Synthesis of (S)-(+)-enantiomers of food-relevant (n-5)-monoenoic and saturated anteiso-fatty acids by a Wittig reaction

Article abstract of DOI:10.1016/j.tet.2006.11.059

We developed an enantioselective synthesis for the food-relevant anteiso-fatty acids a15:0, a16:0, and a17:0. Different (carboxyalkyl)triphenylphosphonium bromide salts were coupled with (S)-3-methylpentanal in a Wittig reaction. Mixtures of the obtained cis-/trans-isomers were separated by Ag⁺-HPLC to give the novel cis-isomers of (S)-(+)-a15:1n-5, (S)-(+)-a16:1n-5, and (S)-(+)-a17:1n-5. Hydrogenation of the monoenoic products led to (S)-(+)-a15:0, (S)-(+)-a16:0, and (S)-(+)-a17:0, which are essential for the assessment of the bioactivity of tests with standards of anteiso-fatty acids.

Full text of DOI:10.1016/j.tet.2006.11.059

Tetrahedron 63 (2007) 1140–1145
Synthesis of (S)-(D)-enantiomers of food-relevant (n-5)-monoenoic
and saturated anteiso-fatty acids by a Wittig reaction
*
Saskia Thurnhofer and Walter Vetter
Institute of Food Chemistry, University of Hohenheim, Garbenstr. 28, D-70599 Stuttgart, Germany
Received 28 September 2006; revised 27 October 2006; accepted 21 November 2006
Available online 15 December 2006
Abstract—We developed an enantioselective synthesis for the food-relevant anteiso-fatty acids a15:0, a16:0, and a17:0. Different (car-
boxyalkyl)triphenylphosphonium bromide salts were coupled with (S)-3-methylpentanal in a Wittig reaction. Mixtures of the obtained
cis-/trans-isomers were separated by Ag+-HPLC to give the novel cis-isomers of (S)-(+)-a15:1n-5, (S)-(+)-a16:1n-5, and (S)-(+)-a17:1n-5.
Hydrogenation of the monoenoic products led to (S)-(+)-a15:0, (S)-(+)-a16:0, and (S)-(+)-a17:0, which are essential for the assessment
of the bioactivity of tests with standards of anteiso-fatty acids.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
species (B. cereus and B. xanthus), Desulfovibrio desulfuri-
cans, and also in Vernix caseosa and adult human skin
lipids.16–19 Furthermore, a17:1, cis-i17:1n-5, and cis-18:
1n-5 was found in B. anthracis, which belongs to the food-
borne bacterial pathogens and aerobic endospore-forming
bacilli.20 Additionally, a15:1 was determined in Brevibacte-
rium fermentans, a biotechnological producer of amino
acids.21 In the cases of a15:1 and a17:1, the positions of
the double bonds could not be assigned due to the lack of
reference standards.
Anteiso-fatty acids (n-2-methyl branched fatty acids, aFAs)
are found as minor lipid components in a wide range of
food (e.g., milk of ruminants, suet, and butterfat) and as major
components in the lipids of diverse bacteria.1–6 At the cellular
level, aFAs play an important role in providing an appropriate
degree of membrane fluidity at low temperatures, presum-
ably through keeping up the fluid–liquid–crystalline state
of membrane lipids.7–9 The dominating aFAs in food and
bacteria are 12-methyltetradecanoic acid (a15:0) and 14-
methylhexadecanoic acid (a17:0).3,10 The contribution of
aFAs to the total amount of fatty acids in food ranges from
approximately 0 to 3%, which is comparable to levels of con-
jugated linoleic acids (CLAs) and trans-fatty acids.11,12
Notably, aFAs are chiral, which is in contrast to iso-fatty
acids (iFAs). Only little data exist on the enantiomer distri-
bution of aFAs in food. About 0.5% of (S)-(+)-a17:0 was
determined in ruminant and ewe-milk fat, wool, as well as
mutton grease. Comparable amounts of (S)-(+)-a15:0 were
also detected in ewe-milk fat.2,13–15 These studies showed
that the (S)-enantiomers of aFAs are food-relevant.
Currently, only a limited number of aFA standards is com-
mercially available, all of which are racemic and saturated.
