Synthesis of wax esters and related trehalose esters from Mycobacterium avium and other mycobacteria

Article abstract of DOI:10.1016/j.tet.2016.05.004

The synthesis of mycobacterial mycolic acid related wax esters and of their trehalose di- and mono-esters is described. The trehalose dimycolates (TDMs) synthesised activated bone marrow derived dendritic cells (BMDCs) in?vitro more strongly than trehalos

Full text of DOI:10.1016/j.tet.2016.05.004

Tetrahedron xxx (2016) 1e14
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Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis of wax esters and related trehalose esters from
Mycobacterium avium and other mycobacteria
,
Salam G. Taher a y, Juma’a R. Al Dulayymi a, H. Giresse Tima b, Hanan M. Ali a,
,
Marta Romano b, Mark S. Baird a
*
a School of Chemistry, Bangor University, Bangor, Gwynedd, Wales, LL57 2UW, UK
b Host-Pathogen Interactions, Scientific Service Immunology, O.D. Communicable and Infectious Diseases, Scientific Institute of Public Health (WIV-ISP),
B1180 Brussels, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of mycobacterial mycolic acid related wax esters and of their trehalose di- and mono-esters
is described. The trehalose dimycolates (TDMs) synthesised activated bone marrow derived dendritic
cells (BMDCs) in vitro more strongly than trehalose dibehenate or the trehalose monomycolates (TMMs).
The inflammatory effects were similar to those of TDM from either a synthetic keto- or methoxy-mycolic
Received 15 February 2016
Received in revised form 17 April 2016
Accepted 3 May 2016
Available online xxx
acid, but somewhat stronger than those of a TDM from an
ester TDM were similar to those of the methoxy-MA and
a-mycolic acid. In vivo, the effects of one wax
-mycolic acid TDMs and trehalose dibehenate.
a
Keywords:
Wax ester
Ó 2016 Elsevier Ltd. All rights reserved.
Mycobacterium avium
TDM
TMM
Trehalose dimycolate
Trehalose monomycolate
Inflammation
1. Introduction
from the hydroxy acid in mycolic acids 2 is S,S,13,19e21 while other
reports identify a R-stereochemistry for the three stereocentres of
Mycobacterial cell walls show unusually low permeability,
a factor which apparently contributes to their resistance to ther-
apeutic agents. This is linked to an exceptionally thick monolayer
formed by the packing of esters of C60eC90 fatty acids.1e7 These
‘mycolic acids’ (MA), exemplified by structures 1e3, contain vari-
the
et al.]22 In the case of Mycobacterium tuberculosis, the MA are of
three main types, -MA (1), methoxy-MA (2) and keto-MA (3)
a
-methyl-trans-epoxy unit.15,16 [but see also Al Kremawi
a
(Scheme 1), though other mycobacteria contain different func-
tional groups in the mero-chain. The detailed composition of the
mixture can provide a fingerprint of the specific mycobacterium,
or even of different strains or stages of development of a single
bacterium. The fingerprint can be used directly to demonstrate
infection.23
In the case of Mycobacterium avium there are no methoxy-MA,
but these are replaced by so called ‘wax-esters’. Over 60 years
ago, Anderson, in an epic series of papers, initiated studies on
mycobacterial lipids and reported the isolation of two new optically
active long-chain alcohols from the neutral fraction of the saponi-
fied waxes of the so called ‘timothy bacillus’,2 later classified as
Mycobacterium phlei.1,2,4,6 These alcohols were identified as d-2-
ous structural features including cis-cyclopropanes,
trans-cyclopropanes, -methyl- -methoxy and -methyl-
groups,8e14 cis-alkene,
-methyl-trans-alkene and -methyl-trans-
a
-methyl-
a
a
b
a
a
b
-keto
epoxy fragments,15,16 and they are generally present as mixtures of
homologues differing by two methylene-units. Each contains
a common R,R-b
-hydroxy acid group,14,17e20 though less is known
about the absolute stereochemistries of the other groups. There is
evidence that the 1-methyl-2-methoxy unit at the distal position
* Corresponding author. Fax: þ44 1248 3702528; e-mail address: chs028@
eicosanol ([
a
]
þ3.5) and d-2-octadecanol ([
a
]
þ5.7). It was
D
D
bangor.ac.uk (M.S. Baird).
noted that the acidic fraction from these saponified waxes con-
tained a high molecular weight component, tentatively identified
y
Current address: Chemistry Department, Faculty of Science and Health, Koya
University, Iraq
http://dx.doi.org/10.1016/j.tet.2016.05.004
0040-4020/Ó 2016 Elsevier Ltd. All rights reserved.
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2
S.G. Taher et al. / Tetrahedron xxx (2016) 1e14
wax ester MA; others, such as M. avium ssp. avium and M. avium ssp.
paratuberculosis, contain , keto and wax ester MA. Others, such as
Mycobacterium komossense and Mycobacterium heckeshornense,
produce
, keto, methoxy and wax ester MA.38,40 HPLC of MA from
a
a
some strains of Mycobacterium gordonae showed double cluster
patterns due to the presence of dicarboxy mycolates after saponi-
fication.41 Wax esters MA are known to be a characteristic com-
ponent in the M. aviumeMycobacterium intracellulare group and
other rapidly growing bacteria.42,43 Their presence in Mycobacte-
rium smegmatis has been implied,44 although not confirmed by
a later study.39 Usually they have been analysed as the corre-
sponding
a
-methyl-alkanol and free acid after hydrolysis.32,39,45
Some such wax esters, such as those from M. aurum, do not ap-
pear to contain cyclopropanes,46 while in other cases the compo-
sition is not clear.5,25e27,40,47e50
The detection of intact trehalose dimycolate (TDM) containing
a wax ester mycolate has been achieved by mass spectrometry.
Three types of TDM were observed, one containing two a-mycolate
residues, one two wax esters, and one having one residue of each
type.34 The analysis of the intact trehalose monomycolate (TMM)
derivatives by MALDI-TOF mass spectrometry shows predominant
ions due to C85 and C87 wax esters from M. aviumeM. intracellulare,
and C80, C81, C82 and C83 for M. phlei and Mycobacterium flavescens.51
Direct observation of the corresponding wax ester trehalose
dimycolate (TDM) species is also reported.42
Yano et al. have reported that TDM isolated from M. tuberculosis
is antigenic to antibodies present in the serum of patients infected
with tuberculosis, and that the sub-class of TDM based on
methoxy-MA is recognised more strongly than those based on a-
MA or keto-MA.52e55 Antibodies in the serum of patients infected
with TB responded less strongly to TDM isolated from M. avium
complex (MAC), while antibodies in serum of MAC infected pa-
tients showed the opposite response pattern. Disease caused by
MAC is an important opportunistic pulmonary infection; the
clinical symptoms of MAC pulmonary disease and TB resemble
each other, but the treatment is different, so rapid distinction
between the infections is desirable. The use of a serodiagnostic
assay based on the selective antigenicity of TDM and TMM offers
a clear opportunity to solve this problem, and considerable work
has been carried out in this area, using extracts from different
types of mycobacterial cell.56e60 Although TDM from M. tubercu-
losis or Mycobacterium bovis induces significant resistance against
influenza or M. tuberculosis infections when combined with mur-
amyl dipeptide, TDM from M. avium, did not show the same effect.
Moreover, while TDM from Mtb confers resistance to Toxoplasma
gondii infections, that from M. avium does not.61 There are major
differences in toxicity and granulomatogenic activity in mice of
TDMs from different mycobacteria, depending on the balance of
different classes of MA and wax ester.62 In addition to the prob-
lems in diagnosing MAC infection in humans, M. avium ssp. para-
tuberculosis is the etiologic agent of Johne’s disease, one of the
most widespread bacterial diseases of domestic animals.63 Given
that one major difference between the constituents of M. tuber-
culosis and M. avium is that the former contains methoxy-MA, but
not normally wax ester MA, while the latter contains wax-ester
MA but not methoxy-MA, the synthesis of both wax esters and
of the corresponding sugar esters may allow a number of issues to
be resolved. We have already reported the synthesis of the di-
Scheme 1. Typical structures of mycolic acids 1e3, and related wax esters 4 and wax
dicarboxylic acid 5.
as being dibasic.24 A careful analysis of the firmly bound lipids from
avian tubercle bacilli (M. avium) again yielded long-chain alcohols,
with d-2-eicosanol as main component.24 These lipid fractions also
produced a long-chain di-acid, recognized for the first time as
a mycolic acid derivative and given the title g-mycolic acid ([a]
D
25e28
þ5.3).24 The pioneering work of Etemadi et al.,
and of Min-
ꢀ
nikin and Polgar,8,9 who isolated methyl avium mycolate ([
a]
D
þ3.05) from M. avium strains Dn, 485 and 7169, and provided mass
spectrometric evidence for structures and molecular weights, led to
a clearer understanding of the presence of esters of these acids,
apparently derived by a biological Baeyer-Villiger reaction on keto-
MA.4,13,29,31,32
Wax esters from Mycobacterium avium ssp. paratuberculosis
were partially characterised as 4 and the corresponding diacids 5,30
and found as a constituent of trehalose mycolates of M. phlei.33,34
The relationship between the keto-MA and the wax ester MA in
M. phlei and Mycobacterium aurum was examined by radio-
labelling, which showed that the keto-MA appeared first in ex-
tractable lipids, and the wax ester MA first in wall-linked de-
rivatives.35 The mass relationship was probed directly by MALDI-
MS.36 In parallel studies, similar alcohols and acids were charac-
terized from an organism claimed to be the causative agent of
leprosy.37 It is clear, however, that this bacterium was not the
leprosy bacillus, as Mycobacterium leprae has not been cultivated to
date and the mycolic acid composition of M. leprae is distinct.38 The
use of two dimensional TLC provided additional evidence of the
presence of wax esters in other mycobacteria.39 In this way, the
distribution of various classes of MA could be characterised. Some
carboxylic acid fragment of a wax ester containing an a-methyl-
trans-cyclopropane;64 we now report the synthesis of three
complete wax esters and of the derived trehalose esters. Com-
pound 6a (Scheme 2) is reported to be the major wax ester of M.
