Biosynthesis of lipstatin. Incorporation of multiply deuterium-labeled (5Z,8Z)-tetradeca-5,8-dienoic acid and octanoic acid

Article abstract of DOI:10.1021/jo010230b

Fermentation experiments with Streptomyces toxytricini were performed using (5Z,8Z)-[10,11,12,12-²H]tetradeca-5,8-dienoic acid or a mixture of [2,2-²H₂]- and [8,8,8-²H₃]octanoic acid as supplements. ²H NMR and mass spectroscopy confirmed the incorporation of (5Z,8Z)-[10,11,12,12-²H]tetradeca-5,8-dienoic acid into the C₁₃ side chain as well as into the C₆ side chain of lipstatin. Moreover, deuterium was incorporated into the C₆ side chain of lipstatin from the 8-position but not from the 2-position of octanoate. The data establish that the β-lactone moiety of lipstatin is formed by condensation of a C₈ and a C₁₄ fatty acid with a concomitant exchange of the H-2 atoms of the C₈ fatty acid.

Full text of DOI:10.1021/jo010230b

J . Org. Chem. 2001, 66, 4673-4678
4673
Biosyn th esis of Lip sta tin . In cor p or a tion of Mu ltip ly
Deu ter iu m -La beled (5Z,8Z)-Tetr a d eca -5,8-d ien oic Acid a n d
Octa n oic Acid
Markus Goese,†,‡ Wolfgang Eisenreich, Ernst Kupfer, Peter Stohler, Wolfgang Weber,
†
§
§
§
§
,†
Hans G. Leuenberger, and Adelbert Bacher*
Lehrstuhl f u¨ r Organische Chemie und Biochemie, Technische Universit a¨ t M u¨ nchen, Lichtenbergstr. 4,
D-85747 Garching, Federal Republic of Germany, and F. Hoffmann-LaRoche AG,
Pharma Research Basel, Discovery Technologies, CH-4070 Basel, Switzerland
adelbert.bacher@ch.tum.de
Received March 2, 2001
Fermentation experiments with Streptomyces toxytricini were performed using (5Z,8Z)-[10,11,12,12-
2
2
2
H]tetradeca-5,8-dienoic acid or a mixture of [2,2- H
H NMR and mass spectroscopy confirmed the incorporation of (5Z,8Z)-[10,11,12,12- H]tetradeca-
,8-dienoic acid into the C13 side chain as well as into the C side chain of lipstatin. Moreover,
deuterium was incorporated into the C side chain of lipstatin from the 8-position but not from the
-position of octanoate. The data establish that the â-lactone moiety of lipstatin is formed by
2 3
]- and [8,8,8- H ]octanoic acid as supplements.
2
2
5
6
6
2
condensation of a C
fatty acid.
8 8
and a C14 fatty acid with a concomitant exchange of the H-2 atoms of the C
In tr od u ction
Ch a r t 1
Lipstatin (1, Chart 1) is a lipophilic â-lactone produced
by Streptomyces toxytricini that inhibits pancreatic lipase
1
-3
by formation of a covalent ester adduct. The tetrahydro
derivative 2 (Orlistat, Xenical; Chart 1) of lipstatin has
been introduced recently for the treatment of severe
obesity and hypercholesterolemia.
Tracer experiments using 13C-labeled lipid mixtures or
D O suggested the â-lactone moiety of lipstatin to be
2
4
,5
biosynthesized from two long-chain fatty acid moieties.
This paper describes incorporation studies designed to
test specific fatty acids as committed precursors of
lipstatin.
Resu lts a n d Discu ssion
On the basis of well-established procedures of Pommier
et al.10 and Cohen et al., a modified method for the
11
*
To whom correspondence should be addressed. Tel: +49-89-289-
1
3360. Fax: +49-89-289-13363.
†
Technische Universit a¨ t M u¨ nchen.
Present address: F. Hoffmann-La Roche Ltd, Vitamins Research
‡
synthesis of multiply 2H-labeled tetradeca-5,8-dienoic
acid was developed. (5Z,8Z)-[10,11,12,12- H]Tetradeca-
Biotechnology, CH-4070 Basel, Switzerland.
2
§
F. Hoffmann-LaRoche AG.
(
1) Weibel, E. K.; Hadvary, P.; Hochuli, E.; Kupfer, E.; Lengsfeld,
H. J . Antibiot. 1987, 40, 1081-1085.
2) Hochuli, E.; Kupfer, E.; Maurer, R.; Meister, W.; Mercadal, Y.;
Schmidt, K. J . Antibiot. 1987, 40, 1086-1091.