Thus, important experiments on the bioavailability and phase
transfer processes could only be performed with racemic
aFAs and thus, most likely not under real life conditions.
Commonly, FAs are either synthesized by Wittig or Grignard
reactions. A Wittig reaction was used to synthesize racemic
12-methylpentadecanoic acid (yield 14%), a15:0 (yield
42%), and cis-i17:1n-5 (yield 19%).22–24 In most cases, how-
ever, the Grignard pathway was used, which led to yields
from 10 to 90%.25,26 Moreover, (R)-12-methylhexadecanoic
acid (yield 74%) and (S)-aFAs were recently obtained in a
total yield of 10%.27,28
Next to the saturated aFAs mentioned above, several mono-
enoic aFAs were detected in different myxobacteria (Stigma-
tella aurantiaca and Myxococcus xanthus), some Bacillus
The goal of this study was to produce both saturated and
monoenoic aFAs as racemates and as pure (S)-enantiomers.
For this reason, we performed the C–C-coupling by a Wittig
reaction, which predominantly leads to cis-monoenoic
aFAs. Aliquots of monoenoic aFAs can be collected for
analytical purposes whereas the remaining part can be con-
verted into the corresponding saturated enantiopure (S)-
aFAs by hydrogenation.
Keywords: Methyl branched fatty acids; Anteiso-fatty acids; Monoenoic
anteiso-fatty acids; Wittig reaction; Enantioselective synthesis; Silver-ion
chromatography.
* Corresponding author. Tel.: +49 711 459 24016; fax: +49 711 459 24377;
e-mail: w-vetter@uni-hohenheim.de
0040–4020/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2006.11.059

S. Thurnhofer, W. Vetter / Tetrahedron 63 (2007) 1140–1145
1141
2. Results and discussion
In the first step of the Wittig reaction, 1a–c were reacted with
equimolar amounts of triphenylphosphine to obtain the cor-
responding triphenylphosphonium bromide salts 2a–c (Fig. 1).
When acetonitrile (ACN) was used as solvent, the resulting
one-phase system provided better yields of 2a–c compared
to the two-phase system with toluene initially used. This
was most likely due to thorough mixing of the solution during
the entire course of reaction. After formation of 2a–c in ACN,
an additional purification step with toluene by heating under
reflux was carried out in order to remove the remaining
educts. Subsequent treatment with anhydrous diethyl ether
converted the oils intowhite salts 2a–c. Even 2awas obtained
as a nearly white powder, which is in contrast to reports in lit-
erature, where only moisture- and air-sensitive glasses could
be obtained.33,34 Purified 2a–c were hygroscopic and had to
be redried directly prior the Wittig-coupling. As a conse-
quence of all efforts, theyields (91–97%) of 2a–c were higher
thanreportedinliterature(62–75%).2,34,35 Thespectraldataof
the synthesized triphenylphosphonium bromide salts (2a–c)
were in agreement with those reported in literature.34,35
Production of monoenoic aFAs 6a–c via a Wittig reaction
required u-bromocarboxylic acids and a racemic or enantio-
pure methyl branched aldehyde (Fig. 1). For enantioselective
synthesis, the methyl branch of the aldehyde must not be in
a-position in order to prevent racemization via the corre-
sponding enol. Hence, the shortest appropriate aldehyde
was 3-methylpentanal, which was also selected since the
required substrates for the preparation of a15:0–a17:0
(1a–c) were commercially available (Fig. 1). However, both
racemic and (S)-3-methylpentanal 5 were not available and
were prepared from racemic and (S)-(+)-3-methylpentanol
4 by mild oxidation using Dess–Martin periodinane (DMP)
(Fig. 1).29 Impurities of 3-methylpentanol in 3-methyl-
pentanal on the basis of GC/EI-MS were <2%. The spectro-
scopic data30 and the specific rotation of enantiopure 5
matched those published for the compound ([a]2D0 ꢀ4.00
and ꢀ2.46).31,32 The resulting 3-methylpentanal was used
without any purification step for the subsequent Wittig
reaction (see below).
