grenz, M.; Bohlmann, F. Phytochem-
istry 1987, 26, 1527-1529.
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3766.
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1539-1542.
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659.
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10.1021/jo015614g CCC: $20.00 © 2001 American Chemical Society
Published on Web 07/28/2001

5650 J . Org. Chem., Vol. 66, No. 17, 2001
Sekiyama et al.
Sch em e 1. Tw o P ossible Mod es in th e
Biosyn th esis of 1
long chain fatty acid such as dodecanoic acid, tetrade-
canoic acid, and oleic acid.19 Although 13C-acetate was
found to be hardly incorporated into 1, feeding [1-13C]-
dodecanoic acid gave rise to 1 labeled at the C-1, -1′, -3′,
-5′, and -7′ positions.20 Feeding experiments of 13C- and
2H-labeled glycerols revealed that the branched C3 moiety
(C-3, -4, and -5 positions) of 1 and 2 originates from a
glycerol metabolite having a carboxylic group, and this
implies that a 3-oxo intermediate would be involved in
acaterin biosynthesis.20 Furthermore, it was suggested
that the glycerol metabolite (C3 precursor) is neither
pyruvate nor lactate, but 1,3-bisphosphoglyceric acid or
its biological equivalent, based on the findings that
feeding of sn-(3R)- and sn-(3S)-[3-2H]glycerols furnished
compound 2 stereospecifically labeled at (E)-H and (Z)-H
on C-5, respectively.21
F igu r e 1. 2H NMR spectra (61 MHz) of 1. (a) Derived from
(4RS)-[4-2H]decanoic acid (3); (b) derived from (3RS,4RS)-[4-
2H]-3-hydroxydecanoic acid (4); (c) derived from ethyl (4RS)-
[4-2H]-3-oxodecanoate (5); (d) derived from (3RS,4RS)-[3,4-
2H2]-3-hydroxydecanoic acid (6); (e) derived from (2RS)-[2-
2H]octanoic acid (8); (f) 1H NMR spectrum (300 MHz) of
nonlabeled 1.
tives in view of the hypothesis that related compounds
2
are biosynthesized via mode A. Potential H-labeled C10
precursors, (4RS)-[4-2H]decanoic acid (3), (3RS,4RS)-[4-
2H]-3-hydroxydecanoic acid (4), and ethyl (4RS)-[4-2H]-
3-oxodecanoate (5) were synthesized and fed to P. sp. A
92, individually. Compounds 3, 4, and 5 (carrying 80%
1
2H at C-4, estimated by H NMR) were readily prepared
by two carbon extension of ethyl (2RS)-[2-2H]octanoate
which was obtained by exchange of R proton of the
Concerning the mode of the formation of acaterin
framework, two distinct modes can be considered: mode
A, introduction of the C3 precursor at the R position of a
decanoate derivative, and mode B, coupling of octanoate
with a C5 unit (hereafter tentatively represented as a C5-
lactone) which is made up of glycerate and acetate (or
malonate) (Scheme 1). It has been proposed that more
than a dozen of secondary metabolites possessing such a
lactone moiety are biosynthesized via mode A.8-16 Fur-
thermore, virginiae butanolide A, a butylolactone auto-
regulator isolated from Streptomyces antibioticus, is
known to be biosynthesized via introduction of a branched
C3 unit (dihydroxyacetone) at the R position of a C9-
polyketide precursor.22 It was also reported that tenu-
azonic acid, a tetramic acid derivative, was biosynthe-
sized via introduction of a branched C6 unit (L-isoleucine)
at the R position of 3-oxobutanoate in Alternaria tenuis.23
In this paper, we report the results of labeling studies
which led to the conclusion that 1 was biosynthesized
via mode B rather than mode A.
2
nonlabeled material with D2O. The H NMR spectra of 1
obtained by feeding 3-5 exhibited a signal at δ 1.71 due
to 2′-2H (Figure 1a-c). These results suggested that the
substrates 3-5 were indifferently incorporated into 1
after conversion to a common precursor. To obtain the
information on the C-3 oxidation level of the postulated
C10 precursor, the metabolic fate of the C-3 hydrogens
was investigated by feeding (3RS,4RS)-[3,4-2H2]-3-hy-
droxydecanoic acid (6) (carrying 80% 2H at C-4 and >98%
2H at C-3, estimated by 1H NMR) and (3RS)-[3-2H]-
2
decanoic acid (7) (carrying 90% H at C-3, estimated by
1H NMR). Compound 6 was prepared by reduction of 5
with NaBD4 and 7 was prepared by one carbon extension
2
of ethyl (2RS)-[2-2H]nonanoate. The H NMR spectrum
of 1 obtained by feeding 6 showed a signal due to 2′-2H
of 1, but not due to 1′-2H (Figure 1d). Furthermore, the
2H NMR spectrum of 1 obtained by feeding 7 exhibited
no deuterium signals. Since decanoic acid is known to
be incorporated into 1 (vide supra), this observation
implies that two hydrogens at the C-3 of decanoate were
completely lost during the transformation. These results
suggested that decanoate and 3-hydroxydecanoate were
not incorporated as such, but incorporated into 1 after
conversion to 3-oxodecanoate. Alternatively, 3-oxode-
canoate was further degraded to octanoate, which was
then incorporated into the C8 side chain of 1 via mode B.
Feeding studies of (2RS)-[2-2H]octanoic acid (8) (carrying
97% 2H at C-2, estimated by 1H NMR) revealed that
octanoate was incorporated into 1 as shown in the 2H
NMR spectrum (Figure 1e) of the resulting 1. Thus, the
candidates of the polyketide precursor of 1 were narrowed
down to 3-oxodecanoate or octanoate (Scheme 2).
Resu lts a n d Discu ssion
F eed in g Exp er im en ts w ith 2H-La beled F a tty Ac-
id s. We initiated feeding studies of C10 fatty acid deriva-
(19) Sekiyama, Y.; Araya, H.; Hasumi. K.; Endo, A.; Fujimoto, Y.