avium;43 compound 6b, with reversed chain lengths is reported to
be the major isomer in M. gordonae.41 In addition, the longer chain
wax ester 6c was prepared in order to study the effects of chain
length on bioactivity (Scheme 2).
mycobacteria, e.g., Mycobacterium vaccae, contain a, a
0, keto and
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S.G. Taher et al. / Tetrahedron xxx (2016) 1e14
3
In order to generate the dicarboxylic acid part of the wax ester,
the aldehyde (12) needed to be coupled to an R,R- -hydroxyacid
b
fragment (15), with a pendant C22 chain. This was prepared from
13,66 using the standard methods (Scheme 5).
Scheme 2. Typical chain lengths of some mycobacterial wax esters and di-acids.
2. Results and discussion
The trans-cyclopropane fragment (9) (Scheme 3), prepared as
described earlier was coupled to the sulfone (8) in a modified Julia-
Kocienski reaction, followed by saturation of the derived mixture of
E/Z-alkenes using di-imide. This led to the chain extended ester
(10).
Scheme 5. (i) 1-phenyl-1H-tetrazole-5-thiol, Ph3P, DEAD, dry THF, (85%) (ii) MCPBA,
CH2Cl2 (86%).
Reaction of the aldehyde (12) with sulfone (15) and base, fol-
lowed by saturation of the derived alkenes provided the fragment
(16) (Scheme 6).
Scheme 3. (i) LHMDS, 8, dry THF (85%); (ii) dipotassium azodicarboxylate, THF-
methanol, CH3COOH (96%).
Replacement of the silyl protecting group of (10) by a tetrahy-
dropyranyl group, was followed by conversion of the ester into
aldehyde (12) (Scheme 4).
Scheme 6. (i) LHMDS, dry THF (78%); (ii) dipotassium azodicarboxylate, THF, metha-
nol, CH3COOH (91%).
The tetrahydropyranyl protecting group was removed from es-
ter (16) and the resulting alcohol was oxidised to produce aldehyde
(17). Coupling of this with the sulfone (18) and base followed by
saturation of the derived alkenes provided the
mono-ester (19) (Scheme 7).
u
-dicarboxylic acid
In order to generate the full wax ester, the
u-dicarboxylic acid
mono-ester (19) needed to be coupled to (S)-2-eicosanol (21). This
was synthesized by reacting the Grignard reagent from
1-bromoheptadecane with (S)-1-epoxypropane (20) to give the
desired alcohol (21) (Scheme 8).
The
u-dicarboxylic acid mono-ester (19) was esterified with S-
2-eicosanol (21) through a Steglisch reaction (Scheme 9). Removal
of the silyl ether and methyl ester protection was achieved in two
steps to produce the free wax ester mycolic acid (6a).
Scheme 4. (i) nBu4NF, dry THF (96%); (ii) pyridinium-p-toluenesulfonate (PTSA), 3,4-
dihydro-2H-pyran, dry CH2Cl2 (98%); (iii) LiAlH4, dry THF (94%); (iv) PCC, CH2Cl2 (93%).
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4
S.G. Taher et al. / Tetrahedron xxx (2016) 1e14
The u-dicarboxylic acid (23) has also been isolated from the cell
wall as a component of a complex mixture.43 Compound (23),
a2D1þ5.8 (c 0.86, CHCl3), was also synthesized starting from the
u-
dicarboxylic acid mono-ester (19) after TBDMS deprotection and
hydrolysis (Scheme 10). The acid showed the typical signals for the
a
-methyl-trans-cyclopropane in the 1H NMR spectrum, together
with the typical signals for the
acid.
a- and b-hydrogens of the hydroxy
Scheme 10. (i) HF-Pyridine, dry THF, 17 h (85%); (ii) LiOH, THF, water-MeOH, 17 h
(85%).
Scheme 7. (i) PTSA, MeOH (84%); (ii) PCC, dichloromethane (93%); (iii) LHMDS, 18, dry
THF (80%); (iv) dipotassium azodicarboxylate, THF, methanol, CH3COOH (80%).
The acid 6a was then converted into the corresponding TDM and
TMM using methods described earlier.67,68 The protected wax ester
mycolate 24 was prepared from the corresponding free hydroxy
wax ester 6a by reaction with an excess of tert-butyldimethylsi-
lylchloride and imidazole in the presence of 4-dimethyl-
aminopyridine for 24 h at 70 ꢀC, followed by hydrolysis of the
TBDMS ester on the acid group by stirring in THF for 15 min with 4%
aqueous tetrabutylammonium hydroxide. Compound 24 was cou-
pled to protected trehalose (25) using 1-(3-dimethylaminopropyl-
3-ethylcarbodiimide hydrochloride, DMAP and molecular sieves in
CH2Cl2 (Scheme 11). This gave the protected TDM 26 (52%) and the
protected TMM 27 (32%).
Scheme 8. (i) CH3(CH2)16MgBr, CuI, dry THF (53%).
The TDM was de-protected in two steps to give 28 and 29, and
the TMM was deprotected in the same way to give 30 and 31
(Schemes 12 and 13).
Using a similar method, the second wax ester, 6b was prepared
and converted into the corresponding TDM (32) and TMM (33)
(Scheme 14), while wax ester 6c was converted into the corre-
sponding TDM (described in full in the Supplementary data).
3. Wax ester TDM and TMM are inflammatory in vitro and
in vivo
The ability of synthetic wax ester TDM and TMM to activate
murine bone marrow derived dendritic cells (BMDCs) in vitro was
examined. For that purpose, BMDCs were stimulated with 1 mM of
the different wax esters and controls (evaporated isopropanol as
a negative control and trehalose dibehenate as a positive control)
for 24 h and the production of pro-inflammatory cytokines (TNF-
IL-6 and IL-1 ) was analysed by ELISA in the supernatant. All the
tested compounds induced the production of pro-inflammatory
cytokines. The levels of TNF- , IL-1 and IL-6 induced by wax es-
a,
b
a
b
Scheme 9. (i) DCC, DMAP, dry CH2Cl2 (80%); (ii) HF-pyridine complex, dry THF (71%);
ter TMMs 31 and 33 were significantly lower compared to the levels
induced by the corresponding wax ester TDMs 29 and 32 and TDB
(Fig. 1A), indicating that the number of mycolate chains bound to
trehalose influences the activation of BMDCs in terms of pro-
inflammatory cytokine secretion. When a dose-response analysis
was performed using pairs of TMM and TDM composed of the same
wax ester, we confirmed that at equimolar concentrations TDM is
more inflammatory than TMM and TDB (Fig. 1B). These data are in
(iii) 5% aq nBu4NOH (60%).
The product 6a, a2D1þ7.6 (c 0.40, CHCl3), showed the character-
istic apparent sextet for the hydrogen adjacent to the ester at
d
4.91
(1H, J 6.2 Hz), as well as the signals for the a- and b-hydrogens of
the hydroxy acid at 3.72 (1H, dt, J 4.8, 9.2 Hz), 2.46 (1H, dt, J 5.3,
9.0 Hz), and a triplet for the methylene group adjacent to the ester
carbonyl at 2.27 (2H, t, J 7.5 Hz).
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S.G. Taher et al. / Tetrahedron xxx (2016) 1e14
5
ꢁ
Scheme 11. EDCI, DMAP, 4 A molecular sieves, CH2Cl2, 6 days at ambient temperature.
Scheme 12. (i) nBu4NF, dry THF; (ii) HF-pyridine complex, dry THF.
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6
S.G. Taher et al. / Tetrahedron xxx (2016) 1e14
Scheme 13. (i) nBu4NF, dry THF; (ii) HF-pyridine complex, dry THF.
Scheme 14. TDM and TMM from 6b.
accordance with our previous findings on trehalose and glucose
mycolate esters.71 Wax ester TDM 31 induced a comparable level of
and TDB can induce a significant footpad swelling 3 days after in-
jection as compared to vehicle control. The level of inflammation
induced was comparable for the four compounds (Fig. 2).
TNF-a to TDMs from a cis-cyclopropane containing methoxy-
mycolic acid (Supplementary data, S1) or a cis-cyclopropane con-
taining keto-mycolic acid (S3). These three compounds induced
more TNF-a than a TDM from a di-cis-cyclopropane containing a-
mycolic acid (S2) in vitro (Fig.1C) (for structures see Supplementary
data).