The Wittig reaction (performed at 0–5 ꢁC) led to 88.6–
91.1% cis- and 8.9–11.4% trans-isomers of aFAs 6a–c,
which were obtained as weak yellow oils. After separation
of impurities (triphenylphosphine oxide and byproducts),
the yields of 6a–c of this step were 36.1–47.0% and the total
yields (including all steps) were 25.0–41.4% (Table 1). The
yields correlated with the chain length of the triphenylphos-
phonium bromide salts, which is most likely owing to the
Br
CH2
CH2
HOOC
x
1a-c
1) PPh3 / ACN
HO
CH3
1
Br-
PPh3+
higher hygroscopy of the shorter ones. Additionally, H
HOOC CH2
CH2
x
CH3
4
NMR data confirmed the identity of 6a–c, which was com-
parable with the reported chemical shifts for the structurally
similar 17-methyl-6(cis)-octadecenoic acid methyl ester.36
2a-c
2) THF / BuLi
DMP
Aliquots of the monoenoic aFAs 6a–c were treated with BF3/
MeOH to produce fatty acid methyl esters (FAMEs). The re-
sulting trans- (first eluted) and cis-isomers (second eluted,
DRRI16:0 Me¼0.0183–0.0288) of monoenoic aFAs showed
identical GC/EI-mass spectra (Fig. 2). The methyl esters
of cis- and trans-monoenoic aFAs showed the expected
molecular ion at low abundance, the base peak at m/z 55,
and further characteristic fragment ions at [Mꢀ61]+ and
[Mꢀ79]+ (Fig. 2).15,37 Furthermore, the GC retention times
were in the expected range.
O
CH3
HOOC
CH2
CH
PPh3
x
CH3
3a-c
5
9 (cis) :1 (trans)
HOOC
CH2
x
CH2
HOOC
CH3
+
CH3
x
Aliquots of the monoenoic (S)-aFAs 6a–c were methylated
and subjected to silver-ion high-performance liquid chroma-
tography (Ag+-HPLC) for the separation of cis- and trans-
isomers.38 After this procedure neat standards of both
cis- and trans-isomers of 6a–c were obtained (Table 1).
CH3
6a-c
CH3
Saturated aFAs 7a–c were finally obtained by hydrogenation
of aFAs 6a–c (Fig. 2). The mass spectra and relative reten-
tion indices (Table 1) of the synthesized methyl esters of
enantiopure aFAs 7a–c were identical with corresponding
data of the commercially available racemates (data not
shown). Additionally, the mass spectra of saturated FAME
showed the typical and major fragment ions m/z 74 and
m/z 87, which contribute at >50% to the total ion current
of saturated fatty acids.39,40 Slight dextrorotation was observed
for the methyl esters of (S)-(+)-a15:0 and (S)-(+)-a17:0,
which was similar to those reported for the free fatty acids
of (S)-(+)-a15:0 and (S)-(+)-a17:0 (Table 1).41,42 Slight
HOOC
CH2
CH3
cis-/trans-separation
x
7a-c
CH3
Figure 1. Synthesis pathway for food-relevant monoenoic and saturated
anteiso-fatty acids via a Wittig-coupling. u-Bromoalkylcarboxylic acids 1
(a: x¼7, b: x¼8 and for c: x¼9) were transferred into the corresponding
triphenylphosphonium bromide salts 2a–c. Addition of either racemic or
(S)-3-methylpentanal 5 obtained from (S)-3-methylpentanol 4 leads to
monoenoic aFAs 6a–c. Aliquots (25%) of cis-/trans-isomers of 6a–c were
separated by Ag+-HPLC whereas hydrogenation of the bulk of 6a–c resulted
in the corresponding saturated aFAs 7a–c.

