A new short synthesis of 10R-tuberculostearic acid and its enantiomer

Article abstract of DOI:10.1016/j.chemphyslip.2006.03.006

Tuberculostearic acid (10R-methyloctadecanoic acid) and its 10S-enantiomer were synthesised by a chiral pool strategy, in four steps from citronellyl bromide.

Full text of DOI:10.1016/j.chemphyslip.2006.03.006

Chemistry and Physics of Lipids 142 (2006) 111–117
A new short synthesis of 10R-tuberculostearic
acid and its enantiomer
Ieuan O. Roberts, Mark S. Baird∗
Department of Chemistry, University of Wales, Bangor, Gwynedd, Wales LL57 2UW, UK
Received 17 December 2005; received in revised form 11 March 2006; accepted 16 March 2006
Available online 29 March 2006
Abstract
Tuberculostearic acid (10R-methyloctadecanoic acid) and its 10S-enantiomer were synthesised by a chiral pool strategy, in four
steps from citronellyl bromide.
© 2006 Elsevier Ireland Ltd. All rights reserved.
Keywords: Tuberculostearic; Tuberculostearate; 10R-Methyloctadecanoic; 10R-Methyloctadecanoate; Tuberculosis
1. Introduction
with stearic acid, it was named tuberculostearic acid.
Investigations into the constitution of other mycobac-
teria showed that TBSA was present in Mycobacterium
bovis (Burt and Anderson, 1931), Mycobacterium leprae
(Anderson and Uyei, 1932), and Mycobacterium “timo-
thy” (phlei) (Pangborn and Anderson, 1931; Pangborn
et al., 1932) but not in Mycobacterium avium (Anderson
and Gilman Roberts, 1930). The study involving M. lep-
rae (Anderson and Uyei, 1932) was the first to show
better agreement to the molecular formula C19H38O2,
though the authors were reluctant to suggest this as
the true formula due to the scarcity of natural fatty
acids possessing odd numbers of carbon atoms known
at that time. The formula was confirmed as C19H38O2
in 1934 (Spielman, 1934); drastic oxidation of TBSA
and analysis of the products led to the proposal of the
structure 10-methylstearic acid (10-methyloctadecanoic
acid). The same study saw the first synthesis of dl-
10-methyloctadecanoic acid, which shared many prop-
erties with the natural sample. Some differences were
observed, which were explained by the suggestion that
TBSA may possess an asymmetric carbon, where the
resultant optical rotation was too small to measure.
During an ongoing project which has seen the synthe-
sis of various naturally occurring chiral fatty acids and
related compounds, including lactobacillic acid (Coxon
et al., 1999, 2003), the enantiomer of grenadamide (Al-
Dulayymi et al., 2004), cascarillic acid (Roberts et al.,
2004), and single enantiomers of ␣-mycolic acids (Al-
Dulayymi et al., 2000), a synthesis of the related methyl-
branched tuberculostearic acid and its enantiomer has
now been completed.
Tuberculostearic acid (TBSA), or 10R-methylstearic
acid, was first reported in 1927 (Anderson, 1927), iso-
lated from the phosphatide fraction of the H.37 strain
of Mycobacterium tuberculosis. A subsequent isola-
tion (Anderson and Chargaff, 1929) and analysis of
the methyl ester, free acid and the silver salt deter-
mined the substance to be optically inactive, and the
formula as C18H36O2, thus, as it was deemed isomeric
∗
Corresponding author. Tel.: +44 1248 382375.
E-mail address: m.baird@bangor.ac.uk (M.S. Baird).
0009-3084/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.chemphyslip.2006.03.006

112
I.O. Roberts, M.S. Baird / Chemistry and Physics of Lipids 142 (2006) 111–117
A new synthesis of racemic TBSA as well as a route
al., 2005). The mode of action of the effective anti-TB
drug, isoxyl, has also been linked to a dose depen-
dent decrease in the synthesis of TBSA and mycolic
acids (Phetsukiri et al., 2003). Early work suggested
that TBSA stimulates the proliferation of monocytes
and epithelial cells and that subcutaneous injection
into healthy animals led to massive tubercular tissue
(Anderson and Chargaff, 1929), while the sodium salt
was reported to act as a bactericide against “Bacillus lep-
rae” (Sabin and Doan, 1927). Moreover, phospholipids
containing racemic TBSA and related branched stearic
acids have been of interest in relation to their chain pack-
ing properties (Menger et al., 1988a,b). In the case of
biological activity, it is clear that this may be affected
by the absolute stereochemistry of TBSA; in the case
of chain packing, the examples reported using racemic
TBSA will clearly involve a combined effect of a number
of diastereomers while in natural membranes any effect
will derive from single enantiomers. We therefore now
report a simple new synthesis of 10-R and 10-S-TBSA,
from readily available starting materials of known stere-
ochemistry and confirm that the former is identical to the
natural material.
to the two single enantiomers of TBSA were reported
in 1948 (Prout et al., 1947, 1948), and it was found
that the single enantiomer possessing a negative [α]D24
(−0.05 to −0.09) was identical in all respects to the
natural compound. TBSA was thus assigned to the L
series. The route involved the resolution of 2-decanol
with brucine followed by six steps to reach the ethyl
ester of TBSA, and included two stages at which a
nucleophilic substitution could lead to partial loss of
stereochemistry. The following years saw two further
syntheses of racemic TBSA, one by ‘normal’ chemical
synthesis involving the coupling of the Grignard reagent
derived from 2-bromodecane with ClCO(CH2)7COOEt
(Schmidt and Shirley, 1949), and the other by elec-
trochemical methods (Linstead et al., 1951). The lat-
ter method could also be used to prepare the single
enantiomers. Thus the electrochemical coupling of the
resolved mono-methyl ester of 3-methylglutaric acid,
firstly with octanoic acid and secondly the mono-methyl
ester of non-anedioc acid, was applied to both enan-
tiomers. However, purification was relatively complex.