Nat. Prod. Lett. 1997, 11, 61-66.
(20) Sekiyama, Y.; Araya, H.; Hasumi. K.; Endo, A.; Fujimoto, Y.
Tetrahedron Lett. 1998, 39, 6233-6236.
(21) Sekiyama, Y.; Fujimoto, Y.; Hasumi. K.; Endo, A. Tetrahedron
Lett. 1999, 40, 4223-4226.
(22) (a) Sakuda, S.; Higashi, A.; Tanaka, S.; Nihira, T.; Yamada, Y.
J . Am. Chem. Soc. 1992, 114, 663-668. (b) Sakuda, S.; Tanaka, S.;
Mizuno, K.; Sukcharoen, O.; Nihira, T.; Yamada, Y. J . Chem. Soc.,
Perkin Trans. 1 1993, 2309-2315.
(23) Gatenbeck, S.; Sierankiewicz, J . Acta Chem. Scand. 1973, 27,
1825-1827.

Biosynthesis of Acaterin
J . Org. Chem., Vol. 66, No. 17, 2001 5651
2
Sch em e 2. In cor p or a tion of H-La beled F a tty
Acid s in to 1
F igu r e 2. 13C NMR spectra (100 MHz, CDCl3) of 1. (a) Derived
from [2,3-13C2]decanoic acid (11); (b) nonlabeled 1. The weak
flanking doublet signals for C-1′ and C-2 may be attributed to
recombination of [1-13C]octanoate and [2-13C]acetate arising
from 11.
Ta ble 1. 13C-En r ich m en ts in 1 Obta in ed by F eed in g
[1-13C]-La beled F a tty Acid s
relative 13C-enrichmenta from
carbon
(δ, ppm) decanoic acid (9) octanoic acid (10) dodecanoic acid
[1-13C]-
[1-13C]-
[1-13C]-
Sch em e 3. P la u sible P a th w a y for th e
Biosyn th esis of 1
C-1 (172.6)
C-2 (136.2)
C-3 (149.3)
C-4 (77.9)
C-5 (18.9)
C-1′ (67.0)
C-2′ (35.4)
C-3′ (25.2)
C-4′ (29.3)
C-5′ (29.1)
C-6′ (31.7)
C-7′ (22.6)
C-8′ (14.0)
13.1
0.8
1.3
1.2
1.0
3.1
1.0
3.0
0.8
2.5
0.7
2.2
1.0
25.2
1.0
0.9
1.0
1.0
43.8
1.0
4.7
0.9
5.2
1.0
3.6
1.0
8.6
0.7
0.9
0.9
1.0
2.7
1.0
4.0
0.9
3.6
0.8
3.6
0.9
This was rigorously established by feeding studies of
[2,3-13C2]decanoic acid (11), which was synthesized from
[1-13C]octanoic acid in 10 steps with K13CN as the label
source. The 13C NMR spectrum of 1 (Figure 2) biosyn-
thesized from 11 showed that the intensity of the C-1′
signal (19.8-fold, normalized as C-5 ) 1.0) is considerably
larger than that of C-2 (10.9-fold). These data clearly
indicated that the doubly labeled substrate was cleaved
between C-2 and C-3, and then the resulting [1-13C]-
octanoate was incorporated into the C8 side chain of 1.
As expected, the C-1 (9.2-fold) and C-2 signals of 1 in
this feeding experiment were considerably enriched,24 due
to the contribution of [1-13C]- and [2-13C]acetate arising
from 11.
In conclusion, we have demonstrated that 1 is biosyn-
thesized via mode B, i.e., coupling of octanoate and a C5
unit. The result is in contrast with the biosynthesis of
structurally related natural compounds. We would like
to propose a pathway for acaterin biosynthesis as shown
in Scheme 3, on the basis of our previous and present
data. Coupling of a hypothesized C5-lactone with oc-
tanoate, elimination of phosphoric acid followed by
reduction at C-3 and C-1′ would produce 2, which is
finally reduced to furnish 1. Investigation of the exact
structure of the C5 unit and the order of the above events
is now in progress in our laboratory.
a
Relative to the abundance at C-5 ) 1.0.
F eed in g E xp er im en t s w it h 13C-La b eled F a t t y
Acid s. To shed light on which fatty acid, C10 or C8, is
the obligatory precursor, feeding studies of [1-13C]de-
canoic acid (9) and [1-13C]octanoic acid (10) were carried
out. 13C-Enrichments in the resulting 1 are listed in Table
1. The 13C NMR spectrum of 1 obtained by feeding 9
showed an enrichment of the C-1 signal (13.1-fold of
natural abundance) accompanied by lower level enrich-
ments of the signals at C-1′, -3′, -5′, and -7′ (2.7-fold,
average of the 4 carbons). These lower level enrichments
were due to the incorporation of [1-13C]acetate arising
from the labeled fatty acid via â-oxidation. On the other
hand, the 13C NMR spectrum of 1 obtained by feeding
10 displayed a remarkable enrichment of the C-1′ signal
(43.8-fold). The specific uptake of 10 indicated that
octanoate was more efficiently utilized in the biosynthesis
of 1 than decanoate, supporting mode B. The spectrum
also showed that the C-3′, -5′, -7′ signals were enriched
to some extent (4.5-fold, average of the three carbons).
Interestingly, the C-1 signal was much more intense
(25.2-fold) than the three signals. These results are in
agreement with our earlier observation that feeding
[1-13C]dodecanoic acid gave rise to 1 which was labeled
at C-1 (8.6-fold) and C-1′, -3′, -5′, and -7′ (3.5-fold, average
of the four carbons).20 It is likely that acetyl CoA produced
by â-oxidation of fatty acids was more efficiently used
for the C5-unit formation, probably via a different pool
of acetate, than the C8 appendage. Therefore, the afore-
mentioned enrichment of the C-1 signal of 1 obtained by
feeding 9 can be rationalized by assuming such acetate
incorporation. Under these experimental conditions, no
enrichment was observed at the C-3, -4, and -5 positions.