3) L u¨ thi-Peng, Q.; M a¨ rki, H.-P.; Hadvary, P. FEBS Lett. 1992, 299,
5
2
,8-dienoic acid (13, Scheme 1) was prepared from
-hexenal (3) as starting material. Catalytic deuteration
(
of 3 afforded hexanal carrying deuterium atoms in
(
2
1
2
positions 2-4 as shown by H NMR and H, H-decoupled
1
11-115.
1
3
C NMR spectroscopy. Positional randomization of the
(
4) Eisenreich, W.; Kupfer, E.; Weber, W.; Bacher, A. J . Biol. Chem.
1
997, 272, 867-874.
deuterium label was observed in line with earlier re-
(
5) Goese, M.; Eisenreich, W.; Kupfer, E.; Weber, W.; Bacher, A. J .
6,7
sults and will be discussed in more detail below. Chain
Biol. Chem. 2000, 275, 21192-21196.
2
elongation of the H-labeled hexanal (4) by two consecu-
(6) Rylander, P. N. Catalytic Hydrogenation over Platinum Metals;
2
Academic Press: New York, 1967; pp 81-120.
tive Wittig reactions afforded H-labeled (5Z,8Z)-tetra-
(
(
(
7) Viala, J . J . Org. Chem. 1993, 58, 1280-1283.
deca-5,8-dienoic acid (13) as summarized in Scheme 1.
8) Huckstep, M.; Taylor, R. J . K. Synthesis 1982, 881-882.
9) Teichmann, H.; Thierfelder, W.; Kochmann, W. East German
Patent 100,960, 12 Oct 1973.
10) Pommier, A.; Pons, J .-M.; Kocienski, P. J . J . Org. Chem. 1995,
0, 7334-7339.
(11) Cohen, N.; Banner, B. L.; Lopresti, R. J .; Wong, F.; Rosenberger,
M.; Liu, Y.-Y.; Thom, E.; Liebman, A. A. J . Am. Chem. Soc. 1983, 105,
3661-3672.
(
6
1
0.1021/jo010230b CCC: $20.00 © 2001 American Chemical Society
Published on Web 06/01/2001

4
674 J . Org. Chem., Vol. 66, No. 13, 2001
Sch em e 1. Syn th esis of (5Z,8Z)-[10,11,12,12- H]Tetr a d eca -5,8-d ien oic Acid (13)a
Goese et al.
2
a
+
-
Reagents and conditions: (a) D2, Pd/C, EtOAc, rt, 96 h; (b) P(Ph)3, (Ph)NH (Me)2 I , MeCN, 60 °C, 6 h; (c) HC(OC3H7-i)3, MeCN/
isoprop, 0 °C to rt, 2 h; (d) NaN(SiMe3)2, THF, -100 °C to rt, 24 h; (e) p-TsOH/H2O, THF, 70 °C, 15 min; (f) (1) LiAlH4, THF, -90 to -20
°
C, (2) HCl, -20 °C to rt; (g) (Ph)3PBr2, MeCN/pyr, -7 °C to rt, 1 h; (h) P(Ph)3, toluene, 90 °C, 48 h; (i) (1) H2SO4, MeOH, 70 °C, 5 h, (2)
PCC, CH2Cl2, rt, 2 h; (j) NaN(SiMe3)2, THF, -100 °C to rt, 24 h; (k) (1) KOH, THF, 70 °C, 4 h, (2) HCl.
2
Ch a r t 2. Isotop om er s of (5Z,8Z)-[10,11,12,12- H]Tetr a d eca -5,8-d ien oic Acid (13) Obta in ed by Ca ta lytic
a
Deu ter a tion of tr a n s-2-Hexen a l
a
Key: 2H-upfield shifts are indicated by values in italics. The relative abundance of each isotopomer (%) estimated from H, H-decoupled
1
2
1
1
3
3
C NMR spectra is also shown.
1
2
The product was analyzed in detail by H, H-decoupled
C NMR spectroscopy. Eight signals were found for C-11
in isotopomers with multiple deuterium substitution. The
fraction of each respective isotopomer in the biosynthetic
mixture could be determined from the signal integrals
in the H, H-decoupled C NMR spectra (Chart 2). The
average deuterium content per product molecule was 1.7
as estimated by NMR spectroscopy and 1.9 as estimated
by mass spectrometry.
and six signals were found for C-13 (Figure 1), reflecting
the presence of 12 different isotopomers carrying up to
four deuterium atoms at C-10, C-11, and/or C-12 (iso-
topomers A-N, Chart 2). H isotope shifts are indicated
in Figure 1. The chemical shift increments are additive
1
2
13
2

Biosynthesis of Lipstatin
J . Org. Chem., Vol. 66, No. 13, 2001 4675
F igu r e 1. 1H, H-decoupled 13C NMR signals of C-11 and C-13
2
2
of (5Z,8Z)-[10,11,12,12- H]tetradeca-5,8-dienoic acid. Capital
2
letters denote isotopomers shown in Chart 2. R-, â-, and γ- H
upfield shifts are indicated by arrows.
