The synthesis of a major α′-mycolic acid of Mycobacterium smegmatis

Article abstract of DOI:10.1016/j.chemphyslip.2010.05.203

The synthesis of (2R,3R,Z)-2-docosyl-3-hydroxytetracont-21-enoic acid, a significant α′-mycolic acid of Mycobacterium smegmatis and other mycobacteria is reported.

Full text of DOI:10.1016/j.chemphyslip.2010.05.203

Chemistry and Physics of Lipids 163 (2010) 678–684
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Chemistry and Physics of Lipids
journal homepage: www.elsevier.com/locate/chemphyslip
The synthesis of a major ␣ꢀ-mycolic acid of Mycobacterium smegmatis
Maged Muzael, Gani Koza, Juma’a J. Al Dulayymi, Mark S. Baird∗
School of Chemistry, Bangor University, Bangor, Gwynedd, LL57 2UW UK
a r t i c l e i n f o
a b s t r a c t
The synthesis of (2R,3R,Z)-2-docosyl-3-hydroxytetracont-21-enoic acid, a significant ␣ꢀ-mycolic acid of
Mycobacterium smegmatis and other mycobacteria is reported.
Article history:
Received 26 March 2010
Received in revised form 19 May 2010
Accepted 26 May 2010
© 2010 Elsevier Ireland Ltd. All rights reserved.
Available online 4 June 2010
Keywords:
Mycobacterium smegmatis
Mycolic acid
cis-alkene
1. Introduction
sistent with 60, 62, 64 and 66 carbons as suggested by Wong et
al. (Laval et al., 2001) Mycobacterium chelonae contains compara-
Mycolic acids 1 (Scheme 1) are characteristic components of
mycobacteria and related taxa (Barry et al., 1998). They are defined
by a R,R-␤-hydroxy acid with two long chain substituents. In
mycobacteria, the ␣-chain is a simple alkyl substituent with a chain
length of either 22 or 24 carbons. In contrast, the mero-chain can
contain a number of substituents—in a number of important bacte-
ria, such as Mycobacterium tuberculosis, these include two or three
functional groups and can be divided into ␣- methoxy and keto-
classes of mycolic acid depending on X and Y. Some years ago,
it was reported that important mycolic acids of Mycobacterium
smegmatis are much simpler and contain just one cis-alkene in the
mero-chain; the mixture of ␣ꢀ-smegmamycolates, m.p. 50–51 ◦C,
[␣]D+2.8, makes up some 25% of the mycolic acids of this species
and the major component mycolic acid was reported to have the
chain lengths identified in structure 2 (a = b = 17) (Etemadi et al.,
1964; Krembel and Etemadi, 1966; Etemadi, 1967).
Some years later, Wong et al. separated three series of mycolic
acids from M. smegmatis, and confirmed that one of these simply
contained one alkene substituent in the mero-chain. However, they
were able to separate this series into component parts by prepar-
ative HPLC and identified six major homologues of this type with
slightly different chain lengths, including 2 (a = b = 17) (Wong and
Gray, 1979; Wong et al., 1979; Gray et al., 1983). Direct analysis of
the mycolic acids by MALDI-TOF mass spectrometry confirmed the
presence of a series of components with molecular weights con-
ble amounts of ␣-mycolates, containing either two cis-alkenes, one
cis and one ␣-methyl-trans-alkene, or one cis-cyclopropane and
one ␣-methyl-trans-alkene at distal and proximal positions and
of shorter chain ␣ꢀ-mycolates containing just one cis-alkene cor-
responding to 2 (a = 15,17, b = 17,19) (Goodfellow and Minnikin,
1981; Minnikin et al., 1982). In other cases, mycobacteria such
as Mycobacterium fortuitum (Minnikin et al., 1980, 1984) and
‘Mycobacterium thamnopheos’ (Lacave et al., 1987; Daffé et al., 1988)
are reported to contain mycolic acid which include two to four
cis-alkenes. ␣ꢀ-mycolates containing 0–2 double bonds and 22–36
carbons have been reported in the genera Corynebacteria, 1–4 dou-
ble bonds and 48–66 carbons in Gordona, 0–3 double bonds and
44–60 carbons in Nocardia, 0–4 double bonds and 34–48 carbons
in Rhodococcus and 1–6 double bonds and 64–78 carbons in Tsuk-
murella (Barry et al., 1998). Unsaturated mycolic acids may be
intermediates in the biosynthesis of other classes of mycolic acid
(Lopez-Marin et al., 1991; Wheeler et al., 1993; Asselineau et al.,
2002).
We have reported the synthesis of examples of a range of ␣-
methoxy- and keto-mycolic acids with structures chosen to match
the chain lengths and, where known the stereochemistry of major
mycobacterial mycolic acids (Al Dulayymi et al., 2005, 2006, 2007;
Baird and Koza, 2007; Koza et al., 2009) We now report the synthe-
sis of the simple cis-alkene mycolic acid 2.