These studies suggested that octanoate is an obligatory
precursor, rather than 3-oxodecanoate.
Exp er im en ta l Section
Gen er a l P r oced u r es a n d Ma ter ia ls. The 1H and 13C
NMR spectra were recorded on J EOL LA-400 or LA-300 or
2
GSX-270 spectrometers in CDCl3 at 298 K. H NMR spectra
(24) Relative 13C-enrichments in 1 obtained by feeding [2,3-13C2]-
decanoic acid are 9.2 (C-1), 10.9 (C-2), 1.4 (C-3), 1.3 (C-4), 1.0 (C-5),
19.8 (C-1′), 4.5 (C-2′), 3.3 (C-3′), 3.7 (C-4′), 4.7 (C-5′), 3.4 (C-6′), 2.7
(C-7′), and 2.9 (C-8′).

5652 J . Org. Chem., Vol. 66, No. 17, 2001
Sekiyama et al.
were recorded on a J EOL LA-400 spectrometer in CHCl3 at
298 K. EI-MS (70 eV), FAB-MS (3-nitrobenzyl alcohol as
matrix), and GC-MS [EI-MS mode, capillary column HEWLETT
PACKARD Ultra 1 (25 m × 0.20 mm i.d., film thickness 0.33
mm), oven temp programmed from 100 °C to 120 °C at the
increasing rate 2 °C/min, injection temp. 200 °C, separator
temp. 200 °C, ion source temp 300 °C] spectra were taken on
a J EOL J MS-AX505HA spectrometer. Melting points were
measured by a Yazawa BY-1 hot-stage microscope and are
uncorrected. Analytical TLC was carried out on Merck silica
gel 60F-254 plates (0.25 mm precoated). Column chromatog-
raphy was performed on 70-230 mesh silica gel from Merck.
Reaction solvents were purified by distillation from appropri-
ate drying agents: THF, ether (LiAlH4), diisopropylamine,
DMSO (NaH), CH2Cl2 (P2O5), and benzene (CaH2). Except for
hydrogenolysis, all reactions were performed under nitrogen
atmosphere. K13CN (99% 13C), [1-13C]octanoic acid (10) (>99%
13C), [1-13C]decanoic acid (9) (>99% 13C) and [1-13C]dodecanoic
acid (>99% 13C) were purchased from Isotec Inc. D2O (>99.8%
D) was purchased from Merck. NaBD4 (98% D) was purchased
from Aldrich.
F er m en ta tion . Pseudomonas. sp. A 92 was maintained at
4 °C on Oxoid Brain Heart Infusion agar slants. The produc-
tion medium contained glucose (0.5%), soybean meal (1.0%),
peptone (0.5%), and CaCO3 (0.2%). The pH of the medium was
adjusted to 7.0 with 1.0 M KOH. The medium was sterilized
for 20 min at 120 °C in a steam autoclave. A loopful of the
organism from a slant culture was inoculated into 100 mL of
the production medium in a 500 mL baffled Erlenmeyer flask.
The inoculated cultures were incubated at 25 °C in the dark
for 48 h on a rotary shaker at 200 rpm.
F eed in g Exp er im en ts w ith La beled F a tty Acid s. In
general, 100 mg each of labeled fatty acid was individually
added to 100 mL of the production medium before sterilization.
In the feeding experiment of [2,3-13C2]decanoic acid, a mixture
of 30 mg of the labeled acid and 70 mg of unlabeled acid was
used. The cultures were worked up, as previously described,19
to give 1 in the amount indicated in parentheses: (4RS)-[4-
2H]decanoic acid (3) (10 mg), (3RS,4RS)-[4-2H]-3-hydroxyde-
canoic acid (4) (12 mg), ethyl (4RS)-[4-2H]-3-oxodecanoate (5)
(4 mg), (3RS,4RS)-[3,4-2H2]-3-hydroxydecanoic acid (6) (12 mg),
(3RS)-[3-2H]decanoic acid (7) (10 mg) [2-2H]octanoic acid (8)
(10 mg), [1-13C]decanoic acid (9) (6 mg), [1-13C]octanoic acid
(10) (6 mg), [2,3-13C2]decanoic acid (11) (17 mg).
mL). The mixture was stirred at room temperature for 10 h
and then diluted with ether (20 mL) and H2O (50 mL). The
separated organic layer was repeatedly extracted with 15%
aqueous NaOH. The combined aqueous layer was acidified
with 2 N HCl and extracted with ether. The organic layer was
washed with brine, dried over anhydrous Na2SO4, and con-
centrated under reduced pressure. Column chromatography
of the resulting crude product with hexane/AcOEt (10:1)
afforded 8 (160 mg, 77%) as a colorless oil: 1H NMR (400 MHz)
δ 0.88 (3H, t, J ) 6.8 Hz), 1.22-1.39 (8H, m), 1.60-1.65 (2H,
m), 2.31-2.37 (1.03H, m, H-2); 13C NMR (100 MHz) δ 14.05,
22.59, 24.54 (C-3 for doubly labeled 8), 24.60 (C-3 for singly
labeled 8), 24.67 (C-3 for unlabeled 8), 28.90, 28.95 (C-4 for
doubly labeled 8), 28.97 (C-4 for singly labeled 8), 29.00 (C-4
for unlabeled 8), 31.60, 33.72 (t, 1J C-D ) 19.4 Hz, C-2 for singly
labeled 8), 34.01 (C-2 for unlabeled 8), 179.95; 2H NMR δ 2.33
(D-2); EI-MS m/z 146 (1.51), 145 (3.23), 144 (M+ for unlabeled
8, 1.14), 62 (34.9) 61 (100), 60 (42.5).
