Synthesis and desaturation of monofluorinated fatty acids

Article abstract of DOI:10.1039/a701571b

A series of monofluoro C₁₆ and C₁₈ fatty acids have been synthesized and used as mechanistic probes for fatty acid desaturation. Only fluoroolefinic products are obtained when these compounds are processed by an in vivo Saccharomyces cerevisiae Δ⁹ desaturating system as determined by ¹H-decoupled ¹⁹F NMR and GC-MS analysis. No evidence for fluorohydrin formation has been found when either methyl (R,S)-9- or 10-fluoropalmitate (stearate) 3a,b and 5a,b was incubated with the Δ⁹ desaturase. On desaturation α- and β-fluorine substituent effects (k_H/k_F) of magnitude 6.2 and 2.4, respectively, have been measured by direct competition experiments between 3a and 3b and between methyl 16-fluoropalmitate 3c and 3b. These results do not support the involvement of discrete hydroxylated and carbocationic intermediates in fatty acid desaturation. Substantial apparent steric effects have been observed for monofluorostearoyl substrates 5c-f bearing a fluorine distal from the site of initial oxidation. In the case of (R,S)-methyl 12-fluorostearate 5f, we show that both enantiomers are desaturated at comparable rates.

Full text of DOI:10.1039/a701571b

View Article Online / Journal Homepage / Table of Contents for this issue
1
Synthesis and desaturation of monofluorinated fatty acids
,
a
a
a
b
Peter H. Buist,* Kostas A. Alexopoulos, Behnaz Behrouzian, Brian Dawson
b
and Bruce Black
a
Ottawa-Carleton Chemistry Institute, Department of Chemistry, Carleton University,
1
125 Colonel By Drive, Ottawa, Ontario, Canada, K1S 5B6
Bureau of Drug Research, Drugs Directorate, Health Protection Branch, Health Canada,
b
Tunney’s Pasture, Ottawa, Ontario, Canada, K1A OL2
A series of monofluoro C16 and C18 fatty acids have been synthesized and used as mechanistic probes for
fatty acid desaturation. Only fluoroolefinic products are obtained when these compounds are processed by
9
1
19
an in vivo Saccharomyces cerevisiae Ä desaturating system as determined by H -decoupled F N M R and
G C–M S analysis. N o evidence for fluorohydrin formation has been found when either methyl (R,S)-9- or
9
1
0-fluoropalmitate (stearate) 3a,b and 5a,b was incubated with the Ä desaturase. On desaturation á- and â-
fluorine substituent effects (k /k ) of magnitude 6.2 and 2.4, respectively, have been measured by direct
H
F
competition experiments between 3a and 3b and between methyl 16-fluoropalmitate 3c and 3b. These
results do not support the involvement of discrete hydroxylated and carbocationic intermediates in fatty
acid desaturation. Substantial apparent steric effects have been observed for monofluorostearoyl
substrates 5c–f bearing a fluorine distal from the site of initial oxidation. In the case of (R,S)-methyl
1
2-fluorostearate 5f, we show that both enantiomers are desaturated at comparable rates.
The biological dehydrogenation (desaturation) of unactivated
fatty acyl hydrocarbon chains as exemplified by the conversion
of stearoyl CoA 1 into oleyl CoA 2 is an essential biochemical
We have obtained a number of results using an in vivo S.
9
cerevisiae (membrane-bound) ∆ desaturase system † which also
5
a–d
point to initial oxidative attack at C-9 of a stearoyl substrate.
Thus, 9-thia fatty acid analogues are more efficiently converted
into the corresponding sulfoxide than are their 10-thia
5
a–c
HR
HR
SCoA
counterparts
and a large primary deuterium kinetic isotope
9
effect has been measured at C-9 whereas C᎐H bond cleavage at
C-10 is essentially insensitive to isotopic substitution. Based
on these results, the fact that hydroxylases and desaturases are
linked at the structural level
dehydrogenation and hydroxylation are occasionally mediated
O
1
0
5
d
1
3
a,b,6
and the observation that
-
2 HR ∆9 desaturase
7
a–c
by the same enzyme,
we have proposed a mechanistic model
SCoA
9
for ∆ desaturases which involves initial rate-determining C᎐H
bond cleavage at C-9 to form a transient carbon-centred radical
followed by collapse to olefin in one of the three possible ways
shown in Scheme 1 (pathways a,b,c).‡ A fascinating question
remains: How do desaturases steer the putative radical
9
O
1
0
2
4
2
a
intermediate to olefin? The X-ray structure of the plant
process for most aerobic lifeforms. It is also an outstanding
example of enzymic regio- and stereo-selectivity and served to
inspire the very early experiments in biomimetic chemistry.
desaturase offers no obvious clues as to how this might be done.
Newcomb et al. have recently presented evidence supporting
the view that the closely related hydroxylation of unactivated
hydrocarbons mediated by sMMO and cytochrome P450 pro-
ceeds by a ‘nonsynchronous, concerted mechanism’ and that
2
b
Recently significant progress has been made in the structural
characterization of the enzymes responsible for these intriguing
3
a,b
transformations. In two seminal papers,
Shanklin and Fox
have shown that the family of O -dependent, non-haem iron-
2
containing enzymes known as desaturases can be divided into
two classes: soluble plant enzymes containing a carboxylate-
bridged diiron active site similar to that of methane monooxy-
† We have elected to work with intact organisms because reconsti-
tuted preparations of membrane-bound ∆ desaturases are notori-
9
ously unstable and have very low activity. Attempts to overexpress the
9
yeast ∆ desaturase have not been successful. Our decade-long experi-
genase (MMO) and
a series of less-well characterized
5
e–f
ence with the in vivo yeast system
has been that methyl esters of our
membrane-bound desaturases which are thought to contain
a diiron active site co-ordinated to histidine groups. A recent
mechanistic probes are incorporated into the cells where they are acted
9
upon by the ∆ desaturase (presumably as the CoA thioesters). If the
9
X-ray structure of the plant ∆ desaturase from castor (Ricinus
product of desaturation is sufficiently nonpolar, it accumulates in the
cell whereas if desaturation is diverted to generate polar, oxygenated
products they are excreted as free acids into the culture medium.
communis) reveals
a tight, crescent-shaped, hydrophobic
substrate binding pocket and computer modelling studies sug-
gest that C-9 of the stearoyl substrate is near the iron atom
‡
For the sake of simplicity, we represent the active oxidizing species as
an iron oxo complex. Although it appears that there are two irons in the
4
responsible for initial oxidation. No detailed mechanistic work
9
3b
∆
yeast desaturase, it is interesting to note that the closely related rat
9 22
on this enzyme has emerged due to the complexity of the
substrate (a fatty acyl ACP thioester) and the lack of a
suitable assay.
liver ∆ desaturase was originally thought to contain one iron atom.