2
(
5Z,8Z)-[10,11,12,12- H]Tetradeca-5,8-dienoic acid (13,
Scheme 1) was proffered to a fed-batch fermentor culture
of S. toxytricini together with an excess of unlabeled
linoleic acid over a period of 1 h as described in the
Experimental Section. The molar ratio of the labeled
compound to unlabeled linoleic acid was 1.6%. Lipstatin
5
was isolated and analyzed by mass spectrometry and
2
H NMR spectroscopy. The deuterium abundance of
lipstatin estimated by mass spectrometry was approxi-
mately 1% indicating that the proffered tetradecadienoic
acid and linoleic acid were used as precursor of lipstatin
2
with similar efficacy. The H NMR spectrum of lipstatin
2
showed two H NMR signals at 2.0 and 1.25 ppm (Figure
2
B).
2
Intact incorporation of (5Z,8Z)-[10,11,12,12- H]tetradeca-
5
,8-dienoic acid should result in deuterium enrichments
at C-12, C-13, and C-14 of lipstatin (Scheme 2). On basis
1
4
of the H NMR signal assignments of lipstatin, the signal
at 2.0 ppm is caused by a deuterium atom at C-12,
whereas the broad signal at 1.25 ppm could reflect
deuterium atoms at positions C-13 and C-14 of lipstatin.
The integral ratio of the C-12 signal and the C-13/C-14
signal was 1:5.2, whereas the corresponding ratio of the
2
2
F igu r e 2. (A) H NMR signals of (5Z,8Z)-[10,11,12,12- H]-
2
tetradeca-5,8-dienoic acid (13). (B) H NMR signals of lipstatin
isolated from a fed-batch culture of S. toxytricini growing with
5Z,8Z)-[10,11,12,12- H]tetradeca-5,8-dienoic acid (13) as supple-
ment. (C) H NMR signals of lipstatin isolated from a fed-batch
2
(
2
culture of S. toxytricini growing with an equimolar mixture of
2
H NMR signals for C-10 and C-11/C-12 of the precursor
[2,2-
2
2
3
H ]- and [8,8,8- H ]octanoic acid as supplement. The
arrow points to the position of the absent deuterium signal at
C-2 (for details see text).
2
1
was 1:2.5 (Figure 2A). Since the H NMR resonances of
positions 2′, 3′, and 4′ of lipstatin are also found at
4
chemical shift values of 1.3-1.2 ppm, it appears likely
2
that the H NMR signal at 1.25 ppm reflects additional
degradation of 13 to acetyl-CoA via â-oxidation with
subsequent reincorporation via de novo synthesis of fatty
acids.
In a second fermentation experiment with S. toxytri-
cini, an equimolar mixture of [2,2- H ]octanoate and
[8,8,8- H ]octanoate was proffered together with an
excess of linoleic acid. The molar ratio of labeled oc-
tanoate to linoleic acid was 4.0%. Lipstatin was again
deuterium atoms at positions 2′-4′ of lipstatin. These
results suggest that approximately 50% of the labeled
lipstatin molecules arose from intact incorporation of the
2
2
2
proffered (5Z,8Z)-[10,11,12,12- H]tetradeca-5,8-dienoic
2
3
acid (13) and that 50% originated from indirect incorpo-
2
ration of 13 via breakdown to [4,5,6,6- H]-2-octenoyl-
2
CoA by â-oxidation. Obviously, [4,5,6,6- H]-2-octenoyl-
2
2
CoA could be reduced to [4,5,6,6- H]octanoyl-CoA. The
isolated and analyzed by mass spectrometry and H NMR
latter could then serve as precursor conducive to the
formation of [2′,3′,4′,4′- H]lipstatin (Scheme 2). It should
spectroscopy (Figure 2C). Mass spectrometry established
an incorporation rate of approximately 2%. The H NMR
2
2
be emphasized that the data clearly rule out total
signal at 0.83 ppm indicates that the position 6′ methyl

4
676 J . Org. Chem., Vol. 66, No. 13, 2001
Sch em e 2. Hyp oth etica l Mech a n ism of Lip sta tin Biosyn th esis in S. toxytr icin ia
Goese et al.
a
Key: H9 denotes atoms that acquired deuterium labeling from (5Z,8Z)-[10,11,12,12- H]tetradeca-5,8-dienoic acid, H° denotes atoms
2
2
that acquired deuterium labeling from [8,8,8- H3]octanoic acid, and H* denotes atoms that were exchanged with the solvent in the course
of biosynthetic transformation into lipstatin. R ) CoA, R′ ) H or formylleucine.