TBSA has been prepared most recently in racemic form
(Wallace et al., 1994).
The detection of TBSA in biological fluids has been
proposed as a method for detecting tuberculosis (Odham
et al., 1979; Larsson et al., 1980; Brooks et al., 1987;
French et al., 1987a,b; Daikos et al., 2004; Stopforth et
2. Results and discussion
A straightforward scheme of synthesis was devised,
using S and R citronellyl bromides (1) and (6) as the
Scheme 1. The synthetic route to TBSA and its enantiomer. (i) CH3(CH2)5MgBr, LiCuCl4, (−78 ◦C) THF; (ii) O3, CH2Cl2 (−78 ◦C), PPh3; (iii)
Br−P+Ph3(CH2)5COOH, LiHMBS (1.03 M in THF), toluene/DMSO; (iv) Pd (10% on carbon), H2, MeOH.

I.O. Roberts, M.S. Baird / Chemistry and Physics of Lipids 142 (2006) 111–117
113
starting materials for 10-R- and 10-S-TBSA, respec-
tively.
lowed by charring. Column chromatography was per-
formed using Fluorochem silica gel 60 (35–70 ␮m)
for flash chromatography (Fluorochem: LC201) eluted
with mixtures of petroleum ether b.p. 40–60 ◦C (petrol)
and Et2O. Starting materials and chemical reagents
were purchased from Aldrich, Avocado and Lancaster
Synthesis.
Dry solvents: THF was used freshly drawn from an
in-house still working according to literature methods
(Furniss et al., 1989); dry toluene was prepared in-
house according to literature methods (Furniss et al.,
1989); dry DMSO was supplied by Aldrich. Solutions in
organic solvents were dried over anhydrous magnesium
sulfate.
A simple Grignard reaction between citronellyl bro-
mide (1) and hexyl magnesium bromide in the presence
of LiCuCl4 produced (2), containing the octyl chain of
the target molecule; the alkene was isolated pure, in good
yield after two fractional distillations and flash column
chromatography. Ozonolysis of the alkene formed the
aldehyde (3), which was quickly coupled via a Wittig
reaction with an acid functionalised phosphonium salt,
to give a diastereomeric mixture of alkenes (4), complet-
ing the carbon skeleton of the product.
Hydrogenation of (4) afforded the free acid (5),
which was converted into the methyl ester by reac-
tion with diazomethane for analytical purposes. 10-
R-TBSA (5) and its methyl ester (5a) were found to
have optical rotations of −0.34 and −0.45, respectively.
In the same way, 10-S-TBSA (10) was prepared from
(R)-citronellyl bromide (6) via the alkene (7) (previ-
ously prepared by reaction of (R)-citronellyl tosylate
with related organometallic species (Mori et al., 1978;
Mori and Tamada, 1979; Kikukawa et al., 1982, 1984))
and the aldehyde (8) (previously reported by ozonoly-
sis of (7) (Odinokov et al., 1992)); the free acid (10)
and the corresponding methyl ester (10a) had optical
rotations of +0.21 and +0.33, in reasonable agreement
with published data for the synthetic material (+0.07
to +0.09) (Prout et al., 1948; Linstead et al., 1951).
Though the values of the rotations are small, they are
convincing due to the fact that the sign of the rotation
changed in the final hydrogenation in both cases; (R)-10-
methyloctadec-6-enoic acid has an [α]2D5 = +2.2, and
gave the levo rotatory product on hydrogenation, (S)-10-
methyloctadec-6-enoic acid possesses [α]2D5 = −3.08
and afforded the dextro rotatory product. This rever-
sal in the signs of the rotations makes it most unlikely
that the final small values are erroneous in any way
(Scheme 1).
3.1. (R)-2,6-Dimethyltetradec-2-ene (2)
All glassware and metal equipment used for the pro-
cedure outlined below were oven dried at 180 ◦C for
atleast48 hpriortouse;septaandplastic-wareweredried
at 90–100 ◦C for the same period. All procedures took
place in an argon environment.
Magnesium turnings (0.72 g, 30 mmol) were refluxed
in dry THF (20 ml) for 30 min. A solution of 1-
bromohexane (3.76 g, 23 mmol) in dry THF (20 ml) was
added dropwise to the suspension over 40 min, with
additional heating via a heat gun during this period;
effervescence of hydrogen was observed after each addi-
tion. The solution was refluxed for 1 h. A sample (1 ml)
was quenched with water (1 ml), extracted with Et2O
(1 ml), dried, and tested by GC for the absence of 1-
bromohexane.
A stirred solution of S-citronellyl bromide (1,
Aldrich) (1.0 g, 4.6 mmol) in dry THF (10 ml) was
cooled to −78 ◦C, and the THF solution of hexyl mag-
nesium bromide was transferred into the same ves-
sel and the resultant mixture was cooled to −78 ◦C.