The mechanistic considerations presented in this paper are not affected
by this uncertainty.
J. Chem. Soc., Perkin Trans. 1, 1997
2617

View Article Online
R1
R2
OCH3
Fe
9
Fe
O
Fe
O
1
0
a
OH
OH
H
H
H
3a,b,c
H
H
H
H
H
H
H
1
0
9
10
9
1
0
9
c
b
R1
R2
a
OCH3
9
O
1
0
Fe
Fe
4
a,b,c
HOH
a
R1 = F, R2 = H, R3 = H
H
OH
H
c
H
H
H
1
0
9
b
c
R1 = H, R2 = F, R3 = H
1
0
9
Scheme 1
R1 = H, R2 = H, R3 = F
the ‘lifetime of the radical be shortened from that of an inter-
mediate to that of a vibrational component in a transition
state’. This would imply that it would be difficult to prevent an
initial hydroxylation event and that desaturases function by
promoting a subsequent Lewis-acid catalyzed dehydration
8
R1
7
5
OCH3
9
R4
O
1
0
R2
(pathway c).§ On the other hand, it is remarkable that biological
12
5
dehydrogenation appears to be favoured when positive charge
7
a–c
stabilizing substituents neighbour the site of initial oxidation.
This suggests that desaturases may utilize a redox switch to
force a 1 electron oxidation/deprotonation route (pathway a).
We decided to explore the possibility of using the fluorine sub-
stituent as a new mechanistic probe for hydroxylated and carbo-
cationic intermediates in desaturation. In this article, we
report the synthesis of some long chain monofluorinated fatty
R1
7
5
9
4
5
R
R
O
1
0
R2
1
2
6
9
acids and the effects of fluorine substitution on the in vivo ∆
desaturation of these substrates.
a
b
c
d
e
f
R1 = F, R2 = H, R3 = H, R4 = H, R5 = H
R1 = H, R2 = F, R3 = H, R4 = H, R5 = H
R1 = H, R2 = H, R3 = F, R4 = H, R5 = H
Results and discussion
We initially chose methyl (R,S)-9-fluoropalmitate 3a and
methyl (R,S)-10-fluoropalmitate 3b as our primary test sub-
strates since the behaviour of these compounds could conveni-
ently be compared with that of a readily accessible, remotely
fluorinated substrate—methyl 16-fluoropalmitate 3c. Thus, 3a
and 3b were synthesized in 8 and 7% overall yield, respectively,
via a sequence of reactions consisting of alkyl Grignard add-
ition to the appropriate ω-alkenal, fluorination using diethyl-
R1 = H, R2 = H, R3 = H, R4 = F, R5 = H
R1 = H, R2 = H, R3 = H, R4 = H, R5 = (S)-F
R1 = H, R2 = H, R3 = H, R4 = H, R5 = (R,S)-F
9
10
aminosulfur trifluoride (DAST), oxidative cleavage and
1
1
methylation (Scheme 2 and Experimental section). Com-
were collected by centrifugation. The cellular fatty acid fraction
from each incubation experiment was isolated by a standard
5d
pound 3c was conveniently prepared by fluorination of methyl
juniperate (methyl 16-hydroxyhexadecanoate). Since the yeast
9
5d
desaturase can act on both C16 and C18 substrates, we also
hydrolysis/methylation procedure and analyzed by GC–MS
∆
1
19
5c
and H-decoupled F NMR. Examination of the fatty acid
profiles (Table 1) revealed that uniformly high incorporation of
the fluorinated substrates was achieved and with one exception
(Expt. 3), no obvious effect on the % desaturation of endogen-
prepared methyl (R,S)-9-fluorostearate 5a and methyl (R,S)-10-
fluorostearate 5b in the same manner as their C16 homologues.
In order to check for possible steric effects of the fluorine sub-
stituent, a further three monofluorinated methyl stearates were
prepared from readily available starting materials. Thus methyl
ous C16
or C18 substrates was identified. (In Expt. 3, only a
modest reduction in the % desaturation of endogenous fatty
acids was noted—this effect was taken into account when we
compared the data of the individual experiments.) Importantly,
a single product (a fluoroolefin) was produced in each case. The
proposed structure of the fluoroalkenes was consistent with the
(
R,S)-5-fluorostearate 5c, methyl (R,S)-7-fluorostearate 5d
and methyl (S)-12-fluorooctadecanoate 5e were synthesized by
DAST fluorination of the corresponding methyl hydroxystear-
ates. All spectral and analytical data obtained for these com-
pounds were in accord with their assigned structure.
ϩ
ϩ
MS data [4a,b,c: m/z 286 (M ); 6a,b: m/z 314 (M ); 6c,d,e: m/z
Each fluorinated fatty acid methyl ester (25 mg) was incu-
3
ϩ
ϩ
bated for 24 h with growing cultures (150 cm ) of S. cerevisiae
ATCC 12341 (Baker’s yeast) as previously described. The
yeast cells grew out to a normal extent in each experiment and
294 (M Ϫ HF); m/z 262 (M Ϫ HF, CH OH)], the expected
3
5
a
GC retention times (ca. 0.1–0.8 min shorter than that of the
19
corresponding substrate) and the
F NMR chemical shifts
[
(CDCl3, CFCl ref.) δ Ϫ105.78 4a; Ϫ105.57 4b; Ϫ218.55 4c;
3
Ϫ105.78 6a; Ϫ105.57 6b; Ϫ181.22 6c; Ϫ179.78 6d; Ϫ179.57 6e].
The E-stereochemistry of the 9- and 10-fluoroolefinic products
4a,b and 6a,b was confirmed by comparison of the observed
§
9
This mechanism was originally discounted by Bloch who found that
-hydroxystearoyl CoA and 10-hydroxystearoyl CoA were not de-
9
23
hydrated to give olefin by the yeast ∆ desaturase. Since it is possible
that these compounds could not bind to the desaturase enzyme, this
result cannot be regarded as entirely conclusive.
1
9
F NMR chemical shifts with literature values obtained for (E)-
1
2
7-fluorotetradec-7-ene (δ Ϫ105.6), (Z)-7-fluorotetradec-7-ene
2
618
J. Chem. Soc., Perkin Trans. 1, 1997

View Article Online
H
H
H
H
F
a,b
F
H
H
F
O
10
9
O
H
H
H
10
9
BrMghexyl
10
9
BrMgheptyl
c
–
H2O
HO
H
OH
O
H
H
F
no reaction
HO
10
9
10
9
–
HF
9
(R)
9(S)
DAST
1
0(R)
10(S)
F
H
H
H
H
a,b
F
F
H
H
H
F
1
0
9
10
9
1
0
9
F
c
c
1
2
KMnO4, H+
BF3–MeOH
–H2O
F
OH
H
H
OH
H
H
F
1
0
9
10
9
F
OCH3
9
O
Scheme 3
3
a
the 3a or 5a incubation militates against the involvement of an
obligatory alcohol intermediate in desaturation.