2
group of lipstatin had been labeled. A H NMR signal at
3
atoms with solvent water (Scheme 2), confirming earlier
results obtained from fermentation experiments with
.16 ppm for the 2-position of lipstatin could not be
detected. Accordingly, octanoic acid had been incorpo-
rated into lipstatin under complete exchange of the H-2
2
2
5
H
2
O and H-labeled lipid mixtures.
The reported data support the biosynthetic formation

Biosynthesis of Lipstatin
J . Org. Chem., Vol. 66, No. 13, 2001 4677
of lipstatin from an unsaturated C14 carboxylic acid (13)
or its thio ester (14) and activated octanoate (15) as
shown in Scheme 2. Decarboxylative condensation could
afford a â-keto thioester (16). Reduction of 16 could yield
the diol 17. Lactonization of 17 could then yield the
â-lactone ring, and formylleucine substitution at C-5
could afford lipstatin.
(3-Oxop r op yl)tr ip h en ylp h osp h on iu m iod id e (6) was
prepared as described:9 1H NMR (360 MHz) δ ppm 9.60 (t, J
3
2
)
1.9 Hz, 1 H-3), 7.75-7.56 (m, 15 Harom.), 3.75 (dt,
J
HP
)
3
3
3
1
2.9 Hz, J ) 7.1 Hz, 2 H-1), 3.07 (dt, J HP ) 13.5 Hz, J ) 7.1
1
3
3
Hz, 2 H-2); C NMR (90.6 MHz) δ ppm 197.0 (C-3, J CP ) 11.3
4
2
Hz), 135.5 (3 Carom., J CP ) 3.3 Hz), 133.7 (6 Carom., J CP ) 10.0
3J CP ) 12.6 Hz), 117.0 (3 Carom., 1
Hz), 130.6 (6 Carom.
86.9 Hz), 35.7 (C-2,
,
J
CP
)
2
1
J CP ) 3.1 Hz), 15.7 (C-1,
PO ext.) δ ppm +25.5.
(3,3-Diisop r op oxyp r op yl)t r ip h en ylp h osp h on iu m io-
J
CP
) 55.9 Hz);
3
1
The absence of deuterium incorporation from the
P NMR (101.3 MHz, H
3
4
2
-position of octanoate puts stringent requirements on
the mechanism of lipstatin biosynthesis. Specifically, one
deuterium atom could be removed by carboxylation of
octanoate leading to hexyl malonyl CoA (15). The ob-
d id e (7). A 92.30 g portion of 6 (0.207 mol) was dissolved in
450 mL of a mixture of dichloromethane and 2-propanol (5:4,
v/v) and cooled to 0 °C in an ice bath. A 116.9 mL portion of
triisopropyl orthoformate (0.414 mol) and 1.5 mL of HCl
concentrated were added. The solution was stirred for 2 h and
allowed to warm to room temperature. Triethylamine was
added until an alkaline pH was reached, and approximately
half of the solvent was removed under reduced pressure. A
500 mL portion of a mixture of dichloromethane, diethyl ether,
and pentane (1:2:2, v/v/v) was added to induce crystallization,
and the mixture was cooled at - 80 °C overnight. The resulting
white crystals were removed by filtration, washed with cold
served removal of the second deuterium atom from [2,2-
2
2
H ]octanoate could proceed at the level of 15 during the
condensation process or at the level of 17 by epimeriza-
5
tion or a dehydration/rehydration process. It should be
noted that epimerization also appears to occur in the
1
3
biosynthesis of the polyketide 6-deoxyerythronolide B.
2
A nonenzymatic loss of H by enolization of 15, 16, or 17
does not appear plausible, since the rates of chemical
exchange are slow in malonyl-CoA derivatives compared
to the typical lifetimes of biochemical intermediates1
1
pentane, and dried in vacuo: yield 116.80 g (97%); H NMR
3
(
360 MHz) δ ppm 7.76-7.64 (m, 15 Harom.), 5.10 (t, J ) 4.7
4-16
3
Hz, 1 H-3), 3.86 (sept, J ) 6.1 Hz, 2 H-1′), 3.62 (m, 2 H-1),
(H. G. Floss, personal communication).