1142
S. Thurnhofer, W. Vetter / Tetrahedron 63 (2007) 1140–1145
Table 1. Relative retention indices (RRI), direction of rotation, and total yields of cis- and trans-monoenoic aFAs and the corresponding saturated (S)-(+)-aFAs
Product
RRIa
[a]2D0, this study
[a]2D0, literature data
Total yieldsb [%]
Monoenoic aFAs
6a cis-(S)-a15:1n-5 ME
6a trans-(S)-a15:1n-5 ME
6b cis-(S)-a16:1n-5 ME
6b trans-(S)-a16:1n-5 ME
6c cis-(S)-a17:1n-5 ME
6c trans-(S)-a17:1n-5 ME
0.9712
0.9529
1.0298
1.0105
1.1016
1.0728
+4.65 (c 1.6 g/100 mL) CHCl3
+4.31 (c 1.9 g/100 mL) CHCl3
+3.82 (c 1.7 g/100 mL) CHCl3
—
—
—
25.0
27.0
41.4
Saturated aFAs
7a (S)-a15:0 ME
7b (S)-a16:0 ME
7c (S)-a17:0 ME
0.9277
0.9822
1.0440
+5.56a (c 3.6 g/100 mL) CHCl3
+5.17a (c 2.9 g/100 mL) CHCl3
+4.29a (c 3.5 g/100 mL) CHCl3
+5.0741 (c 2.08 g/100 mL) CHCl3
+3.328 (c 1.73 g/100 mL) CHCl3
+4.4922,42 neat
25.0
27.0
41.4
a
Retention indices of aFAMEs relative to 16:0 ME (RRI¼1.0000).
b
Including all steps.
(a)
(b)
(c)
70
55
55
Irel
Irel
55
70
70
Irel
83
83
83
97
97
97
109
123
111
123
137
109
M+
M+
254
181
153
M+
268
123
138
185
175
165
135
141
152
221 251
167
179
199
236
282
207
225
203
151
193
[m/z]
[m/z]
[m/z]
60
100
140
180
220
60
70
100
140
180
220
60
100 140 180 220 260
55
55
70
Irel
Irel
Irel
55
69
83
97
83
97
97
83
109
221
123
109
123
138
M+
123
135
M+
M+
153
152
181
203
251
175
109
167
137
199
207
282
141
185
165
225
236
151
254
193
268
179
[m/z]
[m/z]
[m/z]
60
100
140
180
220
60
100
140
180
220
60
100 140 180 220
Figure 2. GC/EI-mass spectra of the methyl esters of cis- (top) and trans-monoenoic aFAs (bottom) of (a) a15:1n-5, (b) a16:1n-5, and (c) a17:1n-5.
clockwise rotation was also found for (S)-(+)-a16:0 methyl
ester. The specific optical rotation of the novel monoenoic
aFAs showed slightly lower dextrorotation compared to the
saturated aFAs as methyl esters and free fatty acids.
of enantioselectivity of aFAs in biological systems. In addi-
tion to the saturated aFAs, our synthesis led for the first time
to enantiopure and racemic (n-5)-monoenoic aFAs. Re-
cently, 16:1n-5 and 14:1n-5 and further fatty acids with
double bonds between (n-7) and (n-4) were identified in
myxobacteria.19 As was mentioned above, a15:1 and a17:1
have been identified in food-related bacteria, but the posi-
tions of the double bonds have not yet been determined.20,21
The monoenoic aFAs synthesized in this study can be used
for a first screening of food samples for these compounds
along with unequivocal structure confirmation. Biosynthesis
of minute amounts of monoenoic aFAs has been linked with
bacterial desaturases.19 Therefore, these monoenoic aFAs
may be suitable markers for the (previous) presence of
bacteria in food.
The performed synthesis pathway is suitable for the pro-
duction of food-relevant aFAs. Both saturated and (n-5)-
cis-monoenoic (w90%) and (n-5)-trans-monoenoic forms
(w10%) were obtained as pure standards.
3. Conclusion
The synthesized (S)-(+)-enantiomers of a15:0, a16:0, and
a17:0 can be used in diverse studies to assess the significance

S. Thurnhofer, W. Vetter / Tetrahedron 63 (2007) 1140–1145
1143
4. Experimental
4.5. Synthesis of aFAs
4.1. General
4.5.1. Preparation of 2a–c. Triphenylphosphine (5.1 g,
0.019 mmol) was dissolved in 20 mL of freshly dried ACN
in a 100 mL round-bottomed flask. An equimolar amount
of u-bromoalkylcarboxylic acid 1a–c in 20 mL of ACN
was slowly added to the stirred triphenylphosphine solution.
The reagents readily dissolved and the orange colored reac-
tion mixture was heated under reflux for 48 h at 90 ꢁC under
argon. The solvent of the reaction mixture was then evapo-
rated and the (carboxyalkyl)triphenylphosphonium bromide
salts 2a–c were obtained as slightly yellow oils, which
proved insoluble in toluene (Fig. 1).44
€
Melting points were determined with a Buchi B-545 instru-
ment and are uncorrected. H NMR spectra were recorded
1
with a Varian Inova 300 MHz instrument in 5-mm tubes
(5–20 mg samples in 700 mL CDCl3). IR spectral data
were recorded with a Nicolet Avata 320 FT-IR spectrometer.