OCH3
This interpretation was substantiated by the results of the
incubation experiments using 10-fluoro substrates 3b and 5b.
Thus (R)-3b or (R)-5b should have given an erythro-1,2-
fluorohydrin || if pathway c was involved in desaturation and
failure to observe this product in our extracts also suggests that
pathway c is not operative. This in turn implies that 10(R)-
O
1
0
F
3
b
Scheme 2
1
2
13
(
δ Ϫ110.6) and (E)-4-fluorooct-4-ene (δ Ϫ105.0). The
stereochemistry of the double bond could be checked for one
of the remotely fluorinated products 6e and shown to be Z as
expected by comparison of the observed F NMR chemical
shift of the biosynthetic product with those of authentic sam-
ples of methyl (Z)-12-fluorooctadec-9-enoate (δ Ϫ179.57) and
methyl (E)-12-fluorooctadec-9-enoate (δ Ϫ179.86).¶
Examination of the methylated dichloromethane extracts of
the culture medium obtained in each experiment revealed
only the presence of starting material. Importantly, none
9
fluoro substrates should be suicide inhibitors of ∆ desaturases
since the formation of the putative carbon-centred radical at C-
9 is almost certainly irreversible. Unfortunately, our method-
ology relies on continuous in vivo production of enzyme and
1
9
9
inactivation of the ∆ desaturases could go undetected under
our conditions. Further experiments with purified enzyme are
necessary to address this point. With respect to the fate of (S)-
3b or (S)-5b, it is not surprising in light of the above discussion
that these substrates were processed cleanly to fluoroolefin with
no evidence of threo-1,2-fluorohydrin || formation.
5
a
(
<0.1% of the total fatty acids) of the possible oxygenated
Taken together, our results do not favour Lewis acid-
catalyzed dehydration of a hydroxylated intermediate as a route
to olefin in desaturation. This led us to consider whether the
effects of fluorine substitution could be used to distinguish
between the two remaining alternatives: the carbocationic and
disproportionation pathways (pathway a,b, Scheme 1). Fluor-
ine substituent effects are legitimate probes for carbocation
formation. That is, a 9-fluorine substituent should favour the
formation of a carbocation at C-9 by overcoming σ-inductive
withdrawal with 2p-2pπ resonance donation while a 10-fluoro
substituent could conceivably shut down neighbouring carbo-
cation formation by a strong, electron-withdrawing inductive
products arising from diverted desaturation (Scheme 3) or their
possible decomposition products/metabolites were detected by
our analytical methods (GC–MS for non-fluorinated com-
1
19
pounds or a combination of GC–MS and H-decoupled
F
NMR for fluorinated products).
We begin our discussion of these results by considering the
possible fate of each enantiomer of the 9- and 10-fluoro sub-
strates if desaturation follows a hydroxylation/dehydration
9
pathway (Scheme 3). Since it is known that the yeast ∆ desatu-
1
4
rase abstracts the pro R hydrogens at C-9 and C-10, and that
5
a–d
oxidation is initiated at C-9,
the (R)-9-fluoro enantiomers
cannot be oxidized. The corresponding (S)-9-fluoro enantio-
mer, however, would give a 9,9-fluorohydrin which should col-
lapse spontaneously to give ketone. The α-hydroxylation of a
1
6a,b
effect.
In our case, one can only use fluorine substituent
effects as evidence for or against carbocation formation if one
makes the following assumptions: (1) effects of (S)-9-fluoro and
1
5a
fluorinated steroid has been shown to give a keto product.
(S)-10-fluoro substitution on non-catalytic steps such as sub-
The fact that we see only fluoroolefin and no methyl 9-
ketopalmitate (stearate) or their metabolites in the extracts of
strate binding are not dramatically different and (2) fluorine
substituent effects on possible carbocation formation are not
completely masked by the effects of fluorine substitution on the
initial hydrogen abstraction step—an event we know from our
¶
1
These compounds were prepared by DAST fluorination of methyl (Z)-
2-hydroxyoctadec-9-enoate and methyl (E)-12-hydroxyoctadec-9-
5d
isotope effect studies to be kinetically important.
enoate. These reactions do not give pure products but the desired com-
pounds are major components which are contaminated with a number
of rearrangement products due to the intermediacy of cyclopropyl car-
|| The fluorohydrins were synthesized according to the method of Muel-
2
4
25
bonium ions as we have previously discussed. The identity of the
desired reference compounds was unambiguously determined using a
bacher and Poulter (see Experimental section) and served as reference
standards in our search for these intermediates. It was shown that these
compounds are stable in solution under physiological conditions.
1
9
13
24
combination of F NMR and C NMR spectroscopy.
J. Chem. Soc., Perkin Trans. 1, 1997
2619

View Article Online
1
19
Fig. 3 H-decoupled F NMR (376.5 MHz) spectrum of cell extract
obtained from incubation of a 1.4:1 mixture of methyl (R,S)-10-
1
9
fluoropalmitate 3b:methyl 16-fluoropalmitate 3c. F resonances due to
b and 3c as well as the products 4b and 4c are assigned as indicated and
3
the relative intensities are given in parentheses.
remotely fluorinated substrate 3c. Thus, a mixture of 3b and 3c
(1.4:1) was incubated with S. cerevisiae, the cellular fatty acids
extracted and the amount of fluoroolefin formed from each
Fig. 1 Dependence of % desaturation of fluorinated substrates as a
function of fluorine position (Table 1). Values for % desaturation of 9-
and 10-fluorinated substrates in Table 1 have been multiplied by a factor
1
substrate in the cellular extract evaluated by H-decoupled
1
9
F NMR spectroscopy (Fig. 3). From this experiment, we can
estimate a k /k = (am’t 4c/am’t 4b) ÷ [(am’t 3c)/am’t (S)-
2
to correct for the fact that only 50% of these racemic substrates can
H
F β
give olefin. The value for % desaturation of methyl 16-fluoropalmitate
has been corrected (×1.3) since the % desaturation of endogenous sub-
strates in this incubation was somewhat lower than normal [83% (ave.)
for C16 and 95% (ave.) for C18].