(4RS)-[4-2H]Deca n oic Acid (3). LiAlH4 (356 mg, 9.38
mmol) was added to a solution of 12 (3.25 g, 18.8 mmol) in
ether (33.0 mL), and the mixture was stirred at room temper-
ature for 30 min. The reaction mixture was diluted with ether
and 2 N HCl. The separated organic layer was washed with
saturated aqueous NaHCO3 and brine, dried over Na2SO4, and
concentrated under reduced pressure. Column chromatogra-
phy of the resulting crude product with hexane/AcOEt (7:1)
gave (2RS)-[2-2H]octanol (13) (1.60 g, 65%) as a colorless oil:
1H NMR (300 MHz, CDCl3 containing a small amount of D2O),
0.88 (3H, t, J ) 6.6 Hz), 1.18-1.40 (10H, m), 1.44-1.66 (1.1H,
m, H-2), 3.60-3.68 (2H, m).
PDC (5.73 g, 15.2 mmol), NaOAc (500 mg, 6.10 mmol), and
molecular sieves 4A powder (5.73 g) were added to the solution
of 13 (1.00 g, 7.62 mmol) in CH2Cl2 (20.0 mL). After being
stirred at room temperature for 2 h, the reaction mixture was
diluted with ether and filtered through a Florisil column. The
filtrate was washed with 2 N HCl, saturated aqueous NaHCO3,
and brine, dried over Na2SO4, and concentrated under reduced
pressure to give a crude (2RS)-[2-2H]octanal (14) (624 mg) as
a yellow oil which was used for the next step without further
purification.
Ethyl (triphenylphosphoranylidene)acetate (1.23 g, 3.53
mmol) was added to the solution of crude 14 (230 mg, 1.78
mmol) in benzene (2.30 mL), and the mixture was stirred at
room temperature for 1 h. The reaction mixture was worked
up as described for 13 to afford ethyl (4RS)-[4-2H]-2-decenoate
(15) (215 mg, 38% from 13) as a colorless oil: 1H NMR (300
MHz) δ 0.88 (3H, t, J ) 6.7 Hz), 1.16-1.38 (11H, m), 1.38-
1.52 (2H, m) 2.14-2.21 (1.1 H, m, H-4), 4.18 (2H, q, J ) 7.1
Hz) 5.81 (1H, d, J ) 15 Hz), 6.92-7.10 (1H, m).
(2RS)-[2-2H]Octa n oic Acid (8). A solution of ethyl oc-
tanoate (5.0 g, 29 mmol) in THF (15 mL) was added dropwise
to a freshly prepared solution of LDA [n-BuLi (23 mL, 1.5 M
solution in hexane, 35 mmol) was added to diisopropylamine
(4.9 mL, 35 mmol) in THF (60 mL) at -78 °C, and the mixture
was stirred at the same temperature for 10 min. The acetone
bath was removed, and the reaction mixture was stirred at
ambient temperature for 10 min and then recooled to -78 °C]
in THF at -78 °C. After the mixture was stirred at this
temperature for 1 h, D2O (1.6 mL, 88 mmol) in THF (13 mL)
was added dropwise to the solution. The resulting mixture was
allowed to warm to room temperature and diluted with ether
and saturated aqueous NH4Cl. The layers were separated, and
the aqueous layer was repeatedly extracted with ether. The
combined organic layer was washed with 2 N HCl, saturated
aqueous NaHCO3, and brine, dried over Na2SO4, and concen-
trated under reduced pressure. Column chromatography of the
resulting crude product with hexane/AcOEt (10:1) afforded
ethyl (2RS)-[2-2H]octanoate (12) (2.0 g, 76%) as a colorless
oil: 1H NMR (400 MHz) δ 0.88 (3H, t, J ) 6.8 Hz), 1.26-1.36
(11H, m), 1.57-1.66 (2H, m), 2.32-2.37 (1.03H, m, H-2), 4.12
(2H, q, J ) 7.1 Hz); 13C NMR (100 MHz) δ 14.05, 14.24, 22.58,
24.84 (C-3 for doubly labeled 12), 24.90 (C-3 for singly labeled
12), 24.96 (C-3 for unlabeled 12), 28.91, 29.02 (C-4 for doubly
labeled 12), 29.05 (C-4 for singly labeled 12), 29.08 (C-4 for
Pd-C (10%, 109 mg) was added to a solution of 15 (543 mg,
2.72 mmol) in MeOH (54.3 mL), and the mixture was stirred
under hydrogen at room temperature for 12 h. The reaction
mixture was filtered through a pad of Celite, and the filtrate
was concentrated under reduced pressure. Column chroma-
tography of the resulting crude product with hexane/AcOEt
(10:1) afforded ethyl (4RS)-[4-2H]decanoate (16) (450 mg, 82%)
as a colorless oil: 1H NMR (270 MHz) δ 0.89 (3H, t, J ) 6.8
Hz), 1.16-1.36 (14.2H, m), 1.50-1.68 (2H, m) 2.28 (2 H, t, J
) 6.1 Hz), 4.12 (2H, q, J ) 7.2 Hz).
Ester 16 (447 mg, 2.22 mmol) was hydrolyzed to 3 (256 mg,
67%) in the same manner as described for 8. 3: white plates;
1
mp 27-29 °C; H NMR (400 MHz) δ 0.88 (3H, t, J ) 6.8 Hz),
1.20-1.38 (11.2H, m), 1.60-1.65 (2H, m), 2.35 (2H, t, J ) 7.6
Hz); 13C NMR (100 MHz) δ: 14.10, 22.65, 24.45 (C-3 for doubly
labeled 3), 24.55 (C-3 for singly labeled 3), 24.65 (C-3 for
1
unlabeled 3), 28.63 (t, J C-D ) 19.0 Hz, C-4 for singly labeled
3), 29.04 (C-4 for unlabeled 3), 29.13 (C-5 for singly labeled
3), 29.24 (C-5 for unlabeled 3 and C-7), 29.32 (C-6 for doubly
labeled 3) 29.35 (C-6 for singly labeled 3), 29.37 (C-6 for
unlabeled 3), 31.84, 34.06 (C-2 for doubly labeled 3) 34.09 (C-2
1
unlabeled 12), 31.64, 34.08 (t, J C-D ) 19.3 Hz, C-2 for singly
2
labeled 12), 34.38 (C-2 for unlabeled 12), 60.13, 173.95; GC-
MS m/z 174 (1.14), 173 (2.98), 172 (M+ for unlabeled 12, 0.57),
129 (14.9), 128 (321), 127 (5.75), 90 (21.1), 89 (100), 88 (21.2).