4. Conclusion
We have described the first syntheses of complete mycobacte-
rial wax esters in a process that can be adjusted to provide any
required absolute stereochemistry or chain length. The wax esters
The in vivo inflammatory potential of synthetic wax ester TDM
(29) was further compared to the synthetic cis-cyclopropane
methoxy and a-TDMs. The compounds were formulated in a water
in oil in water emulsion and the preparations were injected sub-
cutaneously in the two hind footpads of C57BL/6 mice. The footpad
swelling was measured with a caliper as read out for local in-
flammation. The data showed that all synthetic TDM compounds
are initially produced as methyl esters at the
these may be selectively hydrolysed to the free
b
-hydroxy-ester, but
-hydroxy-acid
b
without cleavage of the wax ester part, an eicosanol ester. Selective
protection of the hydroxyl-group using trimethylsilyl allowed the
coupling of the wax ester to a protected trehalose. Following
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S.G. Taher et al. / Tetrahedron xxx (2016) 1e14
7
A
3 0 0 0 0
2 0 0 0 0
1 0 0 0 0
1 5 0 0
1 0 0 0
5 0 0
0
3 0 0
2 0 0
1 0 0
0
bdl
bdl
0
O
O
S
B
9
1
2
B
9
2
3
B
9
1
2
3
S O
2
3
3
3 3
S
3 1
3
2
3 3
D
T
I
I
I
T D
T D
M
M
M
2
M
M
3
M
M
M
3
M
D M
D M
T
M M
M
M
T
T D
T
T D
T D
T D
T M
T
T M
T M
T
B
C
1 5 0 0 0
1 0 0 0 0
5 0 0 0
0
1 5 0 0 0
1 0 0 0 0
5 0 0 0
0
T D M 29
T M M 31
T D B
bdl
O
B
)
9
)
1
3 )
2
IS
S
S 2
T D
S
(
M
(
D
M
(
M
M
D
r
T
T D
T D
e
A
st
A
T
0
1
1
1
1
.
0
e
-
M
M A
0
0
-
-
.
-M
y
x
o
0
.0
h a
x
e t
0
w a
p
o
k
l
-
s
h
t
-a
i
c
s
m e
ci
C o n c e n tra tio n (μ M )
s-
ci
Fig. 1. Wax ester TDM and TMM activate BMDCs in vitro: BMDCs derived from C57BL/6 mice were stimulated for 24 h in triplicate with 1
plate-coated synthetic wax ester TDMs 29 and 32 or TMMs 31 and 33, synthetic TDMs containing cis-cyclopropane methoxy-MA (S1), keto-MA (S3) or
(structures in Supplementary data), or controls (trehalose dibehenate (TDB) or evaporated isopropanol (ISO)). The supernatants were harvested separately and the amount of pro-
inflammatory TNF- , IL-1
mM or the indicated concentration of
a-mycolic acid (S2)
a
b
and IL-6 was determined by sandwich ELISA. Results are expressed as mean pg/ml of cytokines ꢁSD and representative of at least three independent
experiments (bdl: below detection limit).
deprotection, this led to the free trehalose dimycolate and mono-
mycolate of the wax ester.
5
The wax-esters are immuno-stimulatory in vitro and in vivo and
the level of inflammation induced is at least comparable to that
induced by TDB. The applications of the wax ester derivatives as
antigens for the serodiagnosis of diseases caused by mycobacterial
infections will be described elsewhere.
***
***
***
***
4
3
2
1
0
5. Experimental section
5.1. General
n s
n s
n s
Chemicals used were obtained from commercial suppliers
(Sigma, Aldrich, and Alfa Aeser) or prepared from them by the
methods described. Solvents which were required to be dry, e.g.,
ether, THF were dried over sodium wire and benzophenone under
nitrogen, while dichloromethane and HMPA were dried over cal-
cium hydride. All reagents and solvents used were of reagent grade
unless otherwise stated. Silica gel (Merck 7736) and silica gel plates
used for column chromatography and thin layer chromatography
were obtained from Aldrich; separated components were detected
using variously UV light, I2 or phosphomolybdic acid solution in
IMS followed by charring. Anhydrous magnesium sulfate was used
to dry organic solutions. Infra-red (IR) spectra were carried out on
a PerkineElmer 1600 FTIR spectrometer as liquid films or KBr disc
(solid). Melting points were measured using a Gallenkamp melting
e
B
9
2
)
cl
2 )
1
S
h i
T D
S
(
M
D
V e
M
M
(
T
D
D
e r
t
A
T
A
T
e s
M
-
M
-
-
x
a
y
a
h
x
w
p
o
l
a
th
-
e
s
i
c
m
s-
ci
Fig. 2. Wax ester TDM induces inflammation in vivo: Groups of 4e5 C57BL/6 mice
were injected s.c in the hind footpads with w/o/w emulsions composed of 3.2% In-
complete Freund’s Adjuvant and 5
S2. In the emulsion of the vehicle control group no glycolipid was present, in the TDB
control group 5 g/footpad of TDB replaced synthetic glycolipid in the emulsion.
mg/footpad of synthetic mycolate esters 29, S1 and
m
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8
S.G. Taher et al. / Tetrahedron xxx (2016) 1e14
point apparatus. NMR spectra were carried out on a Bruker Avance
400 or 500 spectrometer. Specific rotations were recorded in CHCl3
on a POLAAR 2001 Optical Activity Polarimeter. Mass spectra were
recorded on a Bruker matrix-assisted laser desorption/ionisation-
time of flight mass spectrometry (MALDI-TOF MS) values are
given plus sodium to an accuracy of 1 d.p.; accurate mass values
obtained in Bangor were run on a Bruker LC-MS and those using
MALDI-TOF MS were determined by Dr. Paul Gates in Bristol
University.
29.6, 29.5, 29.3, 29.2, 26.6, 25.9, 25.0,19.9, 18.8,16.9,10.6,10.0; nmax/
cmꢂ1: 3369, 2923, 2853, 1742, 1459, 1170.
(ii) Pyridinium-p-toluenesulfonate (1.01 g, 4.01 mmol) was
added to a stirred solution of the above alcohol (3.05 g, 7.98 mmol)
and 3,4-dihydro-2H-pyran (1.67 g, 2.96 mmol) in dry CH2Cl2
(40 mL) under nitrogen at rt. The reaction was stirred for 30 min,
then quenched with satd aq NaHCO3 (20 mL). The product was
extracted with CH2Cl2 (2ꢃ75 mL) and dried. The solvent was
evaporated and the product was purified by column chromatog-
raphy eluting with petrol/ethyl acetate (10:1) to give the title
compound 11 as a colourless oil, a mixture of diastereoisomers
(3.7 g, 98%), aD21þ19 (c 0.75, CHCl3) [Found (M)þ: 466.4011, C29H54O4
requires: 466.4022]; dH (400 MHz, CDCl3): 4.57 (1H, br m),
3.88e3.79 (2H, m), 3.68 (3H, s), 3.48 (2H, tdd, J 5.8, 10.2, 16.1 Hz),
2.31 (2H, t, J 7.6 Hz), 1.87e1.71 (1H, m), 1.69e1.64 (2H, m), 1.58e1.54
(7H, m), 1.37e1.20 (25H, m), 1.18e1.11 (1H, m), 0.95 (3H, d, J 6.8 Hz),
0.90e0.80 (1H, m), 0.51e0.43 (1H, m), 0.18e0.14 (3H, m); dC
(101 MHz, CDCl3): 174.4, 99.0, 98.9, 66.2, 65.9, 62.4, 51.4, 37.2, 37.1,
35.3, 35.2, 34.4, 34.1, 30.9, 29.74, 29.7, 29.62, 29.6, 29.5, 29.3, 29.2,
26.0, 25.9, 25.5, 25.0, 19.83, 19.8, 18.62, 18.6, 10.6; nmax/cmꢂ1: 2924,
2853, 1743, 1454.