LiCuCl4 (1.0 M in THF, 0.8 ml) was added in one por-
tion and the temperature was seen to rise to −71 ◦C. The
solution was left in the cooling bath overnight, during
which it slowly warmed to ambient temperature. The
bath was removed and the solution stirred for a further
24 h. The reaction was quenched with sat. aq. NH4Cl
(30 ml), the resultant bright blue solution was extracted
with Et2O (3 ml × 50 ml). The combined organic phases
were washed with brine (50 ml), dried and concentrated
by rotary evaporation. The crude product contained
a small amount of an impurity believed to be dode-
cane, which was removed by distillation. Column chro-
matography (petrol/Et2O 9:1) of the residue afforded
pure (R)-2,6-dimethyltetradec-2-ene (2), a colourless oil
(0.98 g, 95%). Compound 2 showed [α]2D5 = +1.15 (c
3. Experimental
1H and 13C NMR spectra were recorded on a Bruker
␭500 NMR spectrometer in CDCl3 at 500 and 125 MHz,
respectively. IR spectra were recorded using a Perkin-
Elmer 1600 FTIR infrared spectrometer. Mass spectra
were recorded on a Micromass GCT instrument. Opti-
cal rotations were obtained using an Optical Activity
POLAAR 2001 automatic polarimeter. Thin layer chro-
matography was performed using Merck Silica gel 60
F254 Glass backed plates (Aldrich Z293024), separated
components were detected using variously UV light,
I2; and 10% ethanolic phosphomolybdic acid acid fol-

114
I.O. Roberts, M.S. Baird / Chemistry and Physics of Lipids 142 (2006) 111–117
1.3, CHCl3); Found M+, 224.2492, C16H32 requires
224.2504; IR (Neat) νmax 2957, 2924, 2853, 1456 and
J = 6.3 Hz); 13C NMR (CDCl3, 125 MHz) δ: 203.2, 41.8,
36.8, 32.5, 32.0, 30.0, 29.8, 29.5, 29.0, 27.1, 22.8, 19.5,
14.2.
1377 cm−1; H NMR (CDCl3, 500 MHz) δ: 5.12 (br t,
1
1× CH, 1H, J = 7.1 Hz, includes J = 1.3 Hz), 1.98 (m,
CH2, 2H), 1.70 (s, CH3, 3H), 1.62 (s, CH3, 3H), 1.4–1.2
(m, 7× CH2 + 1× CH, 15H), 1.12 (m, CH2, 2H), 0.90
(t, CH3, 3H, J = 7.0 Hz), 0.87 (d, CH3, 3H, J = 6.7 Hz);
13C NMR (CDCl3, 125 MHz) δ: 131.0, 125.3, 37.3, 37.2,
32.6, 32.1, 30.2, 29.9, 29.5, 27.2, 25.9, 25.8, 22.9, 19.8,
17.8, 14.3.
3.4. (S)-4-Methyldodecanal (8)
The reaction above was repeated but using, (S)-2,6-
dimethyltetradec-2-ene (7) (0.484 g, 2.16 mmol), PPh3
(0.57 g, 2.16 mmol) and gave (S)-4-methyldodecanal
(8) a fragrant pale yellow oil (Odinokov et al., 1992)
(0.377 g, 88%). Compound 8 showed [α]2D5 = −1.82
(c 1.10, CHCl3); IR (Neat) νmax 2955, 2924, 2854
3.2. (S)-2,6-Dimethyltetradec-2-ene (7)
and 1727 cm−1; H NMR (CDCl3, 500 MHz) δ: 9.78
1
The reaction was completed as above, using
magnesium (0.83 g, 34 mmol), 1-bromohexane (3.76 g,
23 mmol) and R-citronellyl bromide (6, Aldrich) (0.65 g,
2.7 mmol) to give (S)-2,6-dimethyltetradec-2-ene (7),
a colourless liquid (0.57 g, 93%). Compound 7 showed
[α]2D5 = −2.0 (c 1.26, CHCl3) (lit. −1.7 (c 5.5, hexane
(Kikukawa et al., 1982, 1984); −1.5 (c 1.85, CHCl3
(Odinokov et al., 1992)); Found M+, 224.2513, C16H32
requires 224.2504; IR (Neat) νmax 2957, 2921, 2852,
(t, CHO, 1H, J = 1.9 Hz), 2.42 (m, poorly resolved
(expect dddd), CH2, 2H), 1.66 (m including dq, CH,
1H, J = 8.6, 8.5 Hz), 1.44 (m, CH2, 2H), 1.27 (m, 7×
CH2, 1× CH3, 17H), 0.89 (t, CH3, 3H, J = 6.6 Hz);
13C NMR (CDCl3, 125 MHz) δ: 203.1, 41.8, 36.8,
32.5, 32.0, 30.0, 29.8, 29.5, 29.0, 27.1, 22.8, 19.5,
14.2.