Lithium hydroxide monohydrate (604 mg, 14.4 mmol) was
added to the solution of 12 (250 mg, 1.44 mmol) in DME (12.5
for singly labeled 3), 34.11 (C-2 for unlabeled 3), 180.57; H
NMR δ 1.31 (1D, D-4), 1.62 (0.03D, D-3), 2.32 (0.09D, D-2);
EI-MS m/z 173 (M+ for singly labeled 3, 4.67), 144 (5.62), 143
(3.98), 117 (1.57), 116 (8.19), 115 (12.4), 131 (12.2), 130 (19.3),
128 (28.1), 73 (100), 60 (78.2).

Biosynthesis of Acaterin
J . Org. Chem., Vol. 66, No. 17, 2001 5653
(3RS,4RS)-[4-2H]-3-Hyd r oxyd eca n oic Acid (4). Ethyl
bromoacetate (1.28 mL, 11.6 mmol) and zinc powder (760 mg,
11.6 mmol) were added to the solution of crude 14 (300 mg,
2.32 mmol) in benzene (3.00 mL), and the mixture was heated
to 85 °C for 15 min, and then cooled to room temperature. The
reaction mixture was worked up as described for 13 to afford
ethyl (3RS,4RS)-[4-2H]-3-hydroxydecanoate (17) (412 mg, 82%)
as a colorless oil: 1H NMR (300 MHz) δ 0.88 (3H, t, J ) 6.7
Hz), 1.16-1.58 (14.2H, m), 2.39 (1H, dd, J ) 16.4, 8.8 Hz);
2.51 (1H, dd, J ) 16.4, 3.2 Hz), 3.01 (1H, bs), 3.97-4.01 (1H,
m), 4.17 (2H, q, J ) 7.1 Hz).
14.19, 22.60, 24.40 (C-3 for doubly labeled 19), 24.87 (C-3 for
singly labeled 19), 24.94 (C-3 for unlabeled 19), 29.09 (C-4 for
singly labeled 19 and C-6), 29.10 (C-4 for unlabeled 19), 29.19,
1
31.76, 34.04 (t, J C-D ) 19.7 Hz, C-2 for singly labeled 19),
34.33 (C-2 for unlabeled 19), 60.09, 173.89; GC-MS m/z 188
(1.41), 187 (3.24), 186 (M+ for unlabeled 19, 0.34), 158 (8.96),
143 (16.4), 142 (24.22), 141 (3.17), 90 (40.69), 89 (100), 88
(13.5).
Ester 19 (5.0 g, 27 mmol) was converted to (2RS)-[2-2H]-
nonanol (20) (2.9 g, 73%) in the same manner as described for
1
13. 20: colorless oil; H NMR (300 MHz, CDCl3 containing a
Ester 17 (350 mg, 1.61 mmol) was hydrolyzed to 4 (160 mg,
53%) in the same manner as described for 8. 4: white plates;
small amount of D2O) δ 0.88 (3H, t, J ) 6.7 Hz), 1.18-1.40
(12H, m), 1.46-1.62 (1.02H, m), 3.51-3.66 (2H, m).
1
mp 52-53 °C; H NMR (400 MHz) δ 0.88 (3H, t, J ) 6.8 Hz),
p-TsCl (5.7 g, 30 mmol) was added to a solution of alcohol
20 (2.9 g, 20 mmol) in pyridine (15 mL) at 0 °C, and the
mixture was allowed to stand at 4 °C for 10 h. Ice chips and
water were added, and the whole was stirred for 10 min. The
mixture was worked up as described for 13 to afford (2RS)-
[2-2H]nonyl p-toluenesulfonate (21) (5.9 g, 99%) as a colorless
oil: 1H NMR (300 MHz) δ 0.87 (3H, t, J ) 6.7 Hz), 1.14-1.37
(12H, m), 1.55-1.71 (1.1H, m), 2.45 (3H, s), 3.96-4.05 (2H,
m), 7.35 and 7.79 (each 2H, d, J ) 8.3 Hz).
1.19-1.65 (11.2H, m), 2.48 (1H, dd, J ) 16.6, 9.0 Hz), 2.58
(1H, dd, J ) 16.6, 3.4 Hz) 4.00-4.07 (1H, m); 13C NMR (100
MHz) δ 14.06, 22.61, 25.20 (C-5 for doubly labeled 4), 25.31
(C-5 for singly labeled 4), 25.41 (C-5 for unlabeled 4), 29.18,
29.34 (C-6 for doubly labeled 4), 29.37 (C-6 for singly labeled
1
4), 29.39 (C-6 for unlabeled 4), 31.74, 35.85 (t, J C-D ) 19.0
Hz, C-4 for singly labeled 4), 36.42 (C-4 for unlabeled 4), 40.97,
67.87 (C-3 for doubly labeled 4), 67.93 (C-3 for singly labeled
2
4), 177.82; H NMR δ 1.45 and 1.52 (D-4); FAB MS m/z 191
KCN (262 mg, 4.02 mmol) was added to a solution of 21
(1.00 g, 3.35 mmol) in DMSO (12.5 mL), and the mixture was
stirred at 90 °C for 3 h. The reaction mixture was diluted with
saturated aqueous Na2CO3 and ether. The separated aqueous
layer was repeatedly extracted with ether. The combined
organic layer was washed with brine, dried over Na2SO4, and
concentrated under reduced pressure. Column chromatogra-
phy of the resulting crude product with hexane/AcOEt (8:1)
gave (3RS)-[3-2H]decanenitrile (22) (480 mg, 94%) as a color-
less oil: 1H NMR (270 MHz) δ 0.88 (3H, t, J ) 6.6 Hz), 1.16-
1.38 (12H, m), 1.38-1.53 (2H, m), 1.56-1.72 (1.1 H, m), 2.28-
2.37 (2H, m).