Hydrogenation was performed with an Anton Paar Synthos
3000 apparatus. Optical rotations were measured with a Per-
kin Elmer Model 341 digital polarimeter equipped with
a thermostat attuned to 20 ꢁC.
4.2. Materials and chemicals
4.5.2. Purification and isolation of 2a–c. Toluene (20 mL)
was added to each slight yellow oil and heated under reflux
for 30 min. The toluene phase was decanted and the cleaning
process was repeated with 20 mL of toluene. Afterwards,
30 mL of anhydrous diethyl ether was added to the oily resi-
due and recrystallization in the fridge resulted in a slightly
yellow solid. The obtained solid was triturated with diethyl
ether and the precipitate was filtered off and washed several
times with anhydrous diethyl ether. Drying overnight
in a vacuum centrifuge produced white powders of 2a–c
in 91–99% yields. Analytical data of 2a (8-carboxyoctyl)-
triphenylphosphonium bromide: yield 91%; melting point
114–115 ꢁC; IR spectral data (CH2Cl2, cmꢀ1): 2925, 2850,
9-Bromononanoic acid (99%) was purchased from TCI
(Zwijndrecht, Belgium). 10-Bromodecanoic acid and 11-
bromoundecanoic acid were obtained from Aldrich (both
99% purity, Taufkirchen, Germany). Triphenylphosphine,
n-butyllithium solution (2.5 M in n-hexane), sodium in
pieces, benzophenone, and anhydrous sodium sulfate were
from Fluka (Taufkirchen, Germany). (S)-(+)-3-Methylpenta-
nol (>98% purity) and racemic 3-methylpentanol (purity
>99%) were from TCI. Dess–Martin periodinane (DMP,
97% purity) was from Aldrich. Boron-trifluoride–metha-
nol-complex solution (13–15% BF3 in methanol) was from
1
€
Riedel-de-Haen (Seelze, Germany). Toluene, tetrahydrofu-
1710, 1435, 1110, 750, 720, 690; H NMR (CDCl3, ppm):
ran, and diethyl ether were from Roth (Karlsruhe, Germany).
n-Hexane (HPLC grade) and acetonitrile (HPLC grade) were
from Fisher Scientific (Schwerte, Germany). All solvents
used for the reactions were freshly dried over sodium or
molecular sieves and distilled.
7.70–7.86 (m, 15H); 3.71 (m, 2H); 2.41 (t, 2H); 1.30–1.62
(m, 12H). 2b (9-Carboxynonyl)triphenylphosphonium bro-
mide: yield 95%; melting point 93–99 ꢁC; IR spectral data
(CH2Cl2, cmꢀ1): 2925, 2850, 1715, 1435, 1110, 750, 720,
1
685; H NMR (CDCl3, ppm): 7.75–7.89 (m, 15H); 3.73
(m, 2H); 2.42 (t, 2H); 1.27–1.60 (m, 14H). 2c (10-Carboxy-
decyl)triphenylphosphonium bromide: yield 97%; melting
point 104–106 ꢁC; IR spectral data (CH2Cl2, cmꢀ1): 2920,
2850, 1710, 1435, 1110, 750, 725, 690; H NMR (CDCl3,
ppm): 7.75–7.89 (m, 15H); 3.75 (m, 2H); 2.42 (t, 2H);
1.29–1.64 (m, 16H).