1
1461 and 1376 cm−1; H NMR (CDCl3, 500 MHz) δ:
3.5. (R)-10-Methyloctadec-6-enoic acid (4)
5.12 (br t, 1× CH, 1H, J = 7.1 Hz, includes J = 1.3 Hz),
1.98 (m, apparent 10 lines, 1× CH2, 2H), 1.70 (s, CH3,
3H), 1.62 (s, CH3, 3H), 1.4–1.2 (m, 7× CH2 + 1×
CH, 15H), 1.12 (m, CH2, 2H), 0.90 (t, CH3, 3H,
J = 7.0 Hz), 0.87 (d, CH3, 3H, J = 6.6 Hz); 13C NMR
(CDCl3, 125 MHz) δ: 131.0, 125.3, 37.3, 37.2, 32.6,
32.1, 30.2, 29.9, 29.5, 27.2, 25.9, 25.8, 22.9, 19.8, 17.8,
14.3.
(5-Carboxypentyl)triphenylphosphonium bromide
(3.8 g, 8.3 mmol) was dissolved in 3:1 dry toluene/dry
DMSO (75 ml). The solution was cooled to 0 ◦C
and lithium bis-trimethylsilyl amide (1.06 M in THF,
16.4 ml, 17 mmol) was slowly added, maintaining a
temperature of less than 0.3 ◦C. The resultant bright
reddish orange solution was warmed to ambient
temperature over 3 h and stirred at ambient for a
further hour. The solution was cooled to −15 ◦C and
(R)-4-methyldodecanal (3) (0.78 g, 3.92 mmol) was
added as a solution in toluene (8 ml), the temperature
was seen to rise to −10 ◦C on this addition. The solution
was allowed to slowly return to ambient temperature
and was stirred for 48 h. Saturated NH4Cl(aq) (50 ml)
was added, and the mixture was extracted with Et2O
(4 ml × 100 ml), the combined organic phases were
washed with saturated brine (50 ml), dried, and concen-
trated by rotary evaporation. Column chromatography
(petrol/Et2O 1:1) yielded (R)-10-methyloctadec-6-enoic
acid (4) as a yellow oil (0.76 g, 65%) which was an ca.
4:1 mixture of Z and E isomers. Compound 4 showed
[α]2D5 = +2.1 (c 1.23, CHCl3); Found M+, 296.2724,
C19H36O2 requires 296.2715; IR (Neat) νmax 3000
3.3. (R)-4-Methyldodecanal (3)
A stirred solution of (R)-2,6-dimethyltetradec-2-ene
(2) (0.93 g, 4.15 mmol) in CH2Cl2 (40 ml) was cooled
to −78 ◦C prior to treatment with O3 until a blue
colour was seen to persist in the cooled solution.
N2(g) was subsequently bubbled through the solution
to remove excess O3 and avoid danger on warming.
PPh3 (1.09 g, 4.15 mmol) was added to the cold solu-
tion, which was warmed to ambient temperature and
stirred for 1 h. The solvent was removed by rotary
evaporation. Column chromatography (petrol/Et2O 8:2)
on the crude product gave (R)-4-methyldodecanal (3)
a fragrant pale yellow oil (0.78 g, 92%). Compound
3 showed [α]2D5 = +1.40 (c 0.93, CHCl3); IR (Neat)
νmax 2926, 2854 and 1727 cm−1
;
1H NMR (CDCl3,
(br) 2925, 2853 and 1710 cm−1 1H NMR (CDCl3,
;
500 MHz) δ: 9.77 (t, CHO, 1H, J = 1.9 Hz), 2.42 (dddd,
CH2, 2H, J = 1.9, 5.7, 11.1, 17.1 Hz), 1.65 (m includ-
ing dq, CH, 1H, J = 9.5, 8.7 Hz), 1.42 (m, CH2, 2H),
1.26 (m, 7× CH2, 1× CH3, 17H), 0.88 (t, CH3, 3H,
500 MHz) δ: 5.35, (m, 2× CH, 2H), 2.36 (t, CH2, 2H,
J = 7.4 Hz), 2.03 (m, 2× CH2, 4H) 1.66 (m, td apparent,
CH2, 2H, J = 7.7, 7.6 Hz), 1.41 (m, CH2, 2H), 1.27 (m,
7× CH2, 1× CH, 15H), 1.13 (m, CH2, 2H), 0.89 (t,

I.O. Roberts, M.S. Baird / Chemistry and Physics of Lipids 142 (2006) 111–117
115
CH3, 3H, J = 6.9 Hz), 0.86 (d, CH3, 3H, J = 6.3 Hz);
3.8. (S)-10-Methyloctadec-6-enoic acid methyl
ester (9a)
13C NMR (CDCl3, 125 MHz) (major isomer) δ: 180.3,
130.9, 128.9, 37.15, 37.09, 34.1, 32.5, 32.1, 30.2,
29.8, 29.5, 29.2, 27.2, 26.9, 24.9, 24.4, 22.8, 19.7,
14.2.