2. Results and discussion
The aldehyde 3 (Koza et al., 2009) was chain extended by a
modified Julia–Kocienski reaction with 5-(eicosane-1-sulfonyl)-
1-phenyl-1H-tetrazole (prepared from 1-phenyl-1H-tetrazole-5-
∗
Corresponding author at: University of Wales, Bangor, School of Chemistry,
Bangor, Gwynedd, Wales LL57 2UW, UK. Tel.: +44 128382374.
E-mail address: chs028@bangor.ac.uk (M.S. Baird).
0009-3084/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.chemphyslip.2010.05.203

M. Muzael et al. / Chemistry and Physics of Lipids 163 (2010) 678–684
679
Scheme 1.
thiol and 1-bromoeicosane under standard conditions) and base,
followed by hydrogenation of the derived mixture of E and Z-
alkenes to give the ester 4. Removal of the benzyl group led to
alcohol 5 which was in turn oxidised to the aldehyde 6 (Scheme 2).
Reaction of aldehyde 6 with 2,2-dimethyl-propionic acid 16-(1-
phenyl-1H-tetrazole-5-sulfonyl)-hexadecyl ester in the presence
of lithium bis-trimethylsilylamide in a modified Julia–Kocienski
reaction led to a mixture of E and Z-alkenes which was hydro-
genated to give the diester 7 (Scheme 3). Deprotection of the pivolyl
ester led to the alcohol 8 which was oxidized to the aldehyde 9.
Reaction of 9 with 5-(nonadecylsulfonyl)-1-phenyl-1H-tetrazole
and base gave a mixture of E and Z-alkenes 11 and in 70% yield,
providing comparison spectroscopic data for both isomers. In con-
trast, reaction with nondecyltriphenylphosphonium bromide and
sodium bis-trimethylsilylamide gave the required Z-isomer 10
(62%).
typical MD for only chiral element present, the R,R-␤-hydroxy-
acid fragment, has been reported to be around +40 (Quémard
et al., 1997), while other values for natural coryno-mycolic acids
are reported to be +35 (Asselineau and Asselineau, 1966) and
+37.5 (Pudles and Lederer, 1951) and that for synthetic (S)-2-((S)-
1-hydroxytetradecyl)eicosanoic acid is reported to be −23 (the
synthetic R,R-isomer in this case has a value of +13) (Kumaraswamy
and Markondaiah, 2008).
3. Experimental
3.1. General
Chemicals used were obtained from commercial suppliers or
prepared from them by methods described. Solvents which had to
be dry, e.g. ether (which throughout refers to diethyl ether), THF
were dried over sodium wire. Petrol was of boiling point 40–60 ◦C.
Reactions carried under inert conditions, were carried out under
a slow stream of nitrogen. Those carried out at low temperatures
were cooled using a bath of methylated spirit with liquid nitro-
The silyl protecting group was removed by reaction with HF-
pyridine to give the methyl ester 12 as a white solid, m.p. 44–46 ◦C
with a specific rotation of +4.65 (c 1.83, CHCl3). Removal of the
methyl ester using lithium hydroxide in THF–methanol–water then
gave the free mono-alkene mycolic acid 13 as a white solid, m.p.
65–66 ◦C which showed a specific rotation [␣]D of +3.4 in CHCl3
21
gen. Silica gel (Merck 7736) and silica gel plates (Silica Gel 60 F254
)
corresponding to an MD of +33 (Scheme 4).
used for column and thin layer chromatography were obtained
from Aldrich. Organic solutions were dried over anhydrous magne-
sium sulfate. IR spectra were carried out on a Perkin–Elmer 1600
F.T.I.R. spectrometer as liquid films. NMR spectra were recorded
on Bruker AC250 or Advance 500 spectrometers. Specific rotation
values were recorded in CHCl3 on a POLAAR 2001 Optical Activity
polarimeter. Mass spectra were recorded on a Bruker Microtof ESI
instrument.
The value for the specific rotation of 13 is in line with that
reported for ␣ꢀ-smegmamycolates (Etemadi et al., 1964; Krembel
and Etemadi, 1966; Etemadi, 1967), and with the MD predicted
on the basis of the summing of molecular rotations for individ-
ual chiral elements in the molecule, a technique which generally
works well in molecules of this type where the chiral elements
are separated by long chains (Quémard et al., 1997). Thus the
Scheme 2. (i) LiN(SiMe3)2, 5-(eicosane-1-sulfonyl)-1-phenyl-1H-tetrazole, THF, argon, −10 ◦C, then r.t., 1 h (79%); (ii) industrial methylated spirit (IMS), THF, Pd/C, H2, 2 h
(90%); (iii) IMS, THF, Pd/C, H2, 3 days (90%); (iv) pyridinium chlorochromate (PCC), CH2Cl2 (96%).