4.3. Conversion of aFAs into aFAMEs
1
aFAs (4 mg) and 0.5 mL methanolic KOH (0.5 M) were
heated for 5 min at 80 ꢁC. After cooling, 1 mL methanolic
BF3-solution was added and heated for additional 5 min at
80 ꢁC. Then, 2 mL saturated sodium chloride solution and
2 mL n-hexane were added to the cooled solution (ice
bath). The hexane-phase containing the aFAMEs was sepa-
rated and analyzed by GC/EI-MS.43
4.5.3. Oxidation of 3-methylpentanol 4 to 3-methylpenta-
nal 5. Dess–Martin periodinane (3.52 g, 8.29 mmol) was
dissolved in 10 mL of anhydrous methylene chloride. Under
stirring, a solution of 3-methylpentanol 4 (1 mL, 8.06 mmol)
in 10 mL of anhydrous methylene chloride was added
dropwise (Fig. 1). The solution was kept for 1 h at room tem-
perature. Diethyl ether (50 mL) was added, and the reaction
mixture was transferred to a separating funnel. Then, 50 mL
of a 1:1 (v/v) mixture of aqueous 1 M sodium thiosulfate and
a solution of saturated sodium hydrogencarbonate was
added. After 10 min of vigorous shaking, the organic phase
was separated and washed with 50 mL of saturated sodium
hydrogencarbonate solution and 50 mL of distilled water.
The organic phase was dried over anhydrous sodium sulfate,
and the organic solvent was carefully removed by micro-
distillation.29 (S)-(ꢀ)-3-Methylpentanal was obtained in the
above-mentioned way by using (S)-(+)-3-methylpentanol.
The yield was 98%. The identity and purity of the obtained
(S)-(ꢀ)-3-methylpentanal 5 were proved by GC/EI-MS, IR,
and 1H NMR.30 Analytical data of 5: IR spectral data
(CH2Cl2, cmꢀ1): 2960, 2930, 2880, 1720. GC/EI-MS (m/z):
[M]+ 100(1), 82(3), 71(18), 56(100), 43(48), 41(73), 29(42),
4.4. Gas chromatography with electron ionization mass
spectrometry (GC/EI-MS)
A Hewlett–Packard 5890 series II gas chromatograph was
used in combination with a 5971A mass selective detector.
Sample solution (1 mL) was injected with a 7673A auto-
sampler (splitless mode, split opened after 2 min). The injec-
tor and transfer line temperatures were kept at 250 ꢁC and
280 ꢁC. Analyses were performed on a 60 mꢂ0.25 mm i.d.
fused-silica capillary column (CP Sil 88, Supelco, Tauf-
kirchen, Germany). The carrier gas helium (purity 5.0) was
used at a constant flow rate of 1 mL/min. The GC oven
program started at 60 ꢁC (hold time 1 min) was then raised
at 7 ꢁC/min to 180 ꢁC (hold time 2 min), then at 3 ꢁC/min
to 200 ꢁC (hold time 2 min), and finally at 3 ꢁC/min to
220 ꢁC (hold time 10 min). Mass spectra (m/z 50–450)
were recorded at a rate of five scans per second with an ion-
ization energy of 70 eV. The temperature of the ion source
was 170 ꢁC.
1
15(4); H NMR (CDCl3, ppm): 9.80 (s, 1H), 2.40 (m, 2H),

1144
S. Thurnhofer, W. Vetter / Tetrahedron 63 (2007) 1140–1145
1.50–1.00 (m, 9H). Optical rotation [a]2D0 ꢀ2.6 (c 2.1 g/
by GC/EI-MS. The fractionation was repeated several times
and the corresponding fractions were pooled to obtain
sufficient material for further analyses. Analytical data of
cis-(S)-(+)-6a ME: GC/EI-MS (m/z): [M]+ 254(2.0),
[Mꢀ61]+ 193(5.2), [Mꢀ79]+ 175(3.3), 153(14.2), 55(100);
1H NMR (CDCl3, ppm): 5.41–5.34 (m, 2H), 3.68 (s, 3H),
2.34–2.29 (t, 2H), 2.06–1.99 (m, 4H), 1.92–1.85 (m, 2H),
1.65–1.60 (m, 2H), 1.44–1.20 (m, 8H), 1.17–1.13 (m, 1H),
0.91–0.86 (m, 6H). trans-(S)-(+)-6a ME: GC/EI-MS (m/z):
[M]+ 254(2.4), [Mꢀ61]+ 193(5.0), [Mꢀ79]+ 175(1),
153(11.7), 55(100). cis-(S)-(+)-6b ME: GC/EI-MS (m/z):
[M]+ 268(3.5), [Mꢀ61]+ 207(7.1), [Mꢀ79]+ 189(5.7),
167(15.7), 55(100); 1H NMR (CDCl3, ppm): 5.41–5.33
(m, 2H), 3.68 (s, 3H), 2.34–2.29 (t, 2H), 2.06–1.99 (m,
4H), 1.92–1.85 (m, 2H), 1.65–1.60 (m, 2H), 1.44–1.20 (m,
8H), 1.17–1.13 (m, 1H), 0.91–0.86 (m, 6H). trans-(S)-(+)-
6b ME: GC/EI-MS (m/z): [M]+ 268(3.9), [Mꢀ61]+
207(3.9), [Mꢀ79]+ 189(3.4), 167(9.4), 55(100). cis-(S)-
(+)-6c ME: GC/EI-MS (m/z): [M]+ 282(3.4), [Mꢀ61]+
100 mL in CH2Cl2).