The reaction above was repeated with (S)-10-
methyloctadec-6-enoic acid (0.09 g, 0.30 mmol) (9) to
give (S)-10-methyloctadec-6-enoic acid methyl ester
(9a) as a slightly yellow oil (0.079 g, 85%) as an
ca. 4:1 mixture of Z and E isomers. Compound 9a
showed [α]2D5 = −3.08 (c 3.05, CHCl3); Found M+,
310.2877, C20H38O2 requires, 310.2872; IR (Neat) νmax
3.6. (S)-10-Methyloctadec-6-enoic acid (9)
The reaction above was repeated using (5-
carboxypentyl)triphenylphosphonium bromide (1.98 g,
4.32 mmol), lithium bis-trimethylsilyl amide (1.06 M in
THF, 9.5 ml, 9.50 mmol) and (S)-4-methyldodecanal (8)
(0.377 g, 1.90 mmol) to give (S)-10-methyloctadec-6-
enoic acid (9) as a yellow oil (0.24 g, 44%) again as
an ca. 4:1 mixture of Z and E isomers. Compound
9 showed [α]2D5 = −2.28 (c 1.28, CHCl3); Found M+,
296.2723, C19H36O2 requires 296.2715; IR (Neat) νmax
1
2954, 2926, 2855 and 1741 cm−1; H NMR (CDCl3,
500 MHz) δ: 5.34 (m, 2× CH, 2H), 3.66 (s, CH3, 3H)
2.31 (t, CH2, 2H, J = 7.6 Hz), 2.02 (m, 2× CH2, 4H),
1.64 (m, CH2, 2H, includes J = 7.6 Hz), 1.38 (m, CH2,
2H), 1.18–1.32 (m, 7× CH2, 1× CH, 15H), 1.11 (m,
CH2, 2H), 0.86 (m, 2× CH3, 6H); 13C NMR (CDCl3,
125 MHz) (major isomer) δ: 174.30, 130.81, 129.04,
51.57, 37.18, 37.12, 34.15, 32.56, 32.07, 30.17, 29.83,
29.51, 29.38, 27.19, 26.96, 24.95, 24.75, 22.83, 19.71,
14.25.
3000 (br), 2927 and 1708 cm−1 1H NMR (CDCl3,
;
500 MHz) δ: 5.36 (m, 2× CH, 2H), 2.37 (t, CH2, 2H,
J = 7.6 Hz), 2.04 (m, 2× CH2, 4H), 1.66 (m, td appar-
ent, CH2, 2H, J = 7.7, 7.6 Hz), 1.41 (m, CH2, 2H), 1.27
(m, 7× CH2, 1× CH, 15H), 1.14 (m, CH2, 2H), 0.89 (t,
3.9. (R)-10-Methyloctadecanoic acid (5)
CH3, 3H, J = 7.1 Hz), 0.87 (d, CH2, 2H, J = 6.7 Hz); 13
C
NMR (CDCl3, 125 MHz) (major isomer) δ: 180.1, 130.9,
128.9, 37.2, 37.1, 34.1, 32.5, 32.1, 30.2, 29.8, 29.5, 29.2,
27.2, 26.9, 24.9, 24.4, 22.8, 19.7, 14.2.
Palladium on carbon (10%, 0.2 g) was slowly added
under a stream of N2(g) to a stirred solution of (R)-10-
methyloctadec-6-enoic acid (4) (0.2182 g, 0.73 mmol) in
MeOH (20 ml). The flask was connected to a hydrogena-
tion apparatus which was purged of any air by repeated
application of vacuum followed by refilling of the sys-
tem with H2(g), the cycle was finished with the system
full of H2(g). The reaction was monitored by observing
the amount of H2(g) absorbed via a burette that is part
of the apparatus. After 70 min the burette reading was
steady, indicating that the reaction was complete. The
reaction mixture was filtered through a pad of Celite®,
which was washed with copious MeOH. The solvent
was removed by rotary evaporation yielding 0.2267 g
of crude product, which was suspended in CH2Cl2
(30 ml) washed with brine (10 ml), dried and concen-
trated once more, yielding (R)-10-methyloctadecanoic
acid (5) (0.2067 g, 94%). Compound 5 was observed to
be a slightly yellow oil at room temperature, solidify-
ing to an off white, waxy solid at 12–13 ◦C. Compound
5 showed [α]2D3 = −0.34 (c 0.88, CHCl3); Found M+,
298.2863, C19H38O2 requires, 298.2872; IR (Neat) νmax
3000 (br), 2922, 2853, 1712 and 1464 cm−1; 1H NMR
(CDCl3, 500 MHz) δ: 2.35 (t, CH2, 2H, J = 6.8 Hz), 1.64
(m, td apparent, CH2, 2H, J = 6.9, 7.1 Hz), 1.27 (m,
12× CH2, 1× CH, 25H), 1.08 (m, CH2, 2H), 0.89 (t,
CH3, 3H, J = 6.9 Hz), 0.84 (d, CH3, 3H, J = 6.5 Hz);
13C NMR (CDCl3, 125 MHz) δ: 180.5, 37.24, 37.22,
34.3, 32.9, 32.1, 30.2, 30.1, 29.8, 29.6, 29.5, 29.4,
3.7. (R)-10-Methyloctadec-6-enoic acid methyl
ester (4a)
(R)-10-Methyl-octadec-6-enoic acid (4) (0.1 g,
0.34 mmol), was dissolved in Et2O (1 ml). An ethereal
solution of diazomethane (1.5 ml) was added dropwise,
gentle effervescence was observed for 5 min. The
solution was stirred for a further hour. The solvents
were removed by rotary evaporation. The crude (R)-
10-methyloctadec-6-enoic acid methyl ester (4a) was
purified by column chromatography (petrol/Et2O 97.5:
2.5) to yield the pure product as a slightly yellowish oil
(0.092 g, 87%) as an ca. 4:1 mixture of Z and E isomers.