3
b]initial = (54/16) ÷ [1/(0.5)1.4] = 2.4. {This value is somewhat
higher than that calculated from Fig. 1 [% desaturation 3c/%
desaturation (S)-3b = 81/60 = 1.4] but it is more accurate since it
is derived from a direct competition experiment}. An α-fluorine
substituent effect on desaturation can be calculated by combin-
ing the results of the two competition experiments: k /k = kH /
H
F α
kF β × k /k = 2.4 × 2.6 = 6.2.
F β F α
The significance of these values of the α- and β-fluoro sub-
stituent effects on desaturation is that they are in qualitative
agreement with what is known about the effect of fluorine
substitution on related biohydroxylations—rate retardations
are felt most strongly α to the fluorine substituent ** and
1
5a,b
decrease with increasing distance.
Thus, our estimate of the
β-fluorine effect (k /k = 2.4) on desaturation can easily be
H
F β
accounted for by an inductive effect on the initial hydrogen
1
7
abstraction step by a strongly electrophilic agent. If a carbo-
cation was being generated at C-9 during desaturation, one
might have expected to see a far larger β-effect since a rate retar-
dation of k /k = 79 per β-fluorine atom on the formation of
H
F β
1
6a
delocalized (allylic) cations has been observed
and effects
on non-stabilized carbocations should be even greater. Similarly,
one might have expected a much reduced rate-retarding
α-fluorine effect or possibly even an acceleration since a rate
acceleration of k /k = 71 due to an α-fluorine effect on car-
F α
H
1
6a
bocation formation is known. Since we do not see these
sorts of perturbations on the β- and α-fluorine effects, we con-
clude that the intermediacy of carbocations during desatur-
ation is not supported by our data, and thus, by a process
of elimination, a disproportionation mechanism (pathway b,
Scheme 1) must be favoured.
1
19
Fig. 2 H-decoupled F NMR (376.5 MHz) spectrum of cell extract
obtained from incubation of a 1:1 mixture of methyl (R,S)-10-
1
9
fluoropalmitate 3b:methyl (R,S)-9-fluoropalmitate 3a. F resonances
due to 3b and 3a as well as the products 4a and 4b are assigned as
indicated and the relative intensities are given in parentheses.
Finally, we would like to comment on the fact that the %
desaturation of (S)-5a is apparently similar to the values
obtained for stearoyl substrates (5c–e) bearing fluorine sub-
stituents at intermediate distances (γ, δ, ε) from the site of
initial oxidation (Fig. 1). These long-range substituent effects
clearly cannot be due to influences on catalytic steps but are
probably due to unfavourable interactions of the fluorine sub-
stituent with the hydrophobic walls of the desaturase binding
site. We thought it would be interesting to examine this
With these caveats in mind, we examined the data of Table 1 as
represented graphically in Fig. 1. The most striking feature of
Fig. 1 is that 9-fluoroalkenes 4a and 6a are both produced ca. 3
times less efficiently than the corresponding 10-fluoroalkenes 4b
and 6b. This trend was confirmed by performing a direct com-
petition experiment in which a mixture of 3a and 3b (1:1)
was incubated with S. cerevisiae. Analysis of the cell extract
1
19
obtained in this experiment by H-decoupled F NMR spec-
troscopy revealed that a 2.6:1 mixture of 4b:4a was obtained
(
Fig. 2) in good agreement with the value calculated from the
data in Fig. 1 (% desaturation 3b/% desaturation 3a = 60/
4 = 2.5). This result gives the ratio: k /k = 2.6. In order to
1
7a
*
* Stoddart, Nechvatel and Tedder have shown that unlike β-fluorine
effects on ‘chemical’ H atom abstraction which are always rate-retarding,
the corresponding α-fluorine effects can be either accelerating or
decelerating depending on the nature of the reagent. Thus, α-fluorine
substituent effects are a potentially useful tool to evaluate chemical
models of iron-oxo-mediated hydrocarbon activations.
2
F β F α
determine the individual α- and β-fluoro substituent effects on
desaturation, we carried out a direct competition experiment
between one of the fluorinated methyl palmitates 3b and a
2
620
J. Chem. Soc., Perkin Trans. 1, 1997

View Article Online
Table 1 Incubation of fluorinated fatty acids using S. cerevisiae: fatty acid profiles
a
Cellular fatty acid composition
Expt.
#
Compound
incubated
%(F)
incorp.
%(F)
desat.
%(C )
desat.
%(C18)
desat.
1
6
C16 :0
C16 :1
C18 :0
C18 :1
C :0(F)
C :1(F)
n
n
Control
13
7
5
12
6
7
6
4
8
45
35
30
23
29
38
29
38
30
3
1
1
6
1
1
1
1
1
39
24
24
21
20
16
21
19
25
—
29
28
14
41
30
33
32
34
—
4
12
24
3
8
9
6
2
—
33
40
38
44
38
42
38
36
—
78
83
86
66
83
84
83
90
80
93
96
96
78
95
94
95
95
96
1
2
3
4
5
6
7
8
3a
3b
3c
5a
5b
5c
5d
5e
12 (10)
— (30)
62 (62)
7 (7)
21 (20)
21
b
b
17 (20)
6 (5)
a
The relative amounts of each fatty acid component are expressed as a % of the total fatty acids; they were determined using the GC–MS Total Ion
Current (TIC) chromatograms. Each experiment was carried out at least twice and each entry represents an average value (SD = 1–2%). Endogenous
fatty acids include C16 :0 [methyl hexadecanoate], C16 :1 [methyl (Z)-hexadec-9-enoate], C18 :0 (methyl octadecanaoate) and C18 :1 [methyl (Z)-
octadec-9-enoate]. C :0(F) is the saturated, fluorinated substrate, n = 16 (Expt. 1,2) and 18 (Expt. 3,4); C :1(F) is the unsaturated, fluorinated
n
n
product, n = 16 (Expt. 1,2) or 18 (Expt. 3,4). %(F) incorp. = amount of fluorinated fatty acids as a % of the total fatty acids in extract. %(F)
1
19
desat. = % desaturation of fluorinated substrate. Values in brackets were obtained by integration of the relevant peaks in the H-decoupled F NMR
b
spectra of the fatty acid extracts. %(C ) desat. = % desaturation of endogenous C :0 %(C ) desat. = % desaturation of endogenous C :0. These
1
6
16
18
18
1
9
values are based solely on the F NMR data due to overlap of the GC peaks.