5.2. Methyl 16-((1S,2R)-2-((S)-4-((tert-butyldiphenylsilyl)oxy)
butan-2-yl)cyclopropyl)hexadecanoate (10)
LHMDS (17.8 mL, 18.9 mmol, 1.06 M) was added dropwise to
a
stirred solution of (1S,2R)-2-[(S)-3-(tert-butyldiphenylsilany-
loxy)-1-methylpropyl]cyclopropanecarbaldehyde (4.01 g,
9
10.5 mmol)64 with 16-(1-phenyl-1H-tetrazole-5-sulfonyl)-penta-
decanoic acid methyl ester 8 (5.86 g, 12.6 mmol)65 in dry THF
(75 mL) under nitrogen at ꢂ15 ꢀC. The mixture was stirred for 1 h at
room temperature, then cooled to 0 ꢀC and quenched with satd aq
ammonium chloride (30 mL). The organic layer was treated with
petrol/ethyl acetate 10:1 (50 mL), and the aqueous layer was re-
extracted with petrol/ethyl acetate 10:1 (2ꢃ25 mL). The com-
bined organic layers were dried and evaporated; the product was
purified by column chromatography eluting with petrol/ethyl ac-
etate (20:1) to give a colourless oil, methyl (E/Z)-16-((1R,2S)-2-(4-
((tert-butyldiphenylsilyl)oxy)-2-methylbutan-2-yl)cyclopropyl)
hexadec-15-enoate as a 2:1 mixture (5.6 g, 85%). Dipotassium
azodicarboxylate (5.03 g, 26.2 mmol) was added to a stirred solu-
tion of the above alkenes (5.55 g, 8.98 mmol) in THF (75 mL) and
methanol (5 mL) at 5 ꢀC. Glacial acetic acid (5 mL) in THF (5 mL) was
added dropwise over 72 h. The mixture was poured into satd aq
sodium bicarbonate, and then extracted with ethyl acetate
(3ꢃ150 mL). The combined organic layers were dried and evapo-
rated to give a thick oil; column chromatography eluting with
petrol/ethyl acetate (20:1) gave compound 10 as a colourless oil
(5.4 g, 96%), a2D1þ7.9 (c 0.91, CHCl3) [Found (M)þ: 620.4645,
5.4. 16-((1S,2R)-2-((2S)-4-((Tetrahydro-2H-pyran-2-yl)oxy)bu-
tan-2-yl)cyclopropyl)-hexadecanal (12)
(i) The ester 11 (3.63 g, 7.78 mmol) in THF (10 mL) was added to
a stirred suspension of LiAlH4 (0.440 g, 11.7 mmol) in THF (60 mL) at
ꢂ10 ꢀC under nitrogen and then refluxed for 1 h, then quenched
with satd aq sodium sulfate decahydrate at ꢂ10 ꢀC until a white
precipitate had formed. The mixture was stirred at rt for 30 min,
filtered through a pad of Celite and the solvent was evaporated to
give the product which was purified by column chromatography
eluting with petrol/ethyl acetate (3:1) to give 16-((1S,2R)-2-((2S)-4-
((tetrahydro-2H-pyran-2-yl)oxy)butan-2-yl)cyclopropyl)hexa-
decan-1-ol as a colourless oil, a mixture of diastereoisomers (3.4 g,
94%), a2D1þ19 (c 0.94, CHCl3) [Found (M)þ: 438.4084, C28H54O3 re-
quires: 438.4073]; dH (400 MHz, CDCl3): 4.57 (1H, br m), 3.94e3.77
(2H, m), 3.64 (2H, t, J 6.6 Hz), 3.56e3.39 (2H, m), 1.92e1.64 (3H, m),
1.59e1.54 (7H, br m), 1.34e1.26 (28H, br m), 1.17 (1H, m), 0.96 (3H,
d, J 6.7 Hz), 0.90e0.77 (1H, m), 0.58e0.40 (1H, m), 0.28e0.07 (3H,
m); dC (101 MHz, CDCl3): 99.0, 98.9, 67.7, 66.2, 65.9, 63.0, 62.4, 37.2,
37.1, 35.3, 35.2, 34.4, 32.8, 30.9, 30.8, 29.7, 29.65, 29.62, 29.6, 29.4,
C
40H64O3Si requires: 620.4625]; dH (400 MHz, CDCl3): 7.68e7.66
(4H, m), 7.42e7.36 (6H, m), 3.76e3.71 (2H, m), 3.67 (3H, s), 2.30
(2H, t, J 7.6 Hz), 1.7e1.48 (6H, m), 1.43e1.26 (26H, br m), 1.05 (9H, s),
0.89 (3H, s), 0.42 (1H, m), 0.13 (2H, m); dC (101 MHz, CDCl3): 174.2,
135.9, 134.2, 129.57, 127.5, 62.4, 51.4, 40.2, 34.8, 34.4, 34.1, 29.7,
26.0, 25.9, 25.8, 25.5, 19.82, 19.81, 19.8, 18.64, 18.6, 10.6; nmax/cmꢂ1
:
29.63, 29.6, 29.4, 29.3, 29.2, 26.9, 25.9, 25.0, 19.9, 18.6, 10.6; nmax
/
3392, 2923, 2852, 1468.
cmꢂ1: 2925, 2854, 1742, 1428, 1111.
(ii) The above alcohol (3.25 g, 7.41 mmol) in CH2Cl2 (10 mL) was
added to stirred suspension of PCC (3.99 g, 18.6 mmol) in CH2Cl2
(80 mL) and stirred for 2 h, then diluted with petrol/ethyl acetate
10:1 (50 mL), and filtered through a pad of silica gel and Celite. The
solvent was evaporated and the product was purified by column
chromatography eluting with petrol/ethyl acetate (10:1) to give the
title compound 12 as a colourless oil, a mixture of diastereoisomers
(3.0 g, 93%); a2D1þ5.23 (c 1.01, CHCl3) [Found (M)þ: 436.3911,
5.3. Methyl 16-((1S,2R)-2-((2S)-4-((tetrahydro-2H-pyran-2-yl)
oxy)butan-2-yl)cyclopropyl)hexa-decanoate (11)
(i) Tetra-n-butylammonium fluoride (10.2 mL, 10.2 mmol) was
added to a stirred solution of ester 10 (5.30 g, 8.54 mmol) in dry THF
(70 mL) at 0 ꢀC under nitrogen. The mixture was allowed to reach
room temperature and stirred for 5 h, then cooled to 5 ꢀC and
quenched with satd aq ammonium chloride (30 mL). The product
was extracted with ethyl acetate (3ꢃ150 mL), then the combined
organic layers were washed with brine (100 mL), dried and evap-
orated to give a crude product, which was purified by column
chromatography eluting with petrol: ethyl acetate (5:1) to give
methyl 16-((1S,2R)-2-((S)-4-hydroxybutan-2-yl)cyclopropyl)hexa-
decanoate as a colourless oil (3.1 g, 96%), a2D1þ14 (c 0.93, CHCl3)
[Found (M)þ: 382.3467, C24H46O3 requires: 382.3447]; dH
(400 MHz, CDCl3): 3.78e3.69 (2H, m), 3.67 (3H, s), 2.30 (2H, t, J
7.6 Hz), 1.76e1.67 (1H, m), 1.63e1.60 (2H, m), 1.59e1.52 (1H, m),
1.37e1.20 (26H, m), 1.18e1.11 (1H, m), 0.96 (3H, d, J 6.6 Hz),
0.90e0.81 (1H, m), 0.51e0.44 (1H, m), 0.25e0.13 (3H, m); dC
(101 MHz, CDCl3): 174.8, 61.4, 51.4, 40.4, 35.0, 34.4, 34.1, 29.7, 29.63,
C28H52O3 requires: 436.3916]; dH (400 MHz, CDCl3): 9.77 (1H, t, J
1.9 Hz), 4.61e4.53 (1H, br m), 3.95e3.77 (2H, m), 3.56e3.40 (2H,
m), 2.42 (2H, td, J 7.4, 11 Hz), 1.80e1.75 (3H, m), 1.59e1.53 (7H,
br m), 1.34e1.26 (26H, br m), 0.96 (3H, d, J 6.9 Hz), 0.91e0.78 (1H,
m), 0.59e0.41 (1H, m), 0.29e0.10 (3H, m); dC (101 MHz, CDCl3):
203.0, 99.0, 98.9, 66.2, 65.9, 62.4, 37.2, 37.0, 35.3, 35.2, 34.4, 30.9,
30.8, 29.73, 29.7, 29.65, 29.62, 29.6, 29.4, 25.9, 25.5, 19.82, 19.8,
18.61, 18.6, 10.6; nmax/cmꢂ1: 2923, 2852, 1728, 1455.
5.5. Methyl (R)-2-((R)-1-((tert-butyldimethylsilyl)oxy)-3-((1-
phenyl-1H-tetrazol-5-yl)-thio)propyl)tetra-cosanoate (14)
Diethyl azodicarboxylate (DEAD) (2.38 g, 13.7 mmol) in dry THF
(8 mL) was added to a stirred solution of methyl (R)-2-((R)-1-((tert-
Please cite this article in press as: Taher, S. G.; et al., Tetrahedron (2016), http://dx.doi.org/10.1016/j.tet.2016.05.004

S.G. Taher et al. / Tetrahedron xxx (2016) 1e14
9
butyldimethylsilyl)oxy)-3-hydroxypropyl)tetracosanoate 13 66 (6.0 g,
a mixture of diastereoisomers (4.6 g, 91%), a2D1ꢂ8.6 (c 0.91, CHCl3)
[MALDI-Found (MþNa)þ: 997.8949, C62H122NaO5Si requires:
997.8954], dH (400 MHz, CDCl3): 4.58 (1H, br m), 3.97e3.76 (3H, m),
3.67 (3H, s), 3.56e3.40 (2H, m), 2.53 (1H, ddd, J 3.7, 7.1, 10.8 Hz),
1.75e1.64 (3H, m), 1.61e1.06 (84H, br m), 0.96 (3H, d, J 6.6 Hz),
0.90e0.87 (12H, m, including t at 0.87 with J 6.9 Hz), 0.57e0.40 (1H,
m), 0.27e0.11 (3H, m), 0.05 (3H, s), 0.02 (3H, s); dC (101 MHz, CDCl3):
175.2, 99.0, 98.9, 73.2, 66.2, 65.9, 62.4, 51.5, 51.4, 37.4, 37.2, 37.1, 35.3,
35.2, 34.4, 32.8, 31.9, 30.8, 29.8, 29.7, 29.63, 29.60, 29.5, 29.4, 29.3,
27.9, 27.7, 27.6, 25.9, 25.7, 25.5, 23.7, 22.7, 22.6, 20.4, 19.8, 11.2, 10.0,
ꢂ4.4, ꢂ4.9; nmax/cmꢂ1:, 2924, 2853, 1741, 1464.