3
3
1
.85 (m, 2 H-2), 1.15 (d, J ) 6.1 Hz, 6 H-2′), 1.11 (d, J ) 6.1
1
3
4
Hz, 6 H-2′); C NMR (90.6 MHz) δ ppm 135.2 (3 Carom., J CP
)
)
)
Exp er im en ta l Section
2
3
3
.1 Hz), 133.5 (6 Carom., J CP ) 10.0 Hz), 130.5 (6 Carom., J CP
3
12.6 Hz), 118.0 (3 Carom., 1J CP ) 86.3 Hz), 98.3 (C-3, J CP
2
2
2
Ma ter ia ls. [2,2- H
2
]Octanoic acid (98% H) and [8,8,8- H
octanoic acid (98% H) were purchased from Phychem (D u¨ ren,
Germany). Deuterium (D , 99.6%) was from Cambridge Isotope
3
]-
2
2
17.7 Hz), 69.7 (2 C-1′), 29.0 (C-2, J ) 3.8 Hz), 23.2 (2 C-2′),
CP
1
31
2
H
2.9 (2 C-2′), 18.2 (C-1, J CP ) 53.5 Hz); P NMR (101.3 MHz,
2
3
PO
4
ext.) δ ppm +25.5.
Laboratories (Woburn, MA). All other chemicals were from
Aldrich (Steinheim, Germany), Sigma (Deisenhofen, Ger-
many), Fluka (Buchs, Switzerland), and Merck (Darmstadt,
Germany). Solvents were redistilled and dried over molecular
sieves (4 Å) or, in the case of THF and diethyl ether, sodium.
2
[2,3,4,4- H]Hexa n a l (4). A 17.64 g (0.180 mol) portion of
trans-2-hexenal (3) was added to a mixture of 1.7 g of
palladium (5%) on charcoal and 100 mL of ethyl acetate. The
mixture was flushed with nitrogen, evacuated, and then
NMR Sp ectr oscop y. NMR measurements were performed
incubated in a D -atmosphere for 96 h under room temperature
2
2
in CDCl
3
or CHCl
3
( H NMR experiments) at 17 °C using a
and normal pressure. After consumption of 4.1 L of deuterium
(0.183 mol), the reaction was stopped and the mixture filtered
through Celite. The filter cake was washed twice with diethyl
ether. The solvent was removed under reduced pressure at
room temperature. The resulting aldehyde was distilled im-
mediately prior to the subsequent Wittig reaction (bp 135 °C/
Bruker DRX 500 spectrometer operating at 500.13 MHz for
1
13
H experiments, 125.76 MHz for C experiments, and 76.77
2
MHz for H NMR experiments, a Bruker AM 360 spectrometer
1
operating at 360.13 MHz for H experiments and 90.6 MHz
1
3
for C experiments, or a Bruker AC 250 spectrometer operat-
1
13
1
ing at 250.13 MHz for H experiments, 62.9 MHz for
C
1013 mbar): yield 11.00 g (60%); H NMR (360 MHz) δ ppm
3
1
3
experiments, and 101.3 MHz for P experiments. The DRX
00 spectrometer was equipped with a lock-switch unit for H-
9.70 (t, J ) 1.8 Hz, 1 H-1), 2.37 (dt-like, H-2**), 1.56 (quint-
2
3
5
like, H-3**), 1.26 (m, H-4** + H-5), 0.84 (t, J ) 7.7 Hz, 3
H-6); 13C NMR (90.6 MHz) δ ppm 202.6 (C-1), 43.6 (C-2**),
decoupling experiments using the lock channel. NMR experi-
ments were performed with standard Bruker software (XWIN-
NMR 1.3). Prior to Fourier transformation, the free induction
decay was multiplied with a Gaussian function. NMR assign-
2
31.3 (C-4**), 22.4 (C-5**), 21.7 (C-3**), 13.7 (C-6*); H NMR
(76.7 MHz) δ ppm 2.36 (br s, D-2), 1.58 (br s, D-3), 1.24 (br s,
D-4).
1
ments were verified by two-dimensional NMR analysis ( H-
2
(
3Z)-[5,6,7,7- H]Non -3-en a l Diisop r op yl Aceta l (8). An
1
1
1
1
13
1
13
H-TOCSY, H- H-COSY, H- C-HMQC, H- C-HMBC)
8
8.76 g portion of dry 7 (0.162 mol) was suspended in 1200
mL of THF, and 162 mL of sodium bis-trimethyl silyl amide
1 M in THF, 0.162 mol) was added slowly under nitrogen.
3 3 4
and referenced to the CDCl solvent signal or external H PO
3
1
13
1
(
P NMR). C NMR data listed below were achieved by H-
decoupled measurements. ** in the spectroscopic data denotes
NMR signals of atoms that are strongly weakened by 2
(
The orange solution was stirred for 1 h at room temperature
and then cooled to -100 °C using liquid nitrogen in ethanol.