(27.4), 190 (62.3), 189 (MH+ for unlabeled 4, 41.0); EI MS m/z
171 (1.82), 170 (1.02), 89 (100).
Eth yl (4RS)-[4-2H]-3-Oxod eca n oa te (5). Ester 17 (752
mg, 3.46 mmol) was oxidized to 5 (374 mg, 50%) in the same
1
manner as described for 14. 5: pale yellow oil; H NMR (400
MHz) δ 0.88 (3H, t, J ) 6.8 Hz), 1.23-1.34 (11H, m), 1.52-
1.67 (2H, m), 2.14-2.22 (0.1H, m, H-4 for the enol form), 2.47-
2.56 (1.1H, m, H-4 for the keto form), 3.43 (1.9H, s, H-2 for
the keto form), 4.20 (2H, q, J ) 7.2 Hz), 4.97 (0.1H, s, H-2 for
the enol form), 12.1 (0.1H, OH for the enol form); 13C NMR
(100 MHz) δ 14.05, 14.07, 14.25, 22.56, 22.59, 23.37 (C-5 for
singly labeled 5, the keto form), 25.42 (C-5 for unlabeled 5,
the keto form), 26.15 (C-5 for singly labeled 5, the enol form),
26.21 (C-5 for unlabeled 5, the enol form), 28.91 (C-6 for singly
labeled 5), 28.93 (C-6 for unlabeled 5) 29.99, 31.60, 31.65, 34.65
(C-4 for singly labeled 5, the enol form), 35.03 (C-4 for
The mixture of 22 (480 mg, 3.13 mmol), EtOH (24.0 mL),
KOH (1.76 g, 31.4 mmol), and H2O (4.10 mL) was heated for
reflux and stirred for 20 h. The reaction mixture was worked
up as described for 8 to afford 7 (416 mg, 77%) as white
1
plates: mp 26-28 °C; H NMR (400 MHz) δ 0.88 (3H, t, J )
1
unlabeled 5, the enol form), 42.67 (t, J C-D ) 19.4 Hz, C-4 for
6.8 Hz), 1.20-1.38 (12H, m), 1.57-1.68 (1.1H, m), 2.27-2.39
1
singly labeled 5, the keto form), 42.03 (C-4 for unlabeled 5,
the keto form), 42.29, 59.88, 61.32, 88.94, 167.28, 172.77,
179.02, 203.07, 203.15; 2H NMR δ 2.16 (0.2D, D-4 for enol
form), 2.51 (1D, D-4 for keto form); EI MS m/z 215 (M+ for
singly labeled 5, 2.56), 197 (1.66), 196 (2.41), 131 (84.7), 130
(76.3), 128 (51.4), 115 (24.9), 88 (67.2), 85 (32.6), 57 (100).
(3RS,4RS)-[3,4-2H2]-3-Hydr oxydecan oic Acid (6). NaBD4
(111 mg, 2.65 mmol) was added to a solution of 5 (380 mg,
1.77 mmol) in EtOH (3.80 mL), and the mixture was stirred
at room temperature for 5 min. The reaction mixture was
worked up as described for 13 to afford ethyl (3RS,4RS)-[3,4-
2H2]-3-hydroxydecanoate (18) (286 mg, 74%) as a colorless oil:
1H NMR δ 0.88 (3H, t, J ) 6.8 Hz), 1.16-1.56 (14.2H, m), 2.39
(1H, d, J ) 16.4 Hz), 2.50 (1H, d, J ) 16.4 Hz), 4.17 (2H, q, J
) 7.1 Hz).
(2H, m); 13C NMR (100 MHz) δ 14.07, 22.65, 24.28 (t, J C-D
)
19.4 Hz, C-3 for singly labeled 7), 24.64 (C-3 for unlabeled 7),
28.84 (C-4 for doubly labeled 7), 28.94 (C-4 for singly labeled
7), 29.04 (C-4 for unlabeled 7), 29.21 (C-5 for single labeled
7), 29.24 (C-5 for unlabeled 7 and C-7), 29.38, 31.84, 33.96 (C-2
for doubly labeled 7), 34.05 (C-2 for singly labeled 7), 34.13
(C-2 for unlabeled 7), 180.74; 2H NMR δ 1.62 (D-3); EI-MS
m/z 174 (2.49), 173 (8.26), 172 (M+ for unlabeled 7, 2.63), 75
(13.5), 74 (66.0), 73 (24.9), 60 (100).