Compound 4a showed [α]2D5 = +2.2 (c 1.5, CHCl3);
Found M+, 310.2869, C20H38O2 requires, 310.2872;
IR (Neat) νmax 2955, 2925, 2856 and 1742 cm−1; H
1
NMR (CDCl3, 500 MHz) δ: 5.33 (m, 2× CH, 2H), 3.66
(s, CH3, 3H), 2.31 (t, CH2, 2H, J = 7.6 Hz), 2.01 (m, 2×
CH2, 4H), 1.64 (m, CH2, 2H, includes J = 7.6 Hz), 1.37
(m, CH2, 2H), 1.18–1.32 (m, 7× CH2, 1× CH 15H),
1.11 (m, CH2, 2H), 0.86 (m, 2× CH3, 6H); 13C NMR
(CDCl3, 125 MHz) (major isomer) δ: 174.29, 130.80,
129.04, 51.57, 37.18, 37.12, 34.15, 32.56, 32.07, 30.16,
29.83, 29.51, 29.38, 27.19, 26.95, 24.95, 24.75, 22.83,
19.71, 14.24.

116
I.O. Roberts, M.S. Baird / Chemistry and Physics of Lipids 142 (2006) 111–117
29.2, 27.2, 27.1, 24.8, 22.8, 19.8, 14.2. The melt-
ing point and optical rotation of 5 were in agreement
with published data (Prout et al., 1948; Linstead et
al., 1951). The NMR and IR data for 5 were con-
sistent with those of racemic TBSA (Wallace et al.,
1994).
(Prout et al., 1948; Linstead et al., 1951), the NMR and
IR data for 5a were consistent with those of racemic
TBSA (Wallace et al., 1994).
3.12. (S)-10-Methyloctadecanoic acid methyl ester
(10a)
3.10. (S)-10-Methyloctadecanoic acid (10)
As above, using (S)-10-methyloctadecanoic acid
(10) (0.074 g, 0.25 mmol), Et2O (1 ml), diazomethane
(1.5 ml). Affording (S)-10-methyloctadecanoic acid
methyl ester (10a) a colourless oil, 0.071 g, 92%. Com-
pound 10a showed [α]2D5 = +0.33 (c 1.35, CHCl3);
Found M+, 312.3031, C20H40O2 requires, 312.3028;
The procedure described for 5 was repeated
using Pd/C 10% (0.1 g), MeOH (15 ml) and (S)-10-
methyloctadec-6-enoic acid (9), (0.9 g, 3.04 mmol) and
worked up as before to give (S)-10-methyloctadecanoic
acid (10) (0.081 g, 89%). Compound 10 was a slightly
yellow oil at room temperature, solidifying to a yel-
lowish waxy solid at temperatures of less than 12 ◦C.
Compound 10 showed [α]2D4 = +0.21 (c 1.23, CHCl3);
Found M+, 298.2870, C19H38O2 requires, 298.2872; IR
(Neat) νmax 3000 (br) 2922, 2853, 1711, 1464 cm−1; IR
1
IR (Neat) νmax 2923, 2899 and 1741 cm−1; H NMR
(CDCl3, 500 MHz) δ: 3.66 (s, CH3, 3H), 2.29 (t, CH2,
2H, J = 7.6 Hz), 1.61 (m, CH2, 2H, includes J = 7.3 Hz),
1.18–1.39 (m, 12× CH2, 1× CH, 25H) 1.07 (m, CH2,
2H), 0.86 (t, CH3, 3H, J = 7.0 Hz), 0.82 (d, CH3, 3H,
J = 6.6 Hz); 13C NMR (CDCl3, 125 MHz) δ: 174.44,
51.54, 37.24, 37.23, 34.26, 32.89, 32.07, 30.18, 30.08,
29.84, 29.64, 29.51, 29.41, 29.31, 27.23, 27.18, 25.11,
22.83, 19.84, 14.24. The optical rotation of 10a was
in agreement with published data (Prout et al., 1948;
Linstead et al., 1951), the NMR and IR data for 10a
were consistent with those of racemic TBSA (Wallace
et al., 1994).
(film) 3000 (br) 2922, 2853, 1711 and 1464 cm−1; H
1
NMR(CDCl3, 500 MHz)δ:2.35(t, CH2, 2H, J = 7.4 Hz),
1.64 (m, td apparent, CH2, 2H, J = 7.1, 7.3 Hz), 1.27 (m,
12× CH2, 1× CH, 25H), 1.08 (m, CH2, 2H), 0.89 (t,
CH3, 3H, J = 6.6 Hz), 0.84 (d, CH3, 3H, J = 6.7 Hz); 13
C
NMR (CDCl3, 125 MHz) δ: 180.5, 37.24, 37.22, 34.3,
32.9, 32.1, 30.2, 30.1, 29.8, 29.6, 29.5, 29.4, 29.2, 27.2,
27.1, 24.8, 22.8, 19.8, 14.3. The melting point and opti-
cal rotation of 10 were in agreement with published data
(Prout et al., 1948; Linstead et al., 1951), the NMR and
IR data for 10 were consistent with those of racemic
TBSA (Wallace et al., 1994).
References
Al-Dulayymi, J.R., Baird, M.S., Jones, K., 2004. The absolute stereo-
chemistry of grenadamide. Tetrahedron 60, 341–345.