An 11.00 g portion of compound 4 (0.108 mol) was added
dropwise using a syringe, and the solution was allowed to
warm to room temperature overnight. An aqueous saturated
solution of ammonium chloride (250 mL) and 500 mL of diethyl
ether were added. The organic phase was separated, and the
aqueous phase was washed three times with 250 mL of diethyl
ether. The combined organic solutions were washed repeatedly
with aqueous saturated sodium chloride and then dried over
magnesium sulfate. The solvent was removed under reduced
pressure, and the crude product was purified by distillation
H
substitution, * denotes NMR signals of atoms that are slightly
2
weakened by H substitution.
P r ep a r a tion of La beled Com p ou n d s. Meth yl 5-oxo-
8
1
p en ta n oa te (5) was prepared as described: H NMR (250
3
MHz) δ ppm 9.78 (t, J ) 1.3 Hz, 1 H-5), 3.68 (s, 3 H-1′), 2.55
3
3
(
1
2
dt, J ) 7.1 Hz, 1.3 Hz, 2 H-4), 2.39 (t, J ) 7.1 Hz, 2 H-2),
3
13
.96 (quint, J ) 7.1 Hz, 2 H-3); C NMR (62.9 MHz) δ ppm
01.4 (C-5), 173.2 (C-1), 51.5 (C-1′), 42.8 (C-4), 32.8 (C-2), 17.2
(
C-3).
(
12) Bacher, A.; Stohler, P.; Weber, W. European Patent Application
EP 803 576, 29 Oct 1997; Appl. 97106445.6, Filed 18 April 1997.
13) Weissmann, K. J .; Timoney, M.; Bycroft, M.; Grice, P.; Hanefeld,
U.; Staunton, J .; Leadley, P. F. Biochemistry 1997, 36, 13849-13855.
14) Lee, R. E.; Armour, J . W.; Takayama, K.; Brennan, P. J .; Besra,
G. S. Biochim. Biophys. Acta 1997, 1346, 275-284.
15) Sedgwick, B.; Cornforth, J . W. Eur. J . Biochem. 1977, 75, 465-
79.
16) Sedgwick, B.; Cornforth, J . W.; French, S. J .; Gray, R. T.;
Kelstrup, E.; Willadsen, P. Eur. J . Biochem. 1977, 75, 481-495.
1
in vacuo (bp 72 °C/ 0.5 mbar): yield 17.08 g (65%); H NMR
3
(
(500 MHz) δ ppm 5.45 (m, 1 H-4), 5.39 (m, 1 H-3), 4.54 (t, J
3
3
) 5.6 Hz, 1 H-1), 3.87 (sept, J ) 6.2 Hz, 2 H-1′), 2.34 (t, J )
(
6
.2 Hz, 2 H-2), 2.03 (q-like, H-5**), 1.28 (m, H-6** + H-7** +
3
3
H-8), 1.19 (d, J ) 6.2 Hz, 6 H-2′), 1.14 (d, J ) 6.2 Hz, 6 H-2′),
(
3
13
0
.88 (t, J ) 6.6 Hz, 3 H-9); C NMR (125.8 MHz) δ ppm 132.1
C-4*), 124.3 (C-3), 100.1 (C-1), 67.8 (C-1′), 33.8 (C-2), 31.6 (C-
7**), 29.3 (C-6**), 27.5 (C-5**), 23.4 (2 C-2′), 22.6 (2 C-2′), 22.5
4
(
(

4
678 J . Org. Chem., Vol. 66, No. 13, 2001
Goese et al.
(quint, J ) 7.4 Hz, 2 H-3), 1.30 (m, H-11** + H-12** + H-13),
2
3
(
C-8**), 14.1 (C-9*); H NMR (76.7 MHz) δ ppm 2.01 (br s,
3
13
D-5), 1.29 (br s, D-6 + D-7).