[2,3-13C2]Deca n oic Acid (11). [1-13C]Octanoic acid (500 mg,
3.44 mmol) was treated with ethereal diazomethane, and the
mixture was concentrated under reduced pressure. Column
chromatography of the resulting crude product with hexane/
AcOEt (10:1) gave methyl [1-13C]octanoate (23) (540 mg, 99%)
1
as a pale yellow oil. H NMR (300 MHz) δ 0.88 (3H, t, J ) 6.6
Ester 18 (286 mg, 1.31 mmol) was hydrolyzed to 6 (210 mg,
84%) in the same manner as described for 8. 6: white plates;
Hz), 1.18-1.36 (8H, m), 1.50-1.68 (2H, m), 2.30 (2H, q, 2J C-H
3
) 7.3, J H-H ) 7.3 Hz), 3.67 (3H, d, J C-H ) 3.9 Hz); 13C NMR
mp 53-54 °C; H NMR (400 MHz) δ 0.88 (3H, t, J ) 6.6 Hz),
(75 MHz) δ 14.04, 22.57, 24.93 (d, 2J C-C ) 1.9 Hz, C-3), 28.89,
1
3
1
1.20-1.59 (11.2H, m), 2.47 (1H, d, J ) 16.4 Hz), 2.57 (1H, d,
J ) 16.4 Hz); 13C NMR δ (100 MHz) 14.06, 22.63, 25.20 (C-5
for 6 doubly labeled at C-4), 25.30 (C-5 for 6 singly labeled at
C-4), 25.40 (C-5 for 6 unlabeled at C-4), 29.20 29.37 (C-6 for 6
doubly labeled at C-4), 29.39 (C-6 for 6 singly labeled at C-4),
29.41 (C-6 for 6 unlabeled at C-4), 31.77, 35.93 (t, 1J C-D ) 18.5
Hz, C-4 for 6 singly labeled at C-4), 36.33 (C-4 for 6 unlabeled
at C-4), 40.90, 67.53 (m, C-3), 177.85; 2H NMR δ 1.45 and 1.52
(0.8D, D-4), 3.99 (1D, 3-D); FAB MS m/z 192 (16.1), 191 (52.09),
190 (20.43), 189 (MH+ for unlabeled 6, 4.82); EI MS m/z 173
(0.37), 172 (1.39), 171 (1.02), 90 (100).
29.08 (d, J C-C ) 3.8 Hz, C-4), 31.62, 34.07 (d, J C-C ) 56.8
2
Hz, C-2), 51.41, (d, J C-C ) 2.5 Hz), 174.33 (enhanced signal,
C-1).
Ester 23 (540 mg, 3.39 mmol) was converted to [1-13C]-
octanol (24) (440 mg, 99%) in the same manner as described
for 13. 24: colorless oil; 1H NMR(300 MHz) δ 0.88 (3H, t, J )
6.8 H), 1.14-1.38 (10H, m), 1.44-1.64 (2H, m), 3.64 (2H, dt,
1J C-H ) 140.6, J H-H ) 6.6 Hz); 13C NMR (75 MHz), δ 14.09,
22.64, 25.72, 29.26, 29.37 (d, 3J C-C ) 4.4 Hz, C-4), 31.80, 32.78
1
(d, J C-C ) 37.1 Hz, C-2), 63.1 (enhanced signal, C-1).
Alcohol 24 (472 mg, 3.60 mmol) was converted to [1-13C]-
octyl p-toluenesulfonate (25) (896 mg, 87%) in the same
(3RS)-[3-2H]Deca n oic Acid (7). Ethyl nonanoate (5.0 g,
27 mmol) was converted to ethyl (2RS)-[2-2H]nonanoate (19)
(5.0 g, 99%) in the same manner as described for 12. 19:
1
manner as described for 21. 25: colorless oil; H NMR (300
MHz) δ 0.87 (3H, t, J ) 7.3 Hz), 1.12-1.34 (10H, m), 1.50-
1
1
3
colorless oil; H NMR (400 MHz) δ 0.88 (3H, t, J ) 6.8 Hz),
1.68 (2H, m), 2.45 (3H, s), 4.02 (2H, dt, J C-H ) 148.4, J H-H
1.21-1.35 (13H, m), 1.58-1.64 (2H, m), 2.24-2.31 (1.02 H,
) 6.5 Hz), 7.35 and 7.79 (each 2H, d, J ) 8.3 Hz); 13C NMR
(75 MHz) δ 14.05, 21.61, 22.57, 25.29, 28.76 (d, J C-C ) 27.8
m. H-2), 4.13 (2H, q, J ) 7.2 Hz); 13C NMR (100 MHz) δ 14.05,
1

5654 J . Org. Chem., Vol. 66, No. 17, 2001
Sekiyama et al.
3
Hz, C-2), 28.85 (d, J C-C ) 4.3 Hz, C-4), 29.02, 31.66, 70.69
3J C-C ) 5.0 Hz), 29.55 (d, 3J C-C ) 3.7 Hz), 31.86, 32.78 (d, 1J C-C
1
(enhanced signal, C-1), 127.87, 129.77, 133.18, 144.58.
) 37.1 Hz, enhanced signal, C-2), 63.06 (d, J C-C ) 37.1 Hz,
Tosylate 25 (896 mg, 3.14 mmol) was converted to [1,2-13C2]-
nonanenitrile (26) (410 mg, 92%) in the same manner as
described for 22, using K13CN. 26: colorless oil; 1H NMR (300
MHz) δ 0.89 (3H, t, J ) 6.7 Hz), 1.21-1.37 (8H, m), 1.37-
1.51 (2H, m), 1.60-1.73 (2H, m), 2.34 (2H, ddt, 1J C-H ) 134.0,
2J C-H ) 9.6, J H-H )7.2 Hz); 13C NMR (75 MHz) δ 14.05, 17.10
enhanced signal, C-1).
Alcohol 29 (340 mg, 2.32 mmol) was converted to [1,2-13C2]-
nonyl p-toluenesulfonate (30) (639 mg, 92%) in the same
1
manner as described for 21. 30: colorless oil; H NMR (300
MHz) δ: 0.87 (3H, t, J ) 6.8 Hz), 1.13-1.37 (12H, m), 1.63
(2H, dm, 1J C-H ) 123.0 Hz), 2.45 (3H, s), 4.01 (2H, dtd, 1J
C-H
2
1
) 148.2, J H-H ) 6.4, J C-H ) 2.6 Hz), 7.34 and 7.79 (each 2H,
(d, J C-C ) 55.5 Hz, enhanced signal, C-2), 22.58, 25.33, (dd,
d, J ) 8.2 Hz); 13C NMR (75 MHz) δ 14.06, 21.59, 22.60, 25.25
1J C-C ) 33.0, J C-C ) 2.8 Hz, C-3), 28.64 (d, J C-C ) 4.3 Hz),
28.70 (d, 3J C-C ) 4.7 Hz), 28.94, 31.67, 119.88 (d, 1J C-C ) 55.5
Hz, C-1).