10.5 mmol), triphenylphosphine (3.58 g, 13.7 mmol) and 1-phenyl-
1H-tetrazole-5-thiol (2.43 g, 13.9 mmol) in dry THF (70 mL) at 0 ꢀ
C
under nitrogen atmosphere. The mixture was allowed to reach rt and
then stirred for 3 h. The solvent was evaporated and the residue was
stirred with petrol/ethyl acetate (10:1, 150 mL) for 30 min and then
filtered through a pad of Celite. The filtrate was evaporated and the
crude product was purified by column chromatography eluting with
petrol/ethyl acetate (10:1)to give the title compound 14 asa thick pale
yellow oil (6.5 g, 85%), aD21e13.2 (c 1.06, CHCl3) [Found (MꢂBut)þ:
673.4535 calculated for C37H65N4O3SiS: 673.4547]; dH (400 MHz,
CDCl3): 7.59e7.52 (5H, m), 4.07 (1H, m), 3.67 (3H, s), 3.51e3.44 (1H,
m), 3.40e3.34(1H, m), 2.59(1H, ddd,J 4.8,6.9,11.4Hz), 2.16e2.09(1H,
m), 2.01e1.93 (1H, m), 1.59e1.21 (42H, br m), 0.90e0.87 (12H, m,
including t at 0.87, J 6.9 Hz), 0.08 (3H, s), 0.06 (3H, s); dC (101 MHz,
CDCl3): 174.4,154.1,130.1,129.8,123.8, 72.0, 51.5, 51.48, 33.1, 31.9, 29.7,
5.8. Methyl (R)-2-((R)-1-((tert-butyldimethylsilyl)oxy)-19-
((1S,2R)-2-((S)-4-oxobutan-2-yl)cyclo-propyl)nonadecyl)tetra-
cosanoate (17)
29.66, 29.6, 29.5, 29.4, 28.6, 27.9, 27.1, 25.7, 22.7,14.1, ꢂ4.4, ꢂ4.9; nmax
/
(a) Pyridinium-p-toluenesulfonate (0.58 g, 2.3 mmol) was added
to a stirred solution of methyl tetrahydropyranyl acetal 16 (4.51 g,
cmꢂ1: 2918, 2852, 1741, 1498, 1459, 1161.
4.62 mmol) in THF (50 mL), methanol (10 mL) and stirred at 45 ꢀ
C
5.6. Methyl (R)-2-((R)-1-((tert-butyldimethylsilyl)oxy)-3-((1-
phenyl-1H-tetrazol-5-yl)-sulfonyl)propyl)-tetracosanoate (15)
for 6 h. Satd aq NaHCO3 (20 mL) and water (20 mL) were added and
extracted with ethyl acetate (3ꢃ50 mL). The combined organic
layers were dried and the solvent was evaporated. Column
chromatography eluting with petrol/ethyl acetate (12:1) gave
m-Chloroperoxybenzoic acid (7.87 g, 32.1 mmol) was added to
a stirred solution of ester 14 (7.80 g, 10.7 mmol) in CH2Cl2 (50 mL),
followed by addition of NaHCO3 (3.59 g, 42.7 mmol). The mixture
was stirred for 24 h at rt, then poured into sat.aq NaHCO3 (150 mL),
and stirred for 2 h. The product was extracted with CH2Cl2
(3ꢃ100 mL), and the combined organic layers were dried and
evaporated to give a crude product which was purified by column
chromatography eluting with petrol/ethyl acetate (10:1) to give the
title compound 15 as a white solid (5.0 g, 86%), a2D1ꢂ14.4 (c 1.12,
CHCl3) [Found (MꢂBut)þ: 705.4431; calculated for C37H65N4O5SiS:
705.4445]; dH (400 MHz, CDCl3): 7.72e7.7 (2H, m), 7.65e7.61 (3H,
m), 4.15 (1H, m), 3.81 (2H, m), 3.69 (3H, s), 2.53 (1H, ddd, J 4.0, 7.4,
11.0 Hz), 2.16e2.09 (2H, m), 1.59e1.21 (42H, br m), 0.90e0.87 (12H,
m, including t at 0.87, J 6.9 Hz), 0.08 (3H, s), 0.06 (3H, s); dC (101 MHz,
CDCl3): 174.1, 153.2, 133, 131.5, 129.7, 125.0, 70.9, 51.8, 51.6, 51.4, 31.9,
29.7, 29.66, 29.62, 29.6, 29.5, 29.4, 29.3, 27.7, 27.4, 26.2, 25.7, 22.70,
14.1, ꢂ4.5, ꢂ5.1; nmax/cmꢂ1: 2920, 2852, 1729, 1497, 1467, 1155.
methyl
(R)-2-((R)-1-((tert-butyldimethylsilyl)oxy)-19-((1S,2R)-2-
((S)-4-hy-droxybutan-2-yl)cyclopropyl)nonadecyl)tetracosanoate
(3.5 g, 84%), a2D1þ7.9 (c 1.1, CHCl3) [MALDI-Found (MþNa)þ:
913.8369; C57H114NaO4Si requires 913.8384]; dH (400 MHz, CDCl3);
3.92 (1H, ddd, J 4.9, 7.6, 11.8 Hz), 3.80e3.68 (2H, m), 3.66 (3H, s),
2.53 (1H, ddd, J 3.8, 7.1, 10.9 Hz), 1.72 (1H, td, J 6.8, 13.5 Hz),
1.54e1.26 (81H, br m), 0.96 (3H, d, J 6.5 Hz), 0.91e0.84 (12H, m,
including t at 0.87 with J 6.9 Hz), 0.55e0.44 (1H, m), 0.29e0.13 (3H,
m), 0.05 (3H, s), 0.03 (3H, s); dC (10 MHz, CDCl3): 175.2, 73.2, 61.4,
51.6, 51.2, 40.4, 35.0, 34.4, 33.7, 31.9, 31.6, 29.8, 29.72, 29.7, 29.63,
29.6, 29.59, 29.5, 29.4, 29.1, 27.8, 27.7, 27.65, 27.5, 25.9, 25.8, 25.3,
23.7, 22.7, 22.66, 21.0, 20.5, 19.8, 18.8, 17.9, ꢂ4.4, ꢂ4.9; nmax/cmꢂ1
:
3584, 2924, 2853, 1740, 1463, 1066.
(b) The above alcohol (3.25 g, 3.65 mmol) in CH2Cl2 (20 mL) was
added to a stirred suspension of PCC (2.36 g, 10.9 mmol) in CH2Cl2
(120 mL), stirred for 2 h, then diluted with petrol/ethyl acetate
(10:1,100 mL), and filtered through a pad of silica gel and Celite. The
solvent was evaporated and the product was purified by column
chromatography eluting with petrol/ethyl acetate (10:1) to give the
title compound 17 as a colourless oil (3.0 g, 93%), a2D1þ1.6 (c 1.06,
CHCl3) [MALDI-Found (MþNa)þ: 912.2; C57H112NaO4Si requires
911.8]; dH (400 MHz, CDCl3): 9.79 (1H, t, J 2.5 Hz), 3.92 (1H, dt, J 5.9,
7.9 Hz), 3.66 (3H, s), 2.50e2.48 (2H, m), 2.38 (1H, ddd, J 2.5, 7.7,
15.7 Hz), 1.54e1.26 (79H, br m), 1.03 (3H, d, J 6.8 Hz), 0.92e0.80
(12H, m, including t at 0.87 with J 6.9 Hz), 0.50e0.48 (1H, m),
0.38e0.19 (3H, m), 0.05 (3H, s), 0.02 (3H, s); dC (101 MHz, CDCl3):
202.9, 73.2, 51.6, 51.5, 34.1, 33.9, 33.7, 31.9, 29.8, 29.73, 29.7, 29.6,
29.5, 29.4, 27.8, 27.72, 27.7, 27.5, 25.9, 25.8, 25.3, 23.7, 22.7, 22.66,
21.0, 20.5, 19.8, 18.82, 14.1, 11.4, ꢂ4.4, ꢂ4.9; nmax/cmꢂ1: 2924, 2853,
1738, 1464.