0.89 (t, J ) 7.1 Hz, 3 H-14); C NMR (125.8 MHz) δ ppm
174.1 (C-1), 130.3 (C-9*), 129.2 (C-6), 128.6 (C-5), 127.5 (C-
8*), 51.5 (C-1′), 33.4 (C-2), 31.5 (C-12**), 29.3 (C-11**), 27.1
(C-10**), 26.5 (C-4), 25.5 (C-7), 24.7 (C-3), 22.5 (C-13**), 14.1
2
(
3Z)-[5,6,7,7- H]Non -3-en -1-ol (9) was prepared using the
1
0
procedure of Pommier et al. (3Z)-Non-3-enal diisopropyl
acetal was replaced by 8: H NMR (500 MHz) δ ppm 5.51 (m,
1
1
3
3
2
H-4), 5.32 (m, 1 H-3), 3.60 (t, J ) 6.6 Hz, 2 H-1), 2.30 (q, J
(C-14*); H NMR (76.7 MHz) δ ppm 2.02 (br s, D-10), 1.28 (br
-
1
)
6.6 Hz, 2 H-2), 2.01 (t-like, H-5**), 1.78 (br s, OH), 1.26 (m,
s, D-11 + D-12); IR (CCl
4
) ν cm 3009, 2926, 2856, 1742, 1436,
3
13
H-6** + H-7** + H-8), 0.84 (t, J ) 7.1 Hz, 3 H-9); C NMR
1244, 1206, 1156, 722.
(
(
(
125.8 MHz) δ ppm 133.5 (C-4*), 124.7 (C-3), 62.2 (C-1), 31.4
(5Z,8Z)-[10,11,12,12-
2
H]Tetr a d eca -5,8-d ien oic Acid (13).
C-7**), 30.8 (C-2), 29.3 (C-6**), 27.3 (C-5**), 22.5 (C-8**), 14.0
A 1.00 g portion of 12 (0.00417 mol) was added to a mixture
of 30 mL of THF and 10 mL of 2 M KOH. The solution was
stirred for 4 h under gentle reflux and then acidified using 2
M HCl. The product was extracted repeatedly with diethyl
ether. The combined organic layers were washed with aqueous
saturated sodium chloride and water and dried over magne-
sium sulfate, and the solvent was removed under reduced
2
C-9*); H NMR (76.7 MHz) δ ppm 2.02 (br s, D-5), 1.27 (br s,
D-6 + D-7).
2
(
3Z)-[5,6,7,7- H]1-Br om on on -3-en (10) was prepared us-
1
0
ing the procedure of Pommier et al. (3Z)-Non-3-en-1-ol was
replaced by 9: H NMR (500 MHz) δ ppm 5.52 (m, 1 H-4),
5
Hz, 2 H-2), 2.04 (t-like, H-5**), 1.29 (m, H-6** + H-7** + H-8),
0
1
3
3
.35 (m, 1 H-3), 3.36 (t, J ) 7.2 Hz, 2 H-1), 2.61 (q, J ) 7.1
1
pressure: yield 0.63 g (67%);
5.28 (m, 1 H-5 + 1 H-6 + 1 H-8 + 1 H-9), 2.75 (t, J ) 7.0 Hz,
2 H-7), 2.35 (t, J ) 7.5 Hz, 2 H-2), 2.11 (q, J ) 7.2 Hz, 2
H-4), 2.02 (q-like, H-10**), 1.69 (quint, J ) 7.5 Hz, 2 H-3),
1.28 (m, H-11** + H-12** + H-13), 0.86 (t, J ) 7.2 Hz, 3
H NMR (500 MHz) δ ppm 5.37-
3
13
3
.89 (t, J ) 6.6 Hz, 3 H-9); C NMR (125.8 MHz) δ ppm 133.2
(C-4*), 125.7 (C-3), 32.3 (C-1), 31.4 (C-7**), 30.9 (C-2), 29.2
3
3
2
3
(C-6**), 27.3 (C-5**), 22.5 (C-8**), 14.0 (C-9*); H NMR (76.7
MHz) δ ppm 2.01 (br s, D-5), 1.29 (br s, D-6 + D-7).
3
2
13
(
3Z)-[5,6,7,7- H]Tr iph en yln on -3-en ylph osph on iu m Br o-
H-14); C NMR (125.8 MHz) δ ppm 180.3 (C-1), 130.4 (C-9*),
129.4 (C-6), 128.2 (C-5), 127.5 (C-8*), 33.4 (C-2), 31.5 (C-12**),
29.3 (C-11**), 27.2 (C-10**), 26.4 (C-4), 25.6 (C-7), 24.4 (C-3),
m id e (11). The compound was prepared by the procedure of
Cohen et al. (3Z)-1-Iodonon-3-ene was replaced by 10:
NMR (500 MHz) δ ppm 7.86-7.73 (m, 15 Harom.), 5.55 (m, 1
H-3), 5.38 (m, 1 H-4), 3.80 (m, 2 H-1), 2.44 (m, 2 H-2), 1.76
1
1
1
H
2
22.6 (C-13**), 14.1 (C-14*); H NMR (76.7 MHz) δ ppm 2.01
(br s, D-10), 1.28 (br s, D-11 + D-12); IR (CCl ) ν cm-1 3009,
4
3
(
t-like, H-5**), 1.20 (m, H-6** + H-7** + H-8), 0.83 (t, J )
2926, 2857, 1709, 1456, 1413, 1240, 935, 723.