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M. Muzael et al. / Chemistry and Physics of Lipids 163 (2010) 678–684
Scheme 3. (i) LiN(SiMe3)2, 2,2-dimethyl-propionic acid 16-(1-phenyl-1H-tetrazole-5-sulfonyl)-hexadecyl ester, THF, argon, −10 ◦C, then r.t., 1 h (95%), (ii)) IMS, THF, Pd/C,
H2 (85%),(iii) KOH:THF:H2O, 70 ◦C, 3 h (84%), (iv) PCC, CH2Cl2 (92%), (v) nonadecyltriphenyl-phosphonium bromide, sodium bis(trimethylsilyl)amide, THF, argon, r.t., 1 h
(62%), (vi)) LiN(SiMe3)2, 5-(nonadecane-1-sulfonyl)-1-phenyl-1H-tetrazole, THF, argon, −10 ◦C, then r.t., 1 h (70%).
3.2. 5-Eicosylsufanyl-1-phenyl-lH-tetrazole
1-Phenyl-1H-tetrazole-5-thiol (7.76 g,
bromoeicosane (12.5 g, 34.6 mmol), anhydrous potassium
carbonate (15 g, 41.5 mmol), and acetone (500 ml) were mixed.
The mixture was vigorously stirred and refluxed at 60 ◦C for 15 h
when TLC indicated complete reaction of the thiol. The inor-
ganic salts were filtered off and washed well with acetone. The
acetone solution was evaporated to a small bulk and dissolved
in dichloromethane (200 ml). The solution was washed with
water (300 ml) and the aqueous layer was re-extracted with
dichloromethane (2× 50 ml). The combined organic phases were
washed with water (300 ml), dried and the solvent was evapo-
rated. The crude product was recrystallised from acetone (90 ml)
43.5 mmol),
1-
Scheme 4. (i) HF-pyridine, THF, argon, 45 ◦C, 18 h (87%), (ii) LiOH, THF:MeOH:H2O (10:1:1), 40 ◦C, 18 h (81%).

M. Muzael et al. / Chemistry and Physics of Lipids 163 (2010) 678–684
681
and methanol (90 ml) to give a white solid, 5-eicosylsufanyl-1-
66.12, 52.00, 51.19, 33.64, 31.92, 29.69, 29.66, 29.65, 29.63, 29.56,
29.43, 29.35, 27.86, 27.22, 25.7, 22.7, 17.9, 14.08,−4.62,−4.93;
ꢀmax/cm−1: 2928, 2856, 1738, 1665, 1361, 1254, 1192, 1168,
1102. (ii) Palladium 10% on carbon (0.5 g) was added to a stirred
solution of the methyl ester from (i) (4.96 g, 7.52 mmol) in IMS
(60 ml) and THF (60 ml) under a hydrogen atmosphere. Hydro-
genation was carried out for 3 days. The solution was filtered
over a bed of celite and the solvent was evaporated. The crude
product was purified by column chromatography eluting with
petrol/ether (2:1) to give (R)-2-[(R)-1-(tert-butyldimethylsilanyl-
oxy)-3-hydroxypropyl]tetracosanoic acid methyl ester 5 as a
pheny-lH-tetrazole (17.0 g, 89%), m.p. 45–46 ◦C {Found [M+Na]+:
459.3500,
C
27H46N4S requires: 459.3516}. This showed ıH:
7.65–7.52 (5H, m), 3.37 (2H, t, J 7.25 Hz), 1.78 (2H, pent., J 7.25 Hz),
1.43 (2H, pent., J 7.6 Hz), 1.34–1.22 (32H, m), 0.88 (3H, t, J 6.95 Hz,);
ıC: 154.5, 133.8, 130.12, 129.8, 123.9, 33.4, 31.9, 29.68, 29.65,
29.62, 29.54, 29.44, 29.3, 29.09, 28.65, 22.7, 14.1; ꢀmax: 2917, 1501,
1471, 1390, 892, 763, 686 cm−1
3.3. 5-(Eicosane-1-sulfonyl)-1-phenyl-lH-tetrazole
3-Chloroperoxybenzoic acid (9.08 g,
.
white solid (4.77 g, 90%), m.p. 35–37 ◦C, [␣]D −1.76 (c 0.81,
28
377 mmol)
in
dichloromethane (100 ml) was added to a stirred solution of
5-eicosylsulfanyl-1-phenyl-1H-tetrazole (16.82 g, 36.6 mmol) and
NaHCO3 (13.86 g, 164.0 mmol) in dichloromethane (100 ml) at 0 ◦C.
The reaction mixture was stirred at r.t. overnight. The reaction was
quenched with sodium hydroxide solution (200 ml, 5%) and stirred
for 1 h before being extracted with dichloromethane (3 × 200 ml).
The combined organic layers were dried over MgSO4, filtered
and evaporated to a powder. The crude product was purified by
recrystallisation from methanol/acetone (1:1), to give 5-(eicosane-
1-sulfonyl)-1-phenyl-1H-tetrazole as a white solid (14.96 g, 83%),
m.p. 56–58 ◦C {Found [M+Na]+: 491.3403, C27H46O2S requires:
491.3414}. This showed ıH: 7.72–7.70 (2H, m), 7.64–7.60 (3H, m),
3.74 (2H, distorted t, J 7.91 Hz), 1.96 (2H, br, pent., J 7.8 Hz), 1.50
(2H, br, pent., J 7 Hz), 1.36–1.26 (32 H, m), 0.89 (3H, t, J 6.95 Hz); ıC:
153.46, 133.45, 131.45, 129.72, 125.08, 56.04, 31.93, 29.7, 29.63,
CHCl3) {Found [M+H]+: 571.5101, C34H71O4Si requires: 571.5116}.