5.7. Methyl (2R)-2-((1R)-1-((tert-butyldimethylsilyl)oxy)-19-
((1S,2R)-2-((2S)-4-((tetra-hydro-2H-pyran-2-yl)oxy)butan-2-
yl)cyclopropyl)nonadecyl)tetracosanoate (16)
LHMDS (11.1 mL, 11.7 mmol, 1.06 M) was added dropwise to
a stirred solution of sulfone 15 (5.97 g, 7.84 mmol) and aldehyde 12
(2.85 g, 6.53 mmol) in dry THF (50 mL) under nitrogen at ꢂ15 ꢀC. The
mixture was stirred for 1 h at room temperature, then cooled to 0 ꢀC
and quenched with satd aq ammonium chloride (30 mL). The or-
ganic layer was extracted with petrol/ethyl acetate 10:1 (50 mL), and
the aqueous layer was re-extracted with petrol/ethyl acetate (10:1,
2ꢃ25 mL). The combined organic layers were dried and evaporated
to give a crude product, which was purified by column chromatog-
raphy eluting with petrol/ethyl acetate (20:1) to give methyl (E/Z)-
(2R)-2-((1R,E)-1-((tert-butyldimethylsilyl)oxy)-19-((1S,2R)-2-
((2S)-4-((tetrahydro-2H-pyran-2-yl)oxy)butan-2-yl)cyclopropyl)-
nonadec-3-en-1-yl)tetracosanoate as a diastereomeric mixture in
ratio 2:1 (5.0 g, 78%). Dipotassium azodicarboxylate (2.52 g,
13.2 mmol) was added to a stirred solution of the above alkene
mixture (4.95 g, 5.09 mmol) in THF (75 mL) and methanol (5 mL) at
5.9. 15-((1-Phenyl-1H-tetrazol-5-yl)sulfonyl)pentadecanoic
acid (18)
(a) Aq sodium hydroxide (8M) 100 mL was added with stirring
to methyl 15-((1-phenyl-1H-tetrazol-5-yl)thio)pentadecanoate
(10.5 g, 93.8 mmol)69 in THF (100 mL), followed by addition of
MeOH (15 mL). The mixture was refluxed for 30 min, then the
solvent was evaporated, and the residue was dissolved in water
(50 mL), and then acidified with aq HCl (2M) to pH 2. The
aqueous layer was extracted with ethyl acetate (3ꢃ100 mL), and
the combined organic layers were dried and evaporated to give
crude product. Re-crystalization from ethyl acetate gave 15-((1-
5
ꢀC. Glacial acetic acid (5 mL) in THF (5 mL) was added dropwise
over 72 h. The mixture was poured into satd aq sodium bicarbonate
solution, and extracted with ethyl acetate (3ꢃ100 mL). The com-
bined organic layers were dried and evaporated to give a thick oil
residue, which was purified by column chromatography eluting
with petrol/ethyl acetate (20:1) to give the title compound 16 as
Please cite this article in press as: Taher, S. G.; et al., Tetrahedron (2016), http://dx.doi.org/10.1016/j.tet.2016.05.004

10
S.G. Taher et al. / Tetrahedron xxx (2016) 1e14
phenyl-1H-tetrazol-5-yl)thio)pentadecanoic acid as a white solid
(9.0 g, 89%), mp 78e80 ꢀC {[MALDI-Found] (MþNa)þ: 441.4;
5.11. (S)-Eicosan-2-ol (21)
C
22H34N4O2S requires 441.2}; dH (400 MHz, CDCl3): 7.74e7.59
Bromoheptadecane (5.01 g,15.6 mmol) was dissolved in dry THF
(5 mL) and then added slowly to a stirred suspension of magnesium
turnings (2.26 g, 94.1 mmol) in dry THF (8 mL) and warmed gently
until the Grignard reagent started to form. The mixture was
refluxed for 1 h. The Grignard reagent was added slowly to a stirred
suspension of copper iodide (0.980 g, 5.17 mmol) in dry THF
(40 mL) at ꢂ30 ꢀC and stirred for 30 min. (S)-1-Epoxypropane
(0.40 g, 6.89 mmol) in dry THF (5 mL) was added dropwise at
ꢂ30 ꢀC. The mixture allowed to reach room temperature and stir-
red for 16 h, then quenched with ammonium chloride and then
extracted with ethyl acetate (3ꢃ100 mL). The combined organic
layers were dried and evaporated to give the crude product; col-
umn chromatography eluting with petrol/ethyl acetate (10:1) gave
(S)-eicosan-2-ol 21 (1.1 g, 53%). Mp 61e63 ꢀC, aD21þ3.8 (c 1.01,
CHCl3); dH (400 MHZ, CDCl3): 3.80 (1H, sextet, J 6 Hz), 1.48 (2H, m),
1.38e1.25 (32H, br m), 1.2 (3H, d, J 6.2 Hz), 0.88 (3H, t, J 6.7 Hz); dC
(101 MHz, CDCl3): 68.2, 39.4, 31.9, 29.7, 29.66, 29.63, 29.6, 29.4,
25.8, 23.5, 22.5, 14.1; nmax/cmꢂ1: 3386, 2924, 2853, 1464.
(5H, m), 3.40 (2H, t, J 7.4 Hz), 2.36 (2H, t, J 7.5 Hz), 1.82 (2H,
quintet, J 7.4 Hz), 1.66e1.64 (2H, quintet, J 7.4 Hz), 1.48e1.41 (2H,
quintet, J 7.4 Hz), 1.30e1.26 (19H, m); dC (101 Hz, CDCl3): 130.1,
129.8, 123.9, 33.7, 33.4, 29.6, 29.5, 29.49, 29.4, 29.3, 29.28, 29.1,
29.04, 28.6, 24.7; nmax/cmꢂ1: 3436, 2918, 2849, 1725, 1594, 1496,
1463, 1413, 1381, 1172.
(b) A solution of ammonium molybdate (VI) tetrahydrate
(7.60 g, 6.14 mmol) in 35% H2O2 (15 mL) was prepared and cooled
in an ice bath, it was added to a stirred solution of the above acid
(5.01 g, 12.3 mmol) in THF (30 mL) and IMS (30 mL) at 10 ꢀC, and
stirred at rt for 2 h. A further solution of ammonium molybdate
(VI) tetrahydrate (3.80 g, 3.07 mmol) in 35% H2O2 (8 mL) was
added and the mixture was stirred at rt for 18 h. The mixture was
poured into water (250 mL) and extracted with ethyl acetate
(3ꢃ75 mL). The combined organic layers were dried and the sol-
vent was evaporated. The product was purified by recrystallisation
from MeOH/acetone (1:1) to give the title compound 18 aþs a white
solid (4.2 g, 78%), mp 88e89 ꢀC [MALDI-Found (MþNa) : 472.9;
C
22H34N4 O4S requires 473.2]; dH (400 MHz, CDCL3): 7.74e7.70 (2H,
5.12. Methyl (R)-2-((R)-1-((tert-butyldimethylsilyl)oxy)-19-
((1S,2R)-2-((S)-19-((S)-eicosan-2-yloxy)-19-oxoneonadecan-2-
yl)cyclopropyl)nonadecyl)tetracosanoate (22)
m), 7.66e7.61 (3H, m), 3.74 (2H, t, J 8.2 Hz), 2.36 (2H, t, J 7.5 Hz),
1.96 (2H, quintet, J 7.4 Hz), 1.66 (2H, quintet, J 7.4 Hz), 1.48 (2H,
quintet, J 7.4 Hz), 1.30e1.26 (19H, m); dC (101 MHz, CDCl3): 131.5,
129.7, 125.1, 56.1, 33.8, 29.5, 29.42, 29.4, 29.2, 29.17, 29.0, 28.9, 28.1,
24.7, 22.0; nmax/cmꢂ1: 3467, 2921, 2851, 1694, 1597, 1466, 1359,
1152.