1
3
7
.3 Hz, 3 H-9); C NMR (90.6 MHz) δ ppm 135.0 (3 Carom.,
2
F er m en ta tion . Fed-batch fermentation experiments with
S. toxytricini were performed using a 14 L Chemap fermentor
4
J
CP ) 2.7 Hz), 133.5 (6 Carom., J CP ) 10.3 Hz), 132.3 (C-4),
3 3
1
1
30.3 (6 Carom., J CP ) 12.6 Hz), 125.6 (C-3, J CP ) 14.1 Hz),
(
M a¨ nnedorf/Z u¨ rich, Switzerland) with a working volume of 8
1
17.6 (3 Carom., J CP ) 85.8 Hz), 31.0 (C-7**), 28.6 (C-6**), 26.8
12
L according to published procedures. Two hours after feed-
start, a mixture of 0.198 g of carboxylic acid 13 and 0.92 g of
unlabeled linoleic acid (Fluka, Switzerland) was applied at a
rate of 37 mg min . In a second experiment, a mixture of 0.484
2 3
g of [2,2- H ]octanoic acid, 0.479 g of [8,8,8- H ]octanoic acid,
1
2
(
C-5**), 22.7 (C-1, J CP ) 48.8 Hz), 22.1 (C-8**), 20.0 (C-2, J CP
2
)
3.4 Hz), 13.8 (C-9); H NMR (76.7 MHz) δ ppm 1.75 (br s,
31
D-5), 1.18 (br s, D-6 + D-7); P NMR (101.3 MHz, H
3
PO4 ext
)
-1
δ ppm + 24.5.
2
2
2
(
5Z,8Z)-Meth yl [10,11,12,12- H]Tetr a d eca -5,8-d ien oa te
and 4.84 g of unlabeled linoleic acid was inoculated at a rate
(
12). A 5.83 g portion of dry 11 (0.0125 mol) was suspended
-1
of 45 mg min . The molar ratio of the labeled compound to
in 100 mL of THF and cooled to 0 °C. A 12.5 mL portion of
sodium bis-trimethyl silyl amide (1 M in THF, 0.0125 mol) was
added slowly under nitrogen. The orange solution was stirred
for 1 h at room temperature and then cooled to -100 °C. A
the total added fatty acids was 1.6% in the first experiment
and 4.0% in the second experiment. After an incubation time
of 1 h, both experiments were stopped and the fermentation
broth was heated to 70 °C for 10 min.
Isola tion of Lip sta tin . Lipstatin was isolated essentially
as described 4 and was 94.7% pure (first experiment) and 98.8%
pure (second experiment) as judged by high-performance liquid
chromatography (HPLC).
0
.71 g (0.0084 mol) portion of compound 5 was added with a
syringe, and the solution was allowed to warm to room
temperature overnight. A 20 mL portion of aqueous saturated
ammonium chloride was added followed by 50 mL of diethyl
ether. The organic layer was separated from the aqueous layer
,
and the latter washed three times with 25 mL of diethyl ether.
Ack n ow led gm en t. This work was supported by
grants from the Deutsche Forschungsgemeinschaft and
the Fonds der Chemischen Industrie. We thank G.
Oesterhelt for mass spectra and A. Werner for expert
help with the preparation of the manuscript. We thank
H. G. Floss for helpful discussions.
The combined organic layers were washed with aqueous
saturated sodium chloride repeatedly and then dried over
magnesium sulfate. The solvent was removed under reduced
pressure and the crude product purified by distillation in vacuo
(
δ ppm 5.41-5.28 (m, 1 H-5 + 1 H-6 + 1 H-8 + 1 H-9), 3.67 (s,
3
H-2), 2.11 (q, J ) 7.1 Hz, 2 H-4), 2.04 (q-like, H-10**), 1.70
1
bp 92 °C/ 0.4 mbar): yield 1.59 g (65%); H NMR (500 MHz)
3
3
H-1′), 2.76 (t, J ) 6.9 Hz, 2 H-7), 2.32 (t, J ) 7.6 Hz, 2
3
J O010230B