This showed ıH: 4.29 (1H, td, J 6, 4.4 Hz), 3.70–3.66 (2H, m)
3.56 (3H, s), 2.66 (1H, ddd, 10.5, 6.65, 3.8 Hz), 1.98 (1H, br, s),
1.69–1.65 (2H, m), 1.44–1.39 (1H, m), 1.38–1.32 (1H, m), 1.21–1.08
(40H, m), 0.78–0.72 (12H, including a singlet at ı 0.77), 0.00
(3H, s), −0.04 (3H, s); ıC: 174.66, 72.01, 59.47, 51.39, 35.23,
31.92, 29.69, 29.66, 29.64, 29.61, 29.56, 29.55, 29.43, 29.34, 27.85,
27.16, 25.68, 22.67, 17.84, 14.08, −4.35, −4.99; ꢀmax: 3462, 2921,
2857, 1740, 1464, 1362, 1255, 1196, 1166, 1092, 908, 837, 776,
735 cm−1
.
3.5. (R)-2-[(R)-1-(tert-Butyldimethylsilanyloxy)
-3-oxopropyl]tetracosanoic acid methyl ester (6)
(R)-2-[(R)-1-(tert-Butyldimethylsilanyloxy)-3-
29.57, 29.46, 29.36, 29.19, 28.9, 28.15, 22.69, 21.95, 14.12; ꢀmax
:
hydroxypropyl]tetracosanoic acid methyl ester (2.01 g, 3.51 mmol)
in dichloromethane (25 ml) was added in portions at r.t. to a
stirred solution of PCC (1.89 g, 12.29 mmol) in dichloromethane
(100 ml). During the addition a black colour appeared. The reaction
was stirred at r.t. for 2 h, when TLC showed complete reaction,
then ether (300 ml) was added and filtered through a bed of
silica gel. The solvent was evaporated and the crude product was
purified by column chromatography eluting with petrol/ether
(4:1) to give (R)-2-[(R)-1-(tert-butyldimethylsilanyloxy)-3-
oxopropyl]tetracosanoic acid methyl ester 6 (1.92 g, 96%) as
2913, 2846, 1493, 1461, 1337, 1148, 763, 686 cm−1
.
3.4. (R)-2-[(R)-1-(tert-Butyldimethylsilanyloxy)
-3-hydroxypropyl]tetracosanoic acid methyl ester (4)
Lithium bis(trimethylsilyl)amide (1.06 M in THF, 16.44 ml,
17.4 mmol) was added to a stirred solution of 5-benzyloxy-3-(tert-
butyldimethylsilanyloxy)-2-(2-oxo-ethyl)pentanoic acid methyl
ester 3 (Koza et al., 2009) (3.82 g, 9.68 mmol) and 5-(eicosane-
1-sulfonyl)-1-phenyl-1H-tetrazole (5.7 g, 11.6 mmol) in dry THF
(50 ml) at −5 ◦C. The reaction turned bright yellow and was
left to reach r.t. and stirred for 1 h under nitrogen atmosphere,
when TLC showed no starting material was left, the reaction
was quenched by addition of sat. aq. NH4Cl (10 ml). The product
was extracted with petrol/ether (1:2) (3× 150 ml). The combined
organic layers were dried over MgSO4, filtered and evaporated,
to give a crude product which was purified by column chromo-
tography over silica gel, eluting with petrol/ether (20:1) to give
a colourless oil (E/Z)-(R)-2-[(R)-1-(tert-butyldimethylsilanyloxy)-
a colourless oil, [␣]D −4.98 (c 1.23, CHCl3) {Found (M+Na)+:
26
591.4774. C34H68NaO4Si requires: 591.4779}; which showed ıH:
9.72 (1H, t, J 2.2 Hz), 4.36 (1H, br., q, J 6 Hz), 3.59 (3H, s), 2.59 –2.49
(3H, m), 1.57–1.48 (1H, m), 1.46–1.4 (1H, m), 1.35–1.13 (40H, m),
0.80 (3H, t, J 7 Hz), 0.78 (9H, s), 0.002 (3H, s), −0.008 (3H, s); ıC:
201.06, 174.26, 173.95, 68.79, 52.22, 51.43, 48.07, 31.90, 29.68,
29.64, 29.60. 29.52, 29.47, 29.36, 29.34, 27.72, 27.00, 25.58, 22.66,
17.83, 14.06, −4.69, −4.96; ꢀmax: 2924, 2856, 1734, 1466, 1362,
1255, 1198, 1167, 590, 477 cm−1
.