Al-Dulayymi, J.R., Baird, M.S., Roberts, E., 2000. The synthesis of
single enantiomers of a meromycolic acid. Tetrahedron Lett. 41,
7107.
Anderson, R.J., 1927. A study of the phosphatide fraction of tubercule
bacilli. J. Biol. Chem. 74, 537–551.
3.11. (R)-10-Methyloctadecanoic acid methyl ester
(5a)
The method used to prepare 4a was utilised, with
(R)-10-methyloctadecanoic acid (5) (0.1 g, 0.34 mmol),
Et2O (1 ml), and excess diazomethane solution (1.5 ml).
The product was purified by column chromatography
(petrol/Et2O 9:1) affording (R)-10-methyloctadecanoic
acid methyl ester (5a) a colourless oil, 0.09 g, 91%. Com-
pound5ashowed[α]D25 = −0.45(c1.33, CHCl3);Found
M+, 312.3032, C20H40O2 requires, 312.3028; IR (Neat)
νmax 2922 and 1742 cm−1; 1H NMR (CDCl3, 500 MHz)
δ: 3.65 (s, CH3, 3H), 2.29 (t, CH2, 2H, J = 7.6 Hz), 1.61
(m, CH2, 2H, includes J = 7.3 Hz), 1.18–1.39 (m, 12×
CH2, 1× CH, 25H) 1.06 (m, CH2, 2H), 0.87 (t, CH3,
3H, J = 7.0 Hz), 0.82 (d, CH3, 3H, J = 6.6 Hz); 13C NMR
(CDCl3, 125 MHz) δ: 174.45, 51.54, 37.24, 37.22, 34.26,
32.89, 32.07, 30.19, 30.08, 29.84, 29.64, 29.52, 29.41,
29.31, 27.23, 27.18, 25.11, 22.83, 19.84, 14.24. The opti-
cal rotation of 5a was in agreement with published data
Anderson, R.J., Chargaff, E., 1929. The chemistry of the lipoids of
tubercule bacilli VI. Concerning tuberculostearic acid and phthioic
acid from the acetone soluble fat. J. Biol. Chem. 85, 77–88.
Anderson, R.J., Gilman Roberts, E., 1930. The chemistry of the lipoids
of tubercule bacilli XI. The phosphatide fraction of the avian tuber-
cule bacilli. J. Biol. Chem. 85, 519–528.
Anderson, R.J., Uyei, N., 1932. The chemistry of the lipids of tubercule
bacilli XXVII. The composition of the phosphatide fraction of the
bacillus leprae. J. Biol. Chem. 97, 617–637.
Brooks, J.B., Daneshvar, M.I., Fast, D.M., Good, R.C., 1987. Selec-
tive procedures for detecting femtomole quantities of tubercu-
lostearicacidinserumandcerebrospinal-fluidbyfrequency-pulsed
electron-capture gas–liquid-chromatography. J. Clin. Microbiol.
25, 1201–1206.
Burt, M.L., Anderson, R.J., 1931. The chemistry of the lipoids of tuber-
cule bacilli XXIV. Analysis of the acetone-soluble fat of the bovine
tubercule bacillus. J. Biol. Chem. 94, 451–463.
Coxon, G.D., Al-Dulayymi, J.R., Baird, M.S., Knobl, S., Roberts,
E., Minnikin, D.E., 2003. The synthesis of (11R,12S)-lactobacillic
acid and its enantiomer. Tetrahedron: Asymmetr. 14, 1211–1222.

I.O. Roberts, M.S. Baird / Chemistry and Physics of Lipids 142 (2006) 111–117
117
Coxon, G.D., Knobl, S., Roberts, E., Baird, M.S., Al-Dulayymi, J.R.,
Besra, G.S., Brennan, P.J., Minnikin, D.E., 1999. The synthesis of
both enantiomers of lactobacillic acid and mycolic acid analogues.
Tetrahedron Lett. 40, 6689.
Mori, K., Tamada, S., Matsui, M., 1978. Stereocontrolled synthesis
of all of the four possible stereoisomers of erythro-3,7-dimethyl-
pentadec-2-yl acetate and propionate, the sex pheromone of the
pine sawflies. Tetrahedron Lett., 901–904.
Daikos, G.L., Brookes, J.B., Michos, A., Roma, E., Syriopolou, V.,
2004. Detection of tuberculostearic acid in serum and other biolog-
ical fluids from patients with tuberculosis by electron capture-gas
chromatography and chemical ionisation-mass spectrometry. Int.
J. Tuberc. Lung Dis. 8, 1027–1031.
French, G.L., Chan, C.Y., Cheung, S.W., Oo, K.T., 1987a. Diagnosis
of pulmonary tuberculosis by detection of tuberculostearic acid
in sputum by using gas chromatography–mass spectrometry with
selected ion monitoring. J. Infect. Dis. 156, 356–362.
French, G.L., Chan, C.Y., Cheung, S.W., Teoh, R., Humphries, M.J.,
O’Mahony, G., 1987b. Diagnosis of tuberculous meningitis by
detection of tuberculostearic acid in cerebrospinal-fluid. Lancet
2, 117–119.
Odham, G., Larsson, L., Mardh, P.A., 1979. Demonstration of tubercu-
lostearic acid in sputum from patients with pulmonary tuberculosis
by selected ion monitoring. J. Clin. Invest. 63, 813–819.