Acid 19 (2.41 g, 2.16 mmol), (S)-eicosan-2-ol 21 (0.67 g,
2.22 mmol) and DMAP (0.39 g, 3.22 mmol) were dissolved in dry
CH2Cl2 (15 mL) under nitrogen at room temperature. DCC (0.88 g,
4.3 mmol) in dry CH2Cl2 (7 mL) was added dropwise over 15 min
and stirred at rt for 3 h, then diluted with DCM, filtered and
evaporated. Column chromatography eluting with petrol/ethyl ac-
etate (20:1) gave the title compound 22 as a colourless oil (2.4 g,
80%), a2D1þ6.1 (c 0.90, CHCl3) [Found (MþNa)þ: 1418.3659;
5.10. (S)-18-((1R,2S)-2-((19R,20R)-19-((tert-Butyldimethyl-
silyl)oxy)-20-(methoxycarbonyl)dotetracontyl)cyclopropyl)-
nonadecanoic acid (19)
C92H182NaO5Si requires 1418.3654]; dH (400 MHz, CDCl3): 4.91 (1H,
Lithium bis(trimethylsilyl)amide (9.40 mL, 9.97 mmol, 1.06 M)
was added dropwise to a stirred solution of ester 17 (2.95 g,
3.32 mmol) and sulfone 18 (1.79 g, 3.98 mmol) in dry THF (40 mL)
under nitrogen at ꢂ15 ꢀC. The mixture was stirred for 1 h at room
temperature, then cooled to 0 ꢀC and quenched with satd aq am-
monium chloride (30 mL). The organic layer was extracted with
petrol/ethyl acetate (10:1, 40 mL), and the aqueous layer was re-
extracted with petrol/ethyl acetate (10:1, 2ꢃ20 mL). The com-
bined organic layers were dried and evaporated; the product was
purified by column chromatography eluting with 20:1 petrol/ethyl
acetate to give (E/Z)-18-((1R,2S)-2-((19R,20R)-19-((tert-butyldime-
thylsilyl)-oxy)-20-(methoxycarbonyl)dotetracontyl)cyclopropyl)-
nonadec-15-enoic acid (3.0 g, 80%) as a mixture in ratio (2.3:1);
dipotassium azodicarboxylate (3.75 g, 19.5 mmol) was added to
a stirred solution of the above alkene (2.9 g, 2.6 mmol) in THF
(60 mL) and methanol (5 mL) at 5 ꢀC. Glacial acetic acid (5 mL) in
THF (5 mL) was added dropwise over 72 h. The mixture was poured
in to satd aq sodium bicarbonate, then extracted with ethyl acetate
(3ꢃ100 mL). The combined organic layers were dried and evapo-
rated. The product was purified by column chromatography eluting
with petrol/ethyl acetate (10:1) to give the title compound 19 as
a semi-solid (2.6 g, 80%), aD21þ4.1 (c 0.48 CHCl3) {[MALDI-Found]
(MþNa)þ: 1138.0530; C72H142NaO5Si requires 1138.0519}; dH
(400 MHz, CDCl3): 3.92 (1H, dt, J 4.8, 7.1 Hz), 3.66 (3H, s), 2.53 (1H,
ddd, J 3.8, 7.2, 10.9 Hz), 2.36 (2H, t, J 7.5 Hz), 1.64 (3H, m), 1.57e1.03
(106H, br m), 0.94e0.83 (15H, m, including t at 0.87 with J 6.9 Hz),
0.73e0.62 (1H, m), 0.51e0.40 (1H, m), 0.26e0.07 (3H, m), 0.05 (3H,
s), 0.02 (3H, s); dC (101 MHz, CDCl3): 178.8, 175.2, 73.2, 68.2, 60.4,
51.6, 51.2, 38.7, 38.1, 37.4, 33.9, 33.7, 32.7, 31.9, 29.7, 29.6, 29.4, 29.3,
27.6, 27.5, 27.3, 25.8, 25.7, 23.7, 22.7, 21.0, 19.3, 19.2, 18.6, 18.0, 14.10,
14.0, 11.4, 10.5, 10.3, ꢂ4.4, ꢂ4.9; nmax/cmꢂ1: 2924, 2853, 1743, 1711,
1464.
sextet, J 6.2 Hz), 3.92 (1H, dt, J 4.8, 7.1 Hz), 3.66 (3H, s), 2.53 (1H,
ddd, J 3.8, 7.1, 10.9 Hz), 2.27 (2H, t, J 7.5), 1.54e1.26 (142H, br m), 1.20
(3H, d, J 6.2 Hz), 0.91 (3H, d, J 6.8 Hz), 0.91e0.87 (15H, m, including
two t at 0.87 with J 7 Hz), 0.71e0.63 (1H, m), 0.49e0.41 (1H, m),
0.22e0.08 (3H, m), 0.05 (3H, s), 0.02 (3H, s); dC (101 MHz, CDCl3):
175.2, 173.6, 73.2, 70.7, 51.6, 51.2, 38.1, 37.4, 36.0, 34.8, 34.5, 33.7,
31.9, 30.1, 29.83, 29.8, 29.75, 29.73, 29.7, 29.6, 29.57, 29.52, 29.5,
29.4, 29.3, 29.2, 27.8, 27.5, 27.2, 26.2, 25.8, 25.4, 25.1, 23.8, 23.7, 22.7,
20.0, 19.7, 18.6, 18.0, 11.0, 10.5, ꢂ4.4, ꢂ4.9; nmax/cmꢂ1: 2924, 2853,
1738, 1464.
5.13. (R)-2-((R)-1-Hydroxy-19-((1S,2R)-2-((S)-19-((S)-eicosan-
2-yloxy)-19-oxononadecan-2-yl)cyclo-propyl)nonadecyl)tet-
racosanoic acid (6a)
(a) Ester 22 (2.30 g, 1.79 mmol) was dissolved in dry THF (30 mL)
in a dry polyethylene vial equipped with a rubber septum, followed
by addition of pyridine (0.7 mL) at rt under nitrogen. The mixture
was cooled to 10 ꢀC, and then HF-pyridine complex as w70%
(4.0 mL) was added dropwise. The mixture was stirred at 43 ꢀC for
17 h, then neutralized by pouring slowly into satd aq NaHCO3 until
no more CO2 was liberated. The product was extracted with petrol/
ethyl acetate (5:1, 3ꢃ30 mL), then the combined organic layers
were dried and evaporated. Chromatography eluting with petrol/
ethyl acetate (10:1) gave methyl (R)-2-((R)-1-hydroxy-19-((1S,2R)-
2-((S)-19-((S)-eicosan-2-yloxy)-19-oxononadecan-2-yl)cyclopropyl)-
nonadecyl)tetracosanoate as a colourless thick oil (1.5 g, 71%),
a2D1þ7.3 (c 0.11, CHCl3) [MALDI-Found (MþNa)þ: 1304.2782;
C86H168O5Na requires 1304.2784]; dH (400 MHz, CDCl3): 4.91 (1H,
sextet, J 6.2 Hz), 3.71 (3H, s), 3.66 (1H, m), 2.53 (1H, m), 2.27 (2H, t, J
7.5 Hz), 1.54e1.26 (143H, br m), 1.20 (3H, d, J 6.2 Hz), 0.90 (3H, d, J
6.8 Hz), 0.91e0.87 (6H, t, including two t with J 6.9 Hz), 0.71e0.63
Please cite this article in press as: Taher, S. G.; et al., Tetrahedron (2016), http://dx.doi.org/10.1016/j.tet.2016.05.004

S.G. Taher et al. / Tetrahedron xxx (2016) 1e14
11
(1H, m), 0.49e0.41 (1H, m), 0.22e0.08 (3H, m); dC (101 MHz,
5.15. (R)-2-((R)-1-((tert-Butyldimethylsilyl)oxy)-19-((1S,2R)-2-
CDCl3):176.2, 173.6, 72.3, 70.7, 51.6, 51.2, 38.1, 37.4, 36.0, 34.8, 34.5,
33.7, 31.9, 30.1, 29.6, 29.5, 27.8, 27.5, 27.2, 26.1, 25.8, 25.4, 25.1, 23.8,
23.7, 22.7, 20.1, 19.7, 18.6, 18.0, 14.1, 10.5; nmax/cmꢂ1: 3441, 2918,
2850, 1734, 1732, 1466.
((S)-19-((S)-eicosan-2-yloxy)-19-oxo-nonadecan-2-yl)cyclo-
propyl)nonadecyl)tetracosanoic acid (24)
Imidazole (0.302 g, 4.507 mmol) was added to a stirred solution
of acid 6a (0.560 g, 0.448 mmol) in dry DMF (3.5 mL) and dry tol-
uene (5 mL) at rt followed by the addition of TBDMSCl (0.675 g,
4.482 mmol) and DMAP (0.055 g, 0.448 mmol). The mixture was
stirred at 70 ꢀC for 18 h, then the solvent was removed under high
vacuum and the residue was diluted with petrol/ethyl acetate (5:1,
30 mL) and water (20 mL). The organic layer separated and the
aqueous layer was re-extracted with petrol/ethyl acetate (5:1,
2ꢃ20 mL). The combined organic layers were washed with water,
dried and evaporated. The residue was dissolved in THF (8 mL), to
this was added aq nBu4NOH (4.5 mL, 4%). The mixture was stirred
for 15 min at room temperature, and then diluted with water (5 mL)
and petrol/ethyl acetate (2:1, 20 mL). The organic layer was sepa-
rated and the aqueous layer was re-extracted with petrol/ethyl
acetate (2ꢃ20 mL). The combined organic layers were dried and
evaporated to give a crude product; chromatography eluting with
petrol/ethyl acetate (20:1) gave the title compound 24 as a syrup
(0.53 g, 87%), a2D1þ23 (c 0.50, CHCl3) [MALDI-Found (MþNa)þ:
1404.8; C91H180NaO5Si requires 1404.3]; dH (400 MHz, CDCl3): 4.92
(1H, sextet, J 6.2 Hz), 3.83 (1H, m), 2.53 (1H, ddd, J 2.8, 6, 9.1 Hz),
2.27 (2H, t, J 7.4 Hz), 1.81e0.95 (143H, br m), 1.20 (3H, d, J 6.2 Hz),
0.993 (9H, s), 0.90 (3H, d, J 6.8 Hz), 0.91e0.87 (6H, t, including two t
with J 6.9 Hz), 0.70e0.40 (1H, m), 0.48e0.41(1H, m), 0.15 (3H,s),
0.12 (3H, s), 0.22e0.06 (3H, m); dC (101 MHz, CDCl3): 179.7, 173.6,
73.7, 70.7, 60.4, 50.1, 38.1, 37.4, 36.0, 35.5, 34.8, 34.5, 31.9, 30.1, 29.7,
29.5, 29.45, 29.4, 29.36, 29.3, 29.2, 27.3, 27.26, 26.1, 25.8, 25.4, 25.1,
(b) The above alcohol (1.42 g, 1.11 mmol) was dissolved in aq
nBu4NOH (64 mL, 5%) and refluxed at 100 ꢀC for 24 h. The
product was extracted with petroleum/ether (5:2, 3ꢃ75 mL),
dried and evaporated. Chromatography eluting with petrol/ethyl
acetate (5:1) gave the title compound 6a as a white solid (0.84 g,
60%), a2D1þ7.6 (c 0.40, CHCl3), mp 55e56 ꢀC [MALDI-Found
(MþNa)þ: 1290.2617; C85H166O5Na requires 1290.2627]; dH
(400 MHz, CDCl3): 4.91 (1H, sextet, J 6.2 Hz), 3.72 (1H, dt, J 4.8,
9.2 Hz), 2.46 (1H, dt, J 5.3, 9.0 Hz), 2.27 (2H, t, J 7.5 Hz), 1.81e0.95
(144H, br m), 1.20 (3H, d, J 6.2 Hz), 0.90 (3H, d, J 6.8 Hz),
0.91e0.87 (6H, t, including two t with J 6.9 Hz), 0.72e0.63 (1H,
m), 0.50e0.41 (1H, m), 0.22e0.02 (3H, m); dC (101 MHz, CDCl3):
179.6, 173.5, 72.1, 70.8, 50.8, 38.1, 37.4, 36.0, 35.5, 34.8, 34.5, 31.9,
30.1, 29.7, 29.54, 29.5, 29.43, 29.4, 29.3, 29.2, 27.32, 27.3, 26.1,
25.7, 25.4, 25.1, 22.7, 20.0, 19.7, 18.6, 14.1, 10.5; nmax/cmꢂ1: 3432,
2918, 2850, 1721, 1712, 1470.