3-benzyloxy-propyl]-tetracos-4-enoic acid methyl ester as
a
3.6. 2,2-Dimethylpropionic acid
mixture in ratio 2:1 (4.92 g, 79%) {Found [M+H]+: 659.5409,
C41H74O4Si requires: 659.5426}. (i) Palladium 10% on carbon
(2.5 g) was added to a stirred solution of (E/Z)-(R)-2-[(R)-1-(tert-
butyldimethylsilanyloxy)-3-benzyloxypropyl]tetracos-4-enoic
acid methyl ester (5.5 g, 8.34 mmol) in IMS (60 ml) and THF
16-(1-phenyl-1H-tetrazol-5-ylsulfanyl)hexadecyl ester
1-Phenyl-1H-tetrazole-5-thiol (3.66 g, 20.5 mmol),
2,2-
dimethylpropionic acid 16-bromohexadecyl ester (8.34 g,
20.5 mmol), anhydrous potassium carbonate (4.26 g, 30.8 mmol),
and acetone (70 ml) were mixed. The mixture was vigorously
stirred and refluxed at 60 ◦C for 15 h when TLC showed no starting
material was left, the inorganic salts were filtered off and washed
well with acetone. The acetone solution was evaporated to a small
bulk and dissolved in dichloromethane (100 ml). The solution
was washed with water (100 ml) and the aqueous layer was
re-extracted with dichloromethane (2× 50 ml). The combined
organic phases were washed with water (300 ml), dried and
the solvent was evaporated. The crude product was purified by
column chromatography eluting with petrol:ethyl acetate (5:1)
to give 2,2-dimethylpropionic acid 16-(1-phenyl-1H-tetrazol-5-
ylsulfanyl)hexadecyl ester (8.01 g, 77%) (Found [M+Na]+: 525.3356.
C28H46N4O2SNa requires: 525.3234) which showed ıH: 7.53–7.45
(60 ml) under
a hydrogen atmosphere. Hydrogenation was
carried out for 2 h. The solution was filtered over a bed of
celite and the solvent was evaporated. The crude product was
purified by column chromatography eluting with petrol/ether
(2:1) to give (R)-2-[(R)-1-(tert-butyldimethylsilanyloxy)-3-
benzyloxypropyl]tetracosanoic acid methyl ester 4 as a colourless
oil (4.96 g, 90%), [␣]D −5.42 (c 1.13, CHCl3) {Found [M+H]+:
23
661.5570, C41H76O4Si requires: 661.5586} which showed ıH:
7.33–7.31 (4H, m), 7.28–7.25 (1H, m), 4.44 (2H, br., s), 4.13–4.06
(1H, m), 3.65 (3H, s), 3.59–3.54 (2H, m), 2.52 (1H, ddd, J 10.4,
6.6, 3.8 Hz), 1.82 (2H, q, J 6.6 Hz), 1.64–1.51 (2H, m), 1.43–1.15
(40H, br., m), 0.85 (3H, t, J 7 Hz), 0.82 (9H, s), 0.05 (3H, s), 0.04
(3H, s); ıC: 174.65, 138.46, 128.25, 127.5, 127.41, 72.86, 70.71,

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M. Muzael et al. / Chemistry and Physics of Lipids 163 (2010) 678–684
(5H, m), 3.98 (2H, t, J 6.6 Hz), 3.33 (2H, t, J 7.55 Hz), 1.76 (2H,
pent., J 7.9 Hz), 1.55 (2H, pent., J 6.6 Hz),1.38 (2H, pent., J 5.95 Hz),
1.20–1.17(22H, br. m), 1.34 (9H, s); ıC 178.33, 154.27, 133.63,
129.84, 129.57, 123.64, 64.23, 38.7, 33.17, 29.45, 29.43, 29.42,
29.36, 29.32, 29.25, 29.03, 28.92, 28.84, 28.45, 28.43, 27.00 25.72;
ꢀmax: 2923, 2849, 1723, 1597, 1499, 1479, 1386, 1282, 1156, 760,
29.59, 29.57, 29.52, 29.43, 29.35, 29.23, 28.63, 27.83, 27.48,
27.19, 25.91, 25.76, 23.75, 22.67, 17.97, 14.08, −4.37, −4.92;
ꢀmax: 2924, 2856, 1734, 1466, 1362, 1255, 1198, 1167, 590,
477 cm−1
.
3.9. (R)-2-[(R)-1-(tert-Butyldimethylsilanyloxy)
-19-hydroxynonadecyl]tetracosanoic acid methyl ester (8)
693 cm−1
.
3.7. 2,2-Dimethylpropionic acid
(R)-2-[(R)-1-(tert-Butyldimethylsilanyloxy)-19-(2,2-
16-(1-phenyl-1H-tetrazole-5-sulfonyl)-hexadecyl ester
dimethylpropionyloxy)nonadecyl]-tetracosanoic acid methyl
ester (1.81 g, 2.07 mmol) was added to a stirred solution of potas-
sium hydroxide (1.72 g, 30.8 mmol) dissolved in THF:MeOH:H2O
(10:10:1, 21 ml). The mixture was refluxed at 70 ◦C and monitored
by TLC. After 3 h, the TLC showed no starting material was left and
the reaction was cooled down, quenched with water and extracted
with ethyl acetate (3 × 100 ml). The combined organic layers
were dried and the solvent was evaporated. The product was
purified by column chromatography eluting with petrol/ether
Ammonium molybdate (VI) tetrahydrate (8.52 g, 6.9 mmol) in
35% H2O2 (23 ml), prepared and cooled in an ice bath, was added to
a stirred solution of 2,2-dimethylpropionic acid 16-(1-phenyl-1H-
tetrazol-5-ylsulfanyl)-hexadecyl ester (7.71 g, 15.3 mmol) in IMS
(100 ml) and THF (40 ml) at 10 ◦C. After 2 h at room temperature, a
further solution of ammonium molybdate (VI) tetrahydrate (8.52 g,
6.9 mmol) in 35% H2O2 (20 ml) was added and left overnight.