OCH3
9
F
O
1
0
1
2
5
f
KMnO4–NaIO4
O
F
HO
3
(R)-7
+
O
F
HO
3
(S)-7
+
HO
OCH3
O
O
phenomenon further by checking whether there was any
enzymic discrimination between the worst substrate 5e and its
enantiomer. Thus, racemic 12-fluorooctadecanoate 5f was pre-
pared by fluorination of methyl (R,S)-12-hydroxyoctadeca-
noate (see Experimental section) and administered to S. cer-
evisiae in the usual manner. The cell extract was examined by
1
19
H-decoupled F NMR and shown to contain a 95:5 mixture
of starting material:product as expected. The extract was then
1
8
submitted to von Rudloff oxidation conditions and the
1
fluorinated cleavage products 7 were analyzed by H-decoupled
1
9
F NMR spectroscopy. With the aid of the chiral shift reagent
S)-(Ϫ)-1-(naphthyl)ethylamine (4 mg per 0.5 cm ), it was shown
3
(
that the ratio of (R)-3-fluorononanoic acid/(S)-3-fluoro-
Fig. 4 Assessment of enantiomeric purity of 3-fluorononanoic acid
1
9
nonanoic acid derived from biosynthetic 12-fluorooleate was
3:47 (Fig. 4). Thus, it appears that both enantiomers of
obtained by oxidative cleavage of biosynthetic 12-fluorooleate: the
1
9
5
effect of (S)-(Ϫ)-NEA on the F (376.5 MHz) NMR resonances due to
A), (R)- and (S)-3-fluorononanoic acid (R,S)-7 derived from desatur-
(
methyl 12-fluorooctadecanoate are processed at comparable
rates by our desaturating system, and the rate-retarding effect
of 12-fluoro substitution is not stereospecific. These phenom-
ena clearly require more systematic study using a complete set
of fluorinated fatty acids. However, our observations do serve
to emphasize that while monofluorination of fatty acid hydro-
carbon chains may have minimal impact on the bulk bio-
ation of (R,S)-12-fluorooctadecanoate 5f; (B), (S)-3-fluorononanoic
acid (S)-7 derived from desaturation of (S)-12-fluorooctadecanoate
5
e; (C), (S)-3-fluorononanoic acid (S)-7 derived from desaturation
of (S)-12-fluorooctadecanoate 5e spiked with authentic (R)-3-
fluorononanoic acid (R)-7
In summary, we have demonstrated that the use of fluorin-
ated substrates shows considerable promise as a means of test-
ing current mechanistic models for desaturation. We have pro-
vided some evidence against the intermediacy of an alcohol
or carbocation in fatty acid desaturation. Additional work is
2
0
physical properties of condensed monolayers, one cannot
2
1
assume that the ‘isomorphic’ replacement of hydrogen by
fluorine will not result in unexpected effects on enzyme–
substrate interactions.
J. Chem. Soc., Perkin Trans. 1, 1997
2621

View Article Online
required to extend and clarify our observations using purified
enzymes when these become available. It would also be interest-
ing to carry out analogous experiments using the purified plant
NaHCO and water, dried, and then evaporated to give (R,S)-
10-fluoroheptadec-1-ene as a crude yellow solid (1.75 g, 51%,
3
est.); δ 0.89 (3H, t, J 7, CH ),1.2–1.5 (20H, br s, methylenes),
H
3
9
3a
∆
desaturase enzyme system.
1.62 (4H, m, CH CHFCH ), 2.03 (2H, m, CH CH᎐CH ), 4.44
2 2 2 2
(
1H, d of m, JH F 48, CHF ), 4.94 (2H, m, CH᎐CH ) and 5.80
2
1
(1H, m, CH᎐CH ). As judged by the integration of additional H
᎐
2
Experimental
NMR signals at δ 5.37, this material consisted of a 3:1 mixture
of fluoride:diene by-products. This intermediate was used in
the next step without further purification.
General methods
Melting points were determined on a digital Fisher-Johns
melting-point apparatus and are uncorrected.
A mixture of crude (R,S)-10-fluoroheptadec-1-ene (1.75 g,
1
3
Unless otherwise noted, H NMR spectra were obtained at
75% pure, 5.1 mmol) in glacial acetic acid (12 cm ) was oxidized
2
00 MHz on a Varian Gemini 200 spectrometer using dilute
at room temperature for 17 h using aqueous KMnO (40 mmol,
60 cm ) in the presence of hexadecyl(tributyl)phosphonium
4
3
CDCl solutions. Chemical shifts are expressed in ppm and are
3
referenced to tetramethylsilane. Coupling constants (J ) are
bromide (50 mg, 0.1 mmol) and with vigorous stirring. Hexane
(60 cm ) was added to the reaction mixture and excess of oxi-
dant destroyed by the addition of sulfurous acid (prepared ex
situ). The hexane layer was washed with water, dried and evap-
orated to give crude (R,S)-9-fluoropalmitic acid (800 mg) as a
white crystalline product. This compound was methylated with
3
reported in Hz.
1
9
All F NMR spectra were obtained on a Bruker AM400
9.4 T) spectrometer operating at 376.50 MHz with a dedicated
(
1
9
1
19
5
mm F/ H probe and F-specific pre-amplifier. A Bruker 400
MHz band-pass filter and a 376.5 MHz band-stop filter were
used in the proton decoupler channel, and 376.5 MHz band-
pass filter and 400 MHz band-stop channel were used in the
fluorine observe channel for all acquisitions. Proton decoup-
ling was achieved by using low power (composite phase
decoupling). Chemical shifts are reported relative to external
BF –MeOH (pre-prepared reagent) and purifed by flash chro-
3
matography using 2% EtOAc/hexanes to give methyl (R,S)-9-
fluoropalmitate, 3a (456 mg, 31%), mp 26–28 ЊC (from meth-
anol) (Found: C, 70.95; H, 11.53. C H FO requires C, 70.78;
1
7
33
2
H, 11.53%); δH 0.88 (3H, t, J 7, CH ), 1.2–1.5 (20H, br s,
3
trichlorofluoromethane (CFCl ) at 0.00 ppm.
methylenes), 1.63 (4H, m, CH CHFCH ), 2.30 (2H, t, J 7,
3
2
2
Mass spectra were obtained on a Kratos Concept IIH mass
spectrometer equipped with a 70 eV EI ionization chamber.
Samples were introduced using either a direct probe or a gas
chromatograph (HP 5980, 30 m DB-5 capillary column) as
appropriate. Single elemental analyses were performed using a
Perkin-Elmer Elemental Analyzer 240C.
Flash chromatography using silica gel (230–400) mesh was
used to purify substrates prior to incubation experiments. Ana-
lytical TLC was performed using Merck glass plates pre-coated
with silica G/UV 254. Visualization of UV-inactive materials
was accomplished by using a combination of I2 vapour
followed by a water spray.