4.5.4. Wittig-coupling of the aldehyde 5 with 2a–c. Each
(carboxyalkyl)triphenylphosphonium bromide salt 2a–c
(1.0 mmol) was dissolved in 100 mL of freshly distilled
tetrahydrofuran (THF). The solution was cooled to 0–5 ꢁC.
Then a solution of n-BuLi (2.5 M in n-hexane, 0.63 mmol)
in 5 mL THF was added dropwise to produce the bright or-
ange ylide 3 within 20 min at 0–5 ꢁC.45 3-Methylpentanal
or (S)-(ꢀ)-3-methylpentanal 5 (1.0 mmol), dissolved in 5 mL
of THF, was added dropwise and the solution was stirred
for 4 h at room temperature. Afterwards, the mixture was
poured on ice and acidified with 1 M HCl. The aqueous solu-
tion was extracted several times with 20 mL of diethyl ether
and the combined organic phases were washed twice with
saturated sodium chloride solution. The organic phase was
dried over anhydrous sodium sulfate and filtered. After
removal of the solvent, the monoenoic aFAs 6a–c were
obtained as weak yellow oils. For purification,w60% of the
impure oils of aFAs 6a–c were eluted from silica gel with
petroleum ether–ethyl acetate (3:1; v/v). 54.0 mg (36%
yield) of aFA 6a, 51.0 mg (33.8% yield) of aFA 6b, and
70.5 mg (47.0% yield) of aFA 6c were obtained as colorless
oils. The resulting mixtures of cis- and trans-isomers
contained 88.6% (6a), 90.4% (6b), and 91.1% (6c) of the
cis-isomer.
1
221(6.8), [Mꢀ79]+ 203(4.6), 181(12.2), 55(100); H NMR
(CDCl3, ppm): 5.41–5.34 (m, 2H), 3.68 (s, 3H), 2.34–2.29
(t, 2H), 2.06–1.99 (m, 4H), 1.92–1.85 (m, 2H), 1.65–1.60
(m, 2H), 1.44–1.20 (m, 8H), 1.18–1.13 (m, 1H), 0.91–0.86
(m, 6H). trans-(S)-(+)-6c ME: GC/EI-MS (m/z): [M]+
282(2.0), [Mꢀ61]+ 221(5.3), [Mꢀ79]+ 203(6.3), 181(9.5),
1
55(100). The H NMR data of racemic and enantiopure
cis-monoenoic aFAs were identical.
1H NMR (CDCl3, ppm): (S)-(+)-6a: 5.43–5.37 (m, 2H),
2.40–2.35 (t, 2H), 2.10–2.00 (m, 4H), 1.96–1.86 (m, 2H),
1.69–1.60 (m, 2H), 1.47–1.23 (m, 8H), 1.20–1.17 (m, 1H),
0.99–0.88 (m, 6H). (S)-(+)-6b: 5.45–5.37 (m, 2H),
2.41–2.36 (t, 2H), 2.10–2.02 (m, 4H), 1.94–1.85 (m, 2H),
1.69–1.62 (m, 2H), 1.48–1.23 (m, 10H), 1.20–1.13 (m,
1H), 0.99–0.89 (m, 6H). (S)-(+)-6c: 5.44–5.38 (m, 2H),
2.41–2.36 (t, 2H), 2.10–2.02 (m, 4H), 1.94–1.87 (m, 2H),
1.72–1.65 (m, 2H), 1.47–1.23 (m, 12H), 1.20–1.15
4.5.6. Catalytic hydrogenation of the monoenoic aFAs
6a–c to the saturated aFAs 7a–c. The saturated aFAs
7a–c were prepared by catalytic hydrogenation of 30 mg of
monoenoic aFAs 6a–c in 30 mL of absolute methanol
containing 10% palladium on charcoal (10% Pd/C). The
suspension was stirred for 30 min in an H2 atmosphere
(4.8 bar).18,21 The hydrogenated product was filtered through
Celite 545 to remove the catalyst. The resulting solution was
concentrated and analyzed by GC/EI-MS after esterification.