The mixture was poured into water (1L) and extracted with
dichloromethane (2 × 400 ml). The combined organic layers were
dried and evaporated to give a solid residue which was purified by
re-crystallised from petrol giving 2,2-dimethylpropionic acid 16-
(1-phenyl-1H-tetrazole-5-sulfonyl)hexadecyl ester (6.72 g, 81%)
(Found [M+Na]+: 557.3132. C28H46N4O4SNa requires: 557.3132)
which showed ıH: 7.71–7.69 (2H, m), 7.64–7.61 (3H, m), 4.05
(2H, t, J 6.6 Hz), 3.75 (2H, distorted t, J 7.9 Hz), 1.95 (2H, pent.,
J 7.8 Hz), 1.62 (2H, pent., J 6.65 Hz), 1.5 (2H, pent., J 6.65 Hz),
1.35–1.25 (22H, m), 1.2 (9H, s); ıC: 178.67, 153.51, 133.07, 131.46,
129.72, 125.08, 64.23, 56.03, 38.73, 29.62, 29.56, 29.52, 29.46,
(2:1 and then 1:1) to give
a semi-solid (R)-2-[(R)-1-(tert-
butyldimethylsilanyloxy)-19-hydroxynonadecyl]tetracosanoic
20
acid methyl ester 8 (1.38 g, 84%), [␣]D −11.19 (c 1.51, CHCl3)
(Found [M+Na]+: 817.7401. C50H102O4SiNa requires: 817.7440).
This showed ıH: 3.89 (1H, td, J 7.25, 4.7 Hz), 3.63 (3H, s), 3.61 (2H,
t J 6.6 Hz), 2.51 (1H, ddd, J 11.05, 7.25, 3.8 Hz), 1.7 (1H, br, s), 1.54
(2H, pent., J 6.3 Hz), 1.23 (74H, br, s), 0.86 (3H, t J 6.9 Hz), 0.84 (9H,
br, s), 0.02 (3H, s), 0.001(3H, s); ıC: 175.07, 73.18, 62.91, 60.30,
51.52, 33.64, 31.78, 31.89, 29.77, 29.66, 29.61, 29.58, 29.53, 29.51,
29.42, 29.39, 29.32, 27.77, 27.45, 26.86, 22.64, 14.03, −4.43, −5.00;
ꢀmax: 3384, 2923, 2853, 1741, 1464, 1361, 1254, 1195, 1166, 1070,
29.23, 29.2, 28.9, 28.82, 28.62, 28.15, 27.22, 25.91, 21.95; ꢀmax
2928, 2864, 1728, 1498, 1480, 1462, 1344, 1284, 1155, 762,
688 cm−1
:
836, 775, 720 cm−1
.
.
3.10. (R)-2-[(R)-1-(tert-Butyldimethylsilanyloxy)
-19-oxononadecyl]tetracosanoic acid methyl ester
3.8. (R)-2-[(R)–1-(tert-Butyldimethylsilanyloxy)-19-(2,
2-dimethylpropionyloxy)-nonadecyl]-tetracosanoic acid methyl
ester (7)
(R)-2-[(R)-1-(tert-butyldimethylsilanyloxy)-19-
hydroxynonadecyl]tetracosanoic acid methyl ester (0.25 g,
0.314 mmol) in dichloromethane (5 ml) was added in por-
tions at r.t. to a stirred solution of PCC (0.17 g, 0.785 mmol) in
dichloromethane (30 ml). During the addition a black colour
appeared. The reaction was stirred at r.t. for 2 h, when TLC
showed the reaction was complete. The mixture was poured
into petrol/ethyl acetate (50 ml, 10:1) and filtered through a bed
of silica gel. The solvent was evaporated and the product was
purified by column chromatography eluting with petrol/ethyl
acetate (10:1) to give (R)-2-[(R)-1-(tert-butyldimethylsilanyloxy)-
19-oxononadecyl]tetracosanoic acid methyl ester as a colourless
(i) Lithium bis(trimethyl silyl)amide (6.1 ml, 6.65 mmol,
1.06 M) was added to a stirred solution of (R)-2-[(R)-1-(tert-
butyldimethylsilanyloxy)-3-oxopropyl]tetracosanoic acid methyl
ester (1.66 g, 2.91 mmol) and 2,2-dimethylpropionic acid 16-
(1-phenyl-1H-tetrazole-5-sulfonyl)-hexadecyl
ester
(2.34 g,
4.37 mmol) in dry THF (30 ml) at −5 ◦C. The reaction turned
bright yellow and was left to reach r.t. and stirred for 1 h under
nitrogen, when TLC showed no starting material was left. The
reaction was quenched by addition of sat.aq NH4Cl at 0 ◦C. The
product was extracted with petrol/ethyl acetate (10:1) (3× 50 ml).