CH CO), 3.66 (3H, s, CO CH ), 4.45 (1H, d of m, J
48,
2
2
3
H F
ϩ
ϩ
CHF ); δ Ϫ180.50; m/z 268 (M Ϫ HF ) and 236 (M Ϫ HF,
F
CH OH).
3
The route described above was also used to synthesize the
following.
Methyl (R,S)-10-fluoropalmitate (10-fluorohexadecanoate) 3b.
From undec-10-en-1-al: mp 24–25 ЊC (Found: C, 70.71; H,
11.53. C H FO requires C, 70.78; H, 11.53%); δ 0.88 (3H, t,
1
7
33
2
H
J 7, CH ), 1.2–1.5 (20 H, br s, methylenes), 1.63 (4H, m,
3
CH CHFCH ), 2.30 (2H, t, J 7, CH CO), 3.66 (3H, s,
2
2
2
CO CH ) and 4.45 (1H, d of m, J 48, CHF ); δ Ϫ180.49;
2
3
H F
F
ϩ
ϩ
m/z 268 (M Ϫ HF ) and 236 (M Ϫ HF, CH OH).
3
Unless otherwise stated, all reagents and starting materials
were purchased from Aldrich Chemical Company and used
without purification. Chiral materials were used without
improving their optical purity. All air- and moisture-sensitive
Methyl (R,S)-9-fluorostearate (9-fluorooctadecanoate) 5a.
From dec-9-en-1-al: mp 37–39 ЊC (Found: C, 72.08; H, 11.79.
C H FO requires C, 72.10; H, 11.78%); δ 0.88 (3H, t, J 7,
1
9
37
2
H
CH ), 1.2–1.5 (24H, br s, methylenes), 1.63 (4H, m,
3
reactions were performed under N . Unless otherwise noted,
CH CHFCH ), 2.30 (2H, t, J 7, CH CO), 3.66 (3H, s,
2
2
2
2
organic extracts were shaken with sat. NaCl, dried over Na SO
and evaporated on a rotary evaporator. All fluorinated methyl
ester substrates were obtained as white, amorphous solids.
CO CH ) and 4.45 (1H, d of m, J 48, CHF ); δ Ϫ180.49;
2 3 H F F
ϩ ϩ
2
4
m/z 296 (M Ϫ HF ) and 264 (M Ϫ HF, CH OH).
3
Methyl (R,S)-10-fluorostearate (10-fluorooctadecanoate) 5b.
From undec-11-en-1-al: mp 34–36 ЊC (Found: C, 72.03; H,
11.96. C H FO requires C, 72.10; H, 11.78%); δ 0.88 (3H, t,
Synthesis of substrates
1
9
37
2
H
Methyl (R,S)-9-fluoropalmitate (9-fluorohexadecanoate) 3a.
The title compound was prepared using a sequence of
well-established synthetic procedures.
J 7, CH ), 1.2–1.5 (24 H, br s, methylenes), 1.63 (4H, m,
3
CH CHFCH ), 2.31 (2H, t, J 7, CH CO), 3.68 (3H, s,
2
2
2
CO CH ), 4.45 (1H, d of m, J 48, CHF ); δ Ϫ180.44; m/z
2
3
H F
F
2
6
ϩ
ϩ
A solution of dec-9-en-1-al (prepared by Swern oxidation
296 (M Ϫ HF ) and 264 (M Ϫ HF, CH OH).
3
of the commercially available dec-9-en-1-ol) (4.57 g, 29.7 mmol)
in anhydrous diethyl ether (15 cm ) was added to an ethereal
The following compounds were prepared by treating the cor-
responding alcohol with diethylaminosulfur trifluoride essen-
tially as described above. Unless otherwise stated, purification
of the crude product was achieved by flash chromatography
using 2% EtOAc–hexane as eluent.
3
solution of freshly prepared heptylmagnesium bromide (40
mmol) at Ϫ10 ЊC over 30 min. The reaction mixture was poured
onto crushed ice (25 g), acidified with 3  aq. H SO and
2
4
extracted with diethyl ether. The extract was washed with 3 
Methyl 16-fluoropalmitate (16-fluorohexadecanoate) 3c. From
methyl 16-hydroxypalmitate [870 mg, 3.04 mmol; obtained by
aq. H SO , dried and evaporated to give a yellowish solid. This
2
4
1
1
material was purifed by flash chromatography with 5% EtOAc
methylation
of 16-hydroxypalmitic acid (juniperic acid)]
as eluent to yield (R,S)-heptadec-1-en-10-ol as a white solid
(450 mg, 51%), mp 32–34 ЊC (Found: C, 70.42; H, 11.63.
C H FO requires C, 70.78; H, 11.53%); δH 1.2–1.4 (24H,
(
(
(
3.95 g, 52%), mp 41–43 ЊC; δH 0.89 (3H, t, J 7, CH ), 1.2–1.5
3
17 33
2
20H, br s, methylenes), 1.61 (4H, m, CH CHOHCH ), 2.03
br s, methylenes), 1.62 (2H, m, CH CH F ), 2.30 (2H, t, J 7,
2
2
2 2
2H, m, CH CH᎐CH ), 3.57 (1H, br d q, CHOH), 4.93 (2H, m,
CH CO CH ), 3.67 (3H, s, CO CH ) and 4.44 (2H, d of t,
2 2 3 2 3
ϩ
2
2
ϩ
CH᎐CH ) and 5.79 (1H, m, CH᎐CH ); m/z 236 (M Ϫ H O).
JH F 47, JH H 7, CH F ); δ Ϫ218.47; m/z 288 (M ) and 257
᎐
2
2
2
2
F
ϩ
A solution of (R,S)-heptadec-1-en-10-ol (2.3 g, 9.06 mmol) in
(M Ϫ CH O).
3
3
dichloromethane (10 cm ) was treated with diethylaminosulfur
Methyl (R,S)-5-fluorostearate (5-fluorooctadecanoate) 5c.
From methyl (R,S)-5-hydroxystearate (428 mg, 1.36 mmol;
trifluoride (10.2 mmol) at Ϫ45 ЊC. The reaction mixture was
allowed to warm to room temperature whereupon excess of
reagent was carefully quenched with water. The organic layer
2
7
obtained by NaBH reduction of methyl 5-oxooctadecanoate)
4
(134 mg, 29%), mp 39–41 ЊC (Found: C, 72.31; H, 11.81.
C H FO requires C, 72.10; H, 11.78%); δ 0.88 (3H, t, J 7,
3
was diluted with dichloromethane (20 cm ), washed with sat. aq.
1
9
37
2
H
2
622
J. Chem. Soc., Perkin Trans. 1, 1997

View Article Online
CH ), 1.2–1.5 (24H, br s, methylenes), 1.69 (4H, m,
from the cells by a standard hydrolysis–methylation procedure.