Odinokov, V.N., Kukovinets, O.S., Zainullin, R.A., Kasradze, V.G.,
Dolidze, A.V., Tolstikov, G.A., 1992. Ozonolysis of alkenes and
study of reactions of polyfunctional compounds. 51. Synthesis of
potential juvinoids with cyclobutane and cycloporpane fragments
based on (+)-alpha-pinene and (+)-3-Carene ozonolysis products.
Zh. Org. Khim. 28, 1619–1625.
Pangborn, M.C., Anderson, R.J., 1931. The chemistry of the lipoids of
tubercule bacilli XXV. The composition of the phosphatide fraction
of the timothy bacillus. J. Biol. Chem. 94, 465–472.
Pangborn, M.C., Chargaff, E., Anderson, R.J., 1932. The chemistry of
the lipids of tubercule bacilli XXXI. The composition of the ace-
tone soluble fat of the timothy bacillus. J. Biol. Chem. 98, 43–55.
Phetsukiri, B., Jackson, M., Scherman, H., McNeil, M., Besra, G.S.,
Baulard, A.R., Slayden, R.A., De Barber, A.E., Barry, C.E., Baird,
M.S., Crick, D.C., Brennan, P.J., 2003. Unique mechanism of
action of the thiourea drug isoxyl on Mycobacterium tuberculo-
sis. J. Biol. Chem. 278, 53123–53130.
Prout, F.S., Cason, J., Ingersoll, A.W., 1947. The synthesis of tubercu-
lostearic acid. J. Am. Chem. Soc. 69, 1233–11233.
Prout, F.S., Cason, J., Ingersoll, A.W., 1948. Branchedchainfattyacids.
V. The synthesis of optically active 10-methyloctadecanoic acids.
J. Am. Chem. Soc. 70, 298–305.
Furniss, B.S., Hannaford, A.J., Smith, P.W.G., Tatchell, A.R., 1989.
Vogel’s Textbook of Practical Organic Chemistry. Longman, Sin-
gapore.
Kikukawa, T., Imaida, M., Tai, A., 1982. Synthesis of the sex
attractant of pine sawflies (Diprion species)—(2S,3R,7R)-
3,7-dimethylpentadecan-2-ol
and
(2S,3R,7S)-3,7-
dimethylpentadecan-2-ol. Chem. Lett. 11, 1799–1802.
Kikukawa, T., Imaida, M., Tai, A., 1984. Syntheses of the sex attractant
of pine sawflies. Bull. Chem. Soc. Jpn. 57, 1954–1960.
Larsson, L., Mardh, P.A., Odham, G., Westerdahl, G., 1980. Detec-
tion of tuberculostearic acid in biological specimens by means of
glass-capillary gas chromatography–electron and chemical ion-
ization mass spectroscopy, utilising selected ion monitoring. J.
Chromatogr. 182, 402–408.
Roberts, I.O., Baird, M.S., Liu, Y., 2004. The absolute stereochemistry
of cascarillic acid. Tetrahedron Lett. 45, 8685–8686.
Linstead, R.P., Lunt, J.C., Weedon, B.C.L., 1951. Anodic syntheses
Part IV. Synthesis of (+)- and (−)-tuberculostearic acids. J. Chem.
Soc., 1130–1132.
Sabin, F.R., Doan, C.A., 1927. The biological reactions in rabbits to
the protein and phosphatide fractions from the chemical analysis
of human tubercule bacilli. J. Exp. Med. 46, 645.
Menger, F.M., Wood, M.G., Richardson, S., Zhou, Q., Elrington, A.R.,
Sherrod, M.J., 1988a. Chain-substituted lipids in monolayer films.
A study of molecular packing. J. Am. Chem. Soc. 110, 6797–
6803.
Menger, F.M., Wood, M.G., Zhou, Q., Hopkins, H.P., Fumero, J.,
1988b. Thermotropic properties of synthetic chain-substituted
phosphatidylcholines: effect of substituent size, polarity, number,
and location on molecular packing in bilayers. J. Am. Chem. Soc.
110, 6804–6810.
Mori, K., Tamada, S., 1979. Stereocontrolled synthesis of all of the
four possible stereoisomers of erythro-3,7-dimethyl-pentadec-2-yl
acetate and propionate, the sex pheromone of the pine sawflies.
Tetrahedron 35, 1279–1284.
Schmidt, G.A., Shirley, D.A., 1949. A new synthesis of tubercu-
lostearic acid. J. Am. Chem. Soc. 71, 3804–3806.
Spielman, M.A., 1934. The chemistry of the lipids of tubercule bacilli
XXXIX. The constitution of tuberculostearic acid. J. Biol. Chem.
106, 87–96.
Stopforth, A., Tredoux, A., Crouch, A., van Helden, P., Sandra,
P., 2005. A rapid method of diagnosing pulmonary tubercu-
losis using stir bar sorptive extraction–thermal desorption–gas
chromatography–mass spectrometry. J. Chromatogr. A. 1071.
Wallace, P.A., Minnikin, D.E., McCrudden, K., Pizzarello, A., 1994.
Synthesis of (R,S)-10-methyloctadecanoic acid (tuberculostearic
acid) and key chiral 2-methyl branched intermediates. Chem. Phys.
Lipid. 71, 145.