The combined organic layers were dried over MgSO4, filtered
and evaporated; the crude product was purified by column
chromotography over silica gel, eluting with petrol/ethyl acetate
(10:1) to give (E,Z)-(R)- 2-((R-1-(tert-butyldimethylsilyloxy)-19-
(pivaloyloxy)nonadec-3-enyl)tetracosanoic acid methyl ester
(2.45 g, 95%) as a 2:1 mixture of isomers; (ii) Palladium on 10%
carbon (0.5 g, 10%) and the ester from (i) (2.1 g; 2.39 mmol),
in IMS (50 ml) and THF (20 ml) was stirred under hydrogen
atmosphere. Work up as before and the product was purified
by column chromatography eluting with petrol:ethyl acetate
(10:1) to give (R)-2-[(R)-1-(tert-butyldimethylsilanyloxy)-19-(2,2-
dimethylpropionyloxy)nonadecyl]tetracosanoic acid methyl ester
oil (0.23 g, 92%), [␣]D −1.09 (c 1.35, CHCl3) (Found [M+Na]+:
24
816.48, C50H100O4SiNa requires: 816.41 (MALDI)); this showed
ıH: 9.77 (1H, t, J 1.9 Hz), 3.92 (1H, br.td, J 6.6, 4.75 Hz), 3.66 (3H, s),
2.55 (1H, ddd, J 10.7, 6.95, 3.5 Hz), 2.44 (2H, dt, J 7.55, 1.9 Hz), 1.63
(2H, pent., J 7 Hz), 1.55–1.20 (72H, br. m), 0.88 (3H, t, J 6.6 Hz), 0.87
(9H, s), 0.04 (3H, s), 0.02 (3H, s); ıC: 202.9, 175.13, 73.22, 51.58,
51.21, 43.91, 33.68, 31.92, 29.82, 29.69, 29.65, 29.58, 29.43, 29.35,
29.17, 27.83, 27.48, 25.76, 23.71, 22.67, 22.09, 17.97, 14.10, −4.37,
−4.93; ꢀmax: 2924, 2853, 1741, 1464, 1372, 1249, 1167, 1048, 836,
775 cm−1
.
3.11. Methyl (2R,3R,Z)-3-(tert-butyldimethylsilyloxy)
-2-docosyltetracont-21-enoate (10)
20
7 as a colourless oil (1.8 g, 85%), [␣]D −11.19 (c 1.51, CHCl3)
(Found [M+Na]+: 901.8007 C55H110O5SiNa requires: 901.8015).
This showed ıH: 4.04 (2H, t, J 6.6 Hz), 3.9 (1H, br. td, J 7, 4.75 Hz),
3.65 (3H, s), 2.53 (1H, ddd, J 10.7, 6.9, 3.45 Hz), 1.64–1.59 (2H,
m), 1.57–1.24 (74H, m), 1.2 (9H, s), 0.89 (3H, t, J 7 Hz), 0.86
(9H, s), 0.04 (3H, s), 0.02 (3H, s); ıC: 178.57, 175.06, 73.24,
64.44, 51.59, 51.15, 38.71, 33.71, 31.92, 29.82, 29.69, 29.65,
Sodium
bis(trimethylsilyl)amide
(1.75 ml,
1.57 mmol,
1.0 M in THF) was added to a stirred solution of nonadecyl-
triphenylphosphonium bromide (0.476 g, 0.76 mmol) at room
temperature under nitrogen atmosphere. The mixture was stirred
for 30 min then (R)-2-[(R)-1-(tert-butyldimethylsilanyloxy)-19-
oxononadecyl]tetracosanoic acid methyl ester (0.31 g, 0.39 mmol)

M. Muzael et al. / Chemistry and Physics of Lipids 163 (2010) 678–684
683
in dry THF (15 ml) was added. The mixture was stirred for 3 h
3.14. (2R,3R,Z)-2-Docosyl-3-hydroxytetracont-21-enoic acid
(13)
when TLC showed no starting material was left, then the reac-
tion was quenched with sat. aq. NH4Cl (10 ml) and the product
was extracted with petrol:ethyl acetate (20:1) (3 × 20 ml). The
combined organic layers were dried over MgSO4, filtered and
the solvent was evaporated. The product was purified by chro-
matography over silica gel, eluting with petrol/ethyl acetate
(40:1) to give methyl (2R,3R,Z)-3-(tert-butyldimethylsilyloxy)-2-
Lithium hydroxide monohydrate (0.3 g, 5.24 mmol) was added
to a stirred solution of methyl ester 12 (0.32 g, 0.3 mmol) in THF
(10 ml), methanol (1 ml) and water (1.5 ml) at r.t. The mixture was
stirred at 40 ◦C for 18 h, when TLC showed no starting material was
left. The reaction mixture was cooled down to room temperature