2
3
1
1
(d, J C-C ) 33.9 Hz, C-3), 28.75 (d, J C-C ) 37.7 Hz, enhanced
3
3
signal, C-2), 28.88 (d, J C-C ) 4.3 Hz), 29.30 (d, J C-C ) 3.8
1
Hz), 31.76, 70.68 (d, J C-C ) 37.7 Hz, enhanced signal, C-1),
Nitrile 26 (410 mg, 2.90 mmol) was converted to [1,2-13C2]-
nonanoic acid (27) (385 mg, 83%) in the same manner as
127.85, 129.75, 133.19, 144.58.
Tosylate 30 (639 mg, 2.13 mmol) was converted to [2,3-13C2]-
decanenitrile (31) (301 mg, 92%) in the same manner as
1
described for 7. 27: colorless oil; H NMR (400 MHz) δ 0.89
(3H, t, J ) 6.8 Hz), 1.18-1.41 (10H, m), 1.53-1.72 (2H, m),
1
described for 22. 31: colorless oil; H NMR (300 MHz) δ 0.88
2.35 (2H, dq, J C-H ) 128.0, J C-H ) 7.3, J H-H ) 7.3 Hz); 13C
NMR (100 MHz) δ 14.07, 22.62, 24.66 (dd, 1J C-C ) 33.7, 2J C-C
) 1.7 Hz, C-3), 29.05 (d, 3J C-C ) 4.9 Hz), 29.08, 29.18 (d, 3J C-C
1
2
(3H, t, J ) 6.7 Hz), 1.20-1.41 (10H, m), 1.41-1.51 and 1.81-
1
1.93 (4H, m, H-4 and H-3), 2.32 (2H, dm, J C-H ) 143.8 Hz);
13C NMR (75 MHz) δ 14.04, 17.08 (d, J C-C ) 32.7 Hz,
1
1
) 4.1 Hz), 31.78, 33.94 (d, J C-C ) 54.7 Hz, enhanced signal,
C-2), 179.61 (d, J C-C ) 54.7 Hz, enhanced signal, C-1).
1
enhanced signal, C-2), 22.59, 25.32 (d, J C-C ) 32.7 Hz,
1
enhanced signal, C-3), 28.63 (dd, 1J C-C ) 34.6 Hz, 2J C-C ) 2.5
Hz), 28.72 (d, 3J C-C ) 4.4 Hz), 29.12, 29.22 (d, 3J C-C ) 4.3 Hz),
Carboxylic acid 27 (385 mg, 2.40 mmol) was converted to
methyl [1,2-13C2]nonanoate (28) (418 mg, 83%) in the same
1
31.76, 118.09 (d, J C-C ) 41.9 Hz, C-1).
Nitrile 31 (301 mg, 1.95 mmol) was converted to 11 (232
mg, 68%) in the same manner as described for 7. 11: white
1
manner as described for 23. 28: colorless oil; H NMR (400
MHz) δ 0.87 (3H, t, J ) 7.0 Hz), 1.16-1.37 (10H, m), 1.52-
1.67 (2H, m), 2.30 (2H, dq, 1J C-H ) 127.6, 2J C-H ) 7.5, J H-H
)
1
plates; mp 27-28 °C; H NMR (400 MHz) δ 0.88 (3H, t, J )
7.5 Hz), 3.67 (3H, d, J C-H ) 4.0 Hz); 13C NMR (100 MHz) δ
3
6.8 Hz), 1.20-1.39 (12H, m), 1.64 (2H, dm, 1J C-H ) 128.0 Hz),
1
2
14.08, 22.63, 24.93 (dd, J C-C ) 34.6, J C-C ) 1.7 Hz, C-3),
29.10, 29.14 (d, 3J C-C )4.1 Hz), 29.20 (d, 3J C-C ) 4.1 Hz), 31.79,
34.09 (d, 1J C-C ) 56.7 Hz, enhanced signal, C-2), 51.41 (d, 2J C-C
1
2
2.35 (2H, dtd, J C-H ) 127.4 Hz, J H-H ) 7.5 Hz, J C-H ) 4.5
Hz); 13C NMR (100 MHz) δ 14.08, 22.65, 24.63 (d, 1J C-C ) 33.7
1
Hz, enhanced signal, C-3), 29.04 (d, J C-C ) 34.6 Hz, C-4),
1
) 1.7 Hz), 174.35 (d, J C-C ) 56.7 Hz, enhanced signal, C-1).
3
1
29.24, 29.38 (d, J C-C ) 4.1 Hz), 31.84, 34.12 (d, J C-C ) 33.7
Ester 28 (418 mg, 2.34 mmol) was converted to [1,2-13C2]-
nonanol (29) (340 mg, 99%) in the same manner as described
for 13. 29: colorless oil; 1H NMR (300 MHz) δ 0.88 (3H, t, J )
6.7 Hz), 1.14-1.42 and 1.68-1.84 (16H, m, H-3 to H-8 and
1
2
Hz, enhanced signal, C-2), 180.72 (dd, J C-C ) 54.3 Hz, J C-C
) 1.6, C-2); Anal. Calcd for C813C2H20O2: C+13C, 68.93; H,
11.57. Found: C+13C, 69.23; H, 11.85; EI-MS m/z 174 (M+
12.2), 75 (91.94), 61 (100).
H-2), 3.63 (2H, brd, J C-H ) 140.6 Hz); 13C NMR (75 MHz) δ
1
1
14.09, 22.65, 25.70 (d, J C-C ) 34.6 Hz, C-3), 29.24, 29.42 (d,
J O015614G