3
CH CHFCH ), 2.34 (2H, t, J 7, CH CO), 3.67 (3H, s,
Yeast cells from each incubation experiment (ca. 5 g, wet
2
2
2
CO CH ) and 4.46 (1H, d of m, J 48, CHF ); δ Ϫ181.31;
weight) obtained by centrifugation were treated with refluxing
2
3
H F
F
ϩ
ϩ
3
m/z 296 (M Ϫ HF ) and 264 (M Ϫ HF, CH OH).
1  50% ethanolic KOH (50 cm ) for 3 h under N . Cell debris
3
2
Methyl (R,S)-7-fluorostearate (7-fluorooctadecanoate) 5d.
From methyl (R,S)-7-hydroxystearate (1.12 g, 3.5 mmol;
was filtered off and the resultant solution was partially evapor-
ated in vacuo to remove ethanol, diluted with water (100 cm )
3
2
7
obtained by NaBH4 reduction
octadecanoate) (350 mg, 27%), mp 30–32 ЊC [lit., 38.5–
9.9 ЊC (from methanol)] (Found: C, 71.88; H, 12.01. C -
of methyl 7-oxo-
acidified to pH 2 with 50% aq. H SO and extracted with
2
4
2
8
dichloromethane. This extract was washed with water and sat.
brine, dried (Na SO ) and evaporated to give a yellow solid. This
3
1
9
2
4
H FO requires C, 72.10; H, 11.78%); δ 0.88 (3H, t, J 7, CH ),
was treated with a freshly prepared solution of ethereal diazo-
methane to yield fatty acid methyl esters (ca. 25 mg) after
evaporation.
3
7
2
H
3
1
2
.2–1.5 (24H, br s, methylenes), 1.62 (4H, m, CH CHFCH ),
2 2
.30 (2H, t, J 7, CH CO), 3.66 (3H, s, CO CH ), 4.45 (1H, d
2
2
3
ϩ
of m, J 48, CHF ); δ Ϫ180.70; m/z 296 (M Ϫ HF ) and 264
H F
F
ϩ
(M
Ϫ HF, CH OH).
Oxidative cleavage of fatty acids
3
Methyl (S)-12-fluorostearate (12-fluorooctadecanoate) 5e.
From methyl (R)-12-hydroxystearate [400 mg, 1.27 mmol;
A solution of the fatty acid methyl esters (20 mg) in tert-butyl
alcohol–water–0.002  aq. Na CO (20:5:5) was treated with
2
3
1
1
3
obtained by BF -catalyzed methylation
of (R)-12-
an aqueous oxidant (10 cm ) which was 0.0025  in KMnO
3
4
hydroxystearic (octadecanoic) acid]. Purification of the crude
product was accomplished by a 3-fold recrystallization from
methanol (200 mg, 50%), mp 37–39 ЊC (Found: C, 72.09;
H, 11.77. C H FO requires C, 72.10; H, 11.78%); δ 0.88
and 0.01  in KIO with stirring at 28 ЊC for 48 h. Additional
4
aliquots of oxidant were added as needed until the purple
colour persisted. Excess of oxidant was destroyed by addition
of Na S O after which the solution was basified with 1  aq.
1
9
37
2
H
2
2
5
3
t
(
3H, t, J 7, CH ), 1.2–1.5 (24H, br s, methylenes), 1.63
KOH (5 cm ) and the Bu OH removed by roto-evaporation. The
remaining cloudy solution was diluted with water (5 cm ), acid-
3
3
(
4H, CH CHFCH ), 2.30 (2H, t, J 7, CH CO), 3.67 (3H, s,
2
2
2
CO CH ) and 4.45 (1H, d of m, J 48, CHF ); δ Ϫ180.43;
ified to pH 2 with 50% aq. H SO and extracted with hexanes
(4 × 5 cm ).
2
3
H F
F
2
4
ϩ
ϩ
3
m/z 296 (M Ϫ HF ) and 264 (M Ϫ HF, CH OH).
3
Methyl (R,S)-12-fluorostearate (12-fluorooctadecanoate) 5f.
From methyl (R,S)-12-hydroxystearate (370 mg, 1.18 mmol;
Acknowledgements
obtained by NaBH reduction of 12-oxooctadecanoate). The
4
crude fluorinated product was purified by repeated recrystal-
We thank the Natural Sciences and Engineering Research
Council of Canada (NSERC) for financial support of this
work.
lization from methanol (150 mg, 40%), mp 37–38 ЊC (from
2
8
methanol) [lit., 39.5–40 ЊC (from methanol)]. Spectral data
identical with those of 5e.
Methyl erythro-9(10)-fluoro-10(9)-hydroxyoctadecanoate. A
solution of methyl (E)-9,10-epoxyoctadecanoate (200 mg, 0.64
mmol) in CH Cl was treated with pyridine polyhydrofluoride
References
1 Preliminary account of part of this work: P. H. Buist, B. Behrouzian,
K. A. Alexopoulos, B. Dawson and B. Black, J. Chem. Soc., Chem.
Commun., 1996, 2671.
(a) H. Cook, in Biochemistry of Lipids and Membranes, eds. D. E.
Vance and J. E. Vance, The Benjamin Cummings Publishing Co.
Ltd., Menlo Park, CA, 1985, pp. 191–203; (b) R. J. Breslow, Chem.
Soc. Rev., 1972, 1, 553.
3 (a) B. G. Fox, J. Shanklin, J. Ai, T. Loehr and J. Sanders-Loehr,
Biochemistry, 1994, 33, 12 776; (b) J. Shanklin, E. Whittle and
B. G. Fox, Biochemistry, 1994, 33, 12 787.
Y. Linquist, W. Huang, G. Schneider and J. Shanklin, Embo J., 1996,
5, 4081.
5 (a) P. H. Buist and D. M. Marecak, J. Am. Chem. Soc., 1992, 114,
5073; (b) P. H. Buist and D. M. Marecak, Can. J. Chem., 1994, 72,
176; (c) P. Buist, D. Marecak, B. Dawson and B. Black, Can. J.
Chem., 1996, 74, 453; (d) P. H. Buist and B. Behrouzian, J. Am.
Chem. Soc., 1996, 118, 6295; (e) P. H. Buist, H. G. Dallmann, R. R.
Rymerson and P. M. Seigel, Tetrahedron Lett., 1988, 29, 435;
2
2
(0.2 ml) at Ϫ5 ЊC for 15 min. The reaction mixture was diluted
with water to quench the reaction and neutralized with 5% aq.