and diluted with petrol/ethyl acetate (5:2, 10 ml) and acidified to
pH = 1 with 5% HCl. The product was extracted with petrol/ethyl
acetate (5:2) (3 × 10 ml), and the combined organic layers were
dried over MgSO4 and evaporated to give a crude product which
was purified by column chromatography eluting with petrol:ethyl
acetate (7:2) to give (2R,3R,Z)-2-docosyl-3-hydroxytetracont-21-
enoic acid (0.26 g, 81%) 13 as a white solid, m.p. 65–66 ◦C,
[␣]D21+3.43 (c 0.97, CHCl3) (Found [M+Na]+: 937.91, C62H122NaO3
requires: 937.93 (MALDI)) which showed ıH: 5.34–5.25 (2H, m),
3.70 (1H, dt, J 8.2, 4.95 Hz), 2.43 (1H, td, J 8.8, 5.35 Hz), 1.97 (4H,
q, J 6.65 Hz), 1.64–1.48 (16H, m), 1.21 (92H, br.s), 0.85 (6H, t J
6.6 Hz); ıC: 175.14, 129.90, 72.17, 50.52, 35.56, 31.92, 29.77, 29.70,
29.66, 29.57, 29.48, 29.41, 29.35, 29.31, 27.31, 27.21, 25.71, 22.68,
14.10; ꢀmax: 3530, 2915, 2848, 2360, 1684, 1468, 1378, 1208, 965,
24
docosyltetracont-21-enoate 10 (0.255 g, 62%), [␣]D −2.45 (c 1.18,
CHCl3) (Found [M+Na]+: 1066.0319. C69H138O3Si Na requires:
1066.0307); this showed ıH: 5.35 (2H, dt, J 11.65, 5.65 Hz), 3.91
(1H, br.td, J 7, 4.4 Hz), 3.66 (3H, s), 2.53 (1H, ddd, J 11.05, 7.25,
3.8 Hz), 2.02 (4H, br.q, J 7 Hz), 1.6–1.2 (106H, br. m), 0.89 (6H, t, J
6.6 Hz), 0.87 (9H, s), 0.05 (3H, s), 0.02 (3H, s); ıC 175.14, 129.89,
73.22, 51.57, 51.21, 33.67, 31.92, 29.83, 29.77, 29.70, 29.61, 29.58,
29.56, 29.44, 29.36, 29.31, 27.83, 27.50, 27.21, 25.75, 23.68, 22.69,
17.97, 14.11, −4.37, −4.93; ꢀmax: 2923, 2852, 1741, 1468, 1437,
1361, 1179, 1120, 836, 775, 720, 695 cm−1
.
3.12. Methyl (2R,3R,E/Z)-3-(tert-butyldimethylsilyloxy)
-2-docosyltetracont-21-enoate (11)
717 cm−1
.
The same procedure was followed as above in order
to couple aldehyde (0.19 g, 2.44 mmol) and 5-(nonadecane-1-
sulfonyl)-1-phenyl-1H-tetrazole (0.17 g, 3.66 mmol) using lithium
bis(trimethylsilyl)amide (0.5 ml, 5.38 mmol, 0.5 ml), which was
added at −10 ◦C. The product was purified by column chro-
matography eluting with petrol/ethyl acetate (40:1), to give the
title compounds 11 (0.18 g, 70%) (Found [M+Na]+: 1066.0248.
C69H138O3SiNa requires: 1066.0308). This showed ıH: (major iso-
mer) 5.4–5.38 (2H, m), 3.86 (1H, br.dt, J 6.65, 4.7 Hz), 3.61 (3H,
s), 2.48 (1H, ddd, J 11, 7.25, 3.75 Hz), 1.93–1.90 (4H, br.q, J 6 Hz),
1.5–1.15 (104H, m), 0.84 (6H, t, J 7 Hz), 0.82 (9H, s), 0.00 (3H, s),
−0.025 (3H, s); (minor isomer) 5.36–5.34 (2H, m), 1.97 (4H, br.q,
J 6.65 Hz) (the remaining signals obscured by the major isomer);
ıC (both isomers): 175.14, 130.36 (trans isomer), 129.89 (cis iso-
mer), 73.22, 51.57, 51.2, 33.68, 32.6, 31.92, 29.83, 29.78, 29.7, 29.66,
29.6, 29.58, 29.57, 29.54, 29.53, 29.44, 29.36, 29.32, 29.2, 27.82, 27.5,
27.2, 25.75, 23.69, 22.68, 22.6, 17.97, 14.1, −4.38, −4.94; ꢀmax: 2922,
2851, 2360, 1741, 1464, 1361, 1252, 1193, 1165, 1070, 1005, 965,
Acknowledgement
We thank the Government of Iraq for the award of a studentship
to M.M.
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stirred in dry THF (10 ml) in dry polyethylene vial under nitrogen
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added and the mixture was stirred for 18 h at 40 ◦C. The reaction
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