Approximately 30 mg 7a–c corresponding to 100% yield
were obtained (Table 1). IR spectral data (CHCl3, cmꢀ1):
(S)-(+)-7a–c ME: 2927, 2854, 1732. (S)-(+)-7a ME: GC/
EI-MS (m/z): [M]+ 256(1.3), [Mꢀ29]+ 227(1.1), [Mꢀ43]+
1
(m, 1H), 0.99–0.89 (m, 6H); H NMR data of racemic and
enantiopure cis-/trans-monoenoic aFAs were identical.
Despite the dominance of cis-isomers, the presence of
w10% of trans-isomers caused a slight downfield shift
1
1
(w0.02–0.03 ppm) of the olefinic protons in the H NMR
213(4.5), 87(58.8), 74(100); H NMR (CDCl3, ppm): 3.68
spectra. Thus, NMR data is only reported for the neat cis-
monoenoic aFAs obtained after separation of the trans-
isomers (Section 4.5.5).
(s, 3H), 2.34–2.29 (t, 2H), 1.63–1.60 (m, 2H), 1.44–1.27
(m, 18H), 1.15–1.10 (m, 1H), 0.89–0.84 (m, 6H). (S)-(+)-
7b ME: GC/EI-MS (m/z): [M]+ 270(3.4), [Mꢀ29]+
1
241(1.2), [Mꢀ43]+ 227(3.4), 87(58.8), 74(100); H NMR
4.5.5. Separation of cis- and trans-monoenoic aFA 6a–c
by silver-ion high-performance liquid chromatography
(AgD-HPLC). Ag+-HPLC analyses were performed using
a Varian solvent pump equipped with a Rheodyne 7010
injector fitted with a 100 mL loop and a Varian ProStar 325
UV–vis detector with dual wavelength mode set at 206
and 234 nm. After converting the monoenoic aFAs 6a–c
into fatty acid methyl esters (FAMEs), 100 mg of cis- and
trans-monoenoic aFAs in 50 mL n-hexane were injected
and separated using a ChromSpher 5 Lipids column
(250 mmꢂ4.6 mm i.d. stainlees steel, a silver-modified
cation exchange ligand-covered 5 mm spherical silica col-
umn; Varian, Darmstadt, Germany). An isocratic solvent
system of n-hexane with 0.08% ACN was used as mobile
phase at a flow rate of 1.0 mL/min.38 The fractions con-
taining the cis- and trans-monoenoic aFAs 6a–c were
collected with a 701 fraction collector (Varian) and analyzed
(CDCl3, ppm): 3.68 (s, 3H), 2.34–2.29 (t, 2H), 1.65–1.60
(m, 2H), 1.44–1.27 (m, 20H), 1.17–1.10 (m, 1H), 0.89–
0.84 (m, 6H). (S)-(+)-7c ME: GC/EI-MS (m/z): [M]+
284(3.7), [Mꢀ29]+ 255(1.5), [Mꢀ43]+ 241(4), 87(58.9),
1
74(100); H NMR (CDCl3, ppm): 3.68 (s, 3H), 2.34–2.29
(t, 2H), 1.65–1.60 (m, 2H), 1.44–1.27 (m, 22H), 1.15–1.10
(m, 1H), 0.89–0.84 (m, 6H). The H NMR data of racemic
1
and enantiopure aFAs 7a–c were identical and congruent
with the commercially available racemic standards.
Acknowledgements
We are grateful for the financial support from the German
€
Research Foundation (DFG). We thank Gerhard Grobner
and Fredrick Lindstrom from the Department of Chemistry
€
˚
(Biophysical Chemistry), University of Umea in Sweden

S. Thurnhofer, W. Vetter / Tetrahedron 63 (2007) 1140–1145
1145
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