2
NaHCO . The organic layer was dried and evaporated to yield a
3
yellow oil (137 mg). A portion of this material (127 mg) was
purified by flash chromatography using 15% ethyl acetate–
hexane as eluent to give a colourless oil (81 mg, 52%);
δ (400 MHz, Bruker AMX 400) 0.88 (3H, t, J 7, CH ), 1.2–
H
3
1
.5 (20H, br s, methylenes), 1.51 (2H, m, CH CHOH), 1.62
2
4
(
2H, m, CH CHF ), 2.30 (2H, t, J 7, CH CO), 3.67 (3H, s,
2
2
1
CO CH ), 3.71 (1H, m, CHOH), 4.37 (1H, d of m, J
49,
2
3
H F
ϩ
CHF ); δ Ϫ191.48 and Ϫ191.56; m/z 312 (M Ϫ HF ) and 294
F
ϩ
(
M Ϫ HF, H O). This material contained a minor fluorinated
2
19
contaminant of unknown structure which was detected by
NMR and H NMR spectroscopy but not by GC–MS.
F
1
Methyl threo-9(10)-fluoro-10(9)-hydroxyoctadecanoate.
A
(
f ) P. H. Buist, H. G. Dallmann, R. R. Rymerson, P. M. Seigel and
P. Skala, Tetrahedron Lett., 1987, 28, 857.
solution of methyl (Z )-9,10-epoxyoctadecanoate (207 mg,
.66 mmol) in CH Cl was treated with pyridine polyhydro-
0
6 F. J. van de Loo, P. Broun, S. Turner and C. Somerville, Proc. Natl.
Acad. Sci. USA, 1995, 92, 6743.
(a) P. R. Ortiz de Montellano, Trends Pharmacol. Sci., 1989, 10, 354;
b) J. R. Collins, D. L. Camper and G. H. Loew, J. Am. Chem. Soc.,
991, 113, 2736; (c) E. J. Lawlor, S. W. Elson, S. Holland, R. Cassels,
2
2
fluoride (0.2 ml) at Ϫ5 ЊC for 1 h. The reaction was worked
up as described above to yield a crude product (184 mg). A
portion of this material (159 mg) was purified by flash chroma-
tography using 15% ethyl acetate–hexane as eluent to give a low-
7
(
1
J. E. Hodgson, M. Lloyd, J. E. Baldwin and C. J. Schofield,
Tetrahedron, 1994, 50, 8737.
melting waxy solid (112 mg, 52%); δ (400 MHz, Bruker AMX
H
4
1
00) 0.88 (3H, t, J 7, CH ), 1.2–1.5 (20H, br s, methylenes),
8 (a) M. Newcomb, F. H. Le Tadic-Biadetti, D. L. Chestney, E. S.
Roberts and P. F. Hollenberg, J. Am. Chem. Soc., 1995, 117, 12 085.
W. J. Middleton J. Org. Chem., 1975, 40, 574.
0 A. P. Krapcho, J. R. Larson and J. M. Eldridge, J. Org. Chem., 1977,
2, 3749.
1 L. D. Metcalfe and A. A. Schmitz, Anal. Chem., 1961, 33, 363.
2 M. Shimizu and H. Yoshioka, Tetrahedron Lett., 1989, 30, 967.
13 S. H. Lee and J. Schwartz, J. Am. Chem. Soc., 1986, 108, 2445.
14 A. G. McInnes, J. A. Walter and J. L. C. Wright, Tetrahedron, 1983,
3
.48 (2H, m, CH CHOH), 1.62 (2H, m, CH CHF ), 2.30 (2H,
2
2
9
t, J 7, CH CO), 3.66 (3H, s, CO CH ), 3.51 (1H, m, CHOH)
2
2
3
1
and 4.3 (1H, d of m, J 49, CHF ); δ Ϫ195.53 and Ϫ195.66;
H F
F
4
ϩ
ϩ
m/z 312 (M Ϫ HF ) and 294 (M Ϫ HF, H O). This material
2
1
1
also contained a minor fluorinated contaminant of unknown
1
9
1
structure which was detected by F NMR and H NMR
spectroscopy but not by GC–MS.
3
9, 3515.
1
5 (a) H. L. Holland and E. M. Thomas, Can. J. Chem., 1982, 60, 160
and refs. contained therein; (b) T. L. Macdonald, CRC Crit. Rev.
Toxic., 1982, 11, 85.
Biotransformation of fluoro fatty acids using S. cerevisiae
Each fluorinated substrate was incubated with growing cells
of Saccharomyces cerevisiae, # ATTC 12341 as previously
1
6 (a) P. G. Gassman and T. T. Tidwell, Acc. Chem. Res., 1983, 16, 279
and refs. contained therein; (b) X. Creary, Chem. Rev., 1991, 91, 1625.
5
a
described. The desaturated fatty acid products were isolated
J. Chem. Soc., Perkin Trans. 1, 1997
2623

View Article Online
1
7 (a) I. K. Stoddart, A. Nechvatal and J. M. Tedder, J. Chem. Soc.,
Perkin Trans. 2, 1974, 473; (b) W. K. Busfield, I. D. Grice and
I. D. Jenkins, J. Chem. Soc., Perkin Trans. 2, 1994, 1079.
8 E. von Rudloff, Can. J. Chem., 1956, 34, 1413.
24 P. H. Buist and J. M. Findlay, Tetrahedron Lett., 1984, 25, 1433.
25 M. Muehlbacher and C. D. Poulter, J. Org. Chem., 1988, 53, 1026.
26 A. J. Mancuso, S. L. Huang and D. Swern, J. Org. Chem., 1978, 43,
2480.
27 S. W. Chaikin and W. G. Brown, J. Am. Chem. Soc., 1949, 71, 122.
28 N. J. M. Birdsall, Tetrahedron Lett., 1971, 12, 2675.
1
1
2
9 P. H. Buist and R. Adeney, J. Org. Chem., 1991, 56, 3449.
0 L. Dasaradhi, D. O’Hagan, M. C. Petty and C. Pearson, J. Chem.
Soc., Perkin Trans. 1, 1995, 221.
2
2
1 M. Schlosser and D. Michel, Tetrahedron, 1996, 52, 99.
2 P. Strittmatter, L. Spatz, D. Corcoran, M. J. Rogers, B. Setlow and
R. Redline, Proc. Natl. Acad. Sci. USA, 1975, 71, 4565.
3 R. J. Light, W. J. Lennarz and K. Bloch, J. Biol. Chem., 1962, 237,
Paper 7/01571B
Received 15th March 1997
Accepted 12th May 1997
2
1
793.
2
624
J. Chem. Soc., Perkin Trans. 1, 1997