5.14. (R)-2-((R)-19-((1S,2R)-2-((S)-18-Carboxyoctadecan-2-yl)-
cyclopropyl)-1-hydroxynonadecyl)tetracosanoic acid (23)
(a)
(S)-18-((1R,2S)-2-((19R,20R)-19-((tert-Butyldimethylsilyl)
oxy)-20-(methoxycarbonyl)dotetracontyl)-cyclopropyl)nona-
decanoic acid (0.10 g, 0.09 mmol) was dissolved in dry THF (5 mL) in
a dry polyethylene vial equipped with a rubber septum, and fol-
lowed by addition of pyridine (0.1 mL) at rt under nitrogen. The
mixture was cooled to 10 ꢀC, and then HF-pyridine complex (w70%,
0.4 mL) was added dropwise. The mixture was stirred at 43 ꢀC for
17 h, then poured slowly into satd aq sodium bicarbonate until no
more CO2 was liberated. The product was extracted with petrol/
ethyl acetate (5:1, 3ꢃ10 mL), dried and evaporated; chromatogra-
phy eluting with petrol/ethyl acetate (4:1) gave (S)-18-((1R,2S)-2-
((19R,20R)-19-Hydroxy-20-(methoxycarbonyl)dotetracontyl)-cyclo-
propyl)nona-decanoic acid as a viscous oil (75 mg, 85%), a2D1þ7.8 (c
1.6, CHCl3) [MALDI-Found (MþNa)þ: 1024.6; C66H128NaO5 requires
1023.9]; dH (400 MHz, CDCl3): 3.71 (3H, s), 3.68e3.65 (1H, m), 2.53
(1H, dt, J 4.7, 9.8 Hz), 2.36 (2H, t, J 7.5 Hz), 1.74e1.62 (4H, m),
1.55e1.26 (106 H, m), 0.91e0.86 (6H, d, J 5.4 Hz, including t with J
5.7 Hz), 0.69e0.62 (1H, m), 0.49e0.41 (1H, m), 0.22e0.08 (3H, m);
dC (101 MHz, CDCl3): 178.6, 176.3, 72.3, 60.4, 51.6, 51.0, 38.2, 37.4,
35.7, 34.5, 33.9, 31.9, 30.1, 29.76, 29.73, 29.7, 29.63, 29.6, 29.58,
29.54, 29.5, 29.47, 29.43, 29.4, 27.3, 29.1, 27.4, 27.2, 25.7, 24.7, 22.7,
21.1, 19.7, 18.6, 14.2; nmax/cmꢂ1: 2917, 2849, 1733, 1701, 1464.
(b) The above acid (0.071 g, 0.072 mmol) was added to
a stirred solution of THF (8 mL), water (1 mL) and MeOH (0.8 mL),
followed by the addition of lithium hydroxide monohydrate
(0.044 g, 1.073 mmol, 15 mol eq.). The mixture was heated at
45 ꢀC for 16 h, then diluted with petrol/ethyl acetate (5:1, 4 mL),
and acidified to pH 2 using satd aq KHSO4. The aqueous layer was
extracted with petrol/ethyl acetate (5:1, 3ꢃ5 mL), and the com-
bined organic layers were dried and evaporated to give a crude
product; chromatography eluting with petrol/ethyl acetate (1:3)
gave the title compound 23 as a white solid (59 mg, 85%), mp
76e78 ꢀC, aD21þ5.8 (c 0.86, CHCl3) [MALDI-Found (MþNa)þ:
1009.9520; C65H126NaO5 requires 1009.9497]; dH (400 MHz,
CDCl3): 3.74e3.70 (1H, m), 2.53 (1H, dt, J 4.3, 9.8 Hz), 2.35 (2H, t, J
7.5 Hz), 1.77e1.62 (4H, m), 1.59e1.26 (107 H, m), 0.91e0.86 (6H,
d, J 5.4 Hz, including t with J 5.7 Hz), 0.73e0.68 (1H, m),
0.50e0.44 (1H, m), 0.24e0.10 (3H, m); dC (101 MHz, CDCl3):
180.1, 179.1, 72.2, 38.1, 37.5, 35.6, 34.5, 34.0, 31.9, 30.8, 29.7, 29.6,
29.5, 29.4, 29.1, 27.4, 27.3, 26.2, 25.7, 24.7, 22.7, 19.7, 18.6, 14.0,
10.5; nmax/cmꢂ1: 2917, 2849, 1733, 1701, 1464.
22.7, 22.3, 20.0, 19.7, 18.6, 17.9, 14.1, 14.05, 10.5, ꢂ4.2, ꢂ4.9; nmax
/
cmꢂ1: 3432, 2918, 2850, 1721, 1712, 1470.
5.16. 6-O-[(R)-2-((R)-1-Hydroxy-19-((1S,2R)-2-((S)-19-((S)-
eicosan-2-yloxy)-19-oxononadecan-2-yl)-cyclopropyl)non-
adecyl)tetracosanoate]-a-D
-glucopyranosyl-(1-10)-60-O-[(R)-2-
((R)-1-hydroxy-19-((1S,2R)-2-((S)-19-((S)-eicosan-2-yloxy)-19-
oxononadecan-2-yl)cyclopropyl)nonadecyl)tetracosanoate]-
a-
D
-glucopyranoside (29) and 6-O-[(R)-2-((R)-1-hydroxy)-19-
((1S,2R)-2-((S)-19-((S)-eicosan-2-yloxy)-19-oxononadecan-2-
yl)cyclopropyl)nonadecyl)tetracosanoate]- -glucopyran-
osyl-(1-10)-
-glucopyranoside (31)
a-D
a-D
(i) 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydro-
chloride (EDCI) (242 mg, 1.26 mmol) and DMAP (154 mg,
1.26 mmol) were added to a stirred solution of acid 24 (500 mg,
0.360 mmol), protected trehalose 25 (141 mg, 0.18 mmol)67,70 and
ꢁ
powdered 4 A molecular sieves in dry CH2Cl2 (4 mL) at rt under
nitrogen. The mixture was stirred for 5 days then three spatulas of
silica gel was added and the solvent was evaporated under reduced
pressure to give a residue; chromatography on silica eluting with
petroleum ether/ethyl acetate (25:1) gave first compound 26
(0.33 g, 52%), a2D1þ21 (c 0.50, CHCl3) [MALDI-Found (MþNa)þ
3524.0; C212H426NaO19Si8 requires 3524.0]; dH (400 MHz, CDCl3):
4.92 (2H, sextet, J 6.3 Hz), 4.85 (2H, d, J 3.0 Hz), 4.38 (2H, br, d, J
10.0 Hz), 4.04e3.98 (4H, m), 4.0e3.96 (2H, m) 3.93 (2H, m), 3.52
(2H, t, J 8.9 Hz), 3.38 (2H, dd, J 2.9, 9.3 Hz), 2.55 (2H, ddd, J 3.5, 4.8,
10.1 Hz), 2.26 (4H, t, J 7.5 Hz), 1.56e1.21 (284H, m), 1.20 (6H, d, J
6.2 Hz), 0.88 (18H, s), 0.90 (6H, d, J 6.8 Hz), 0.91e0.87 (12H, t, in-
cluding two t with J 6.9 Hz), 0.72e0.65 (2H, m), 0.48e0.41 (2H, m),
0.22e0.06 (6H, m), 0.16 (18H, s), 0.145 (18H, s), 0.138 (18H, s), 0.062
(12H, s); dC (101 MHz, CDCl3): 173.8, 173.6, 94.8, 73.5, 73.4, 72.8,
71.8, 70.7, 62.4, 60.4, 51.9, 41.3, 38.1, 37.4, 36.0, 34.5, 33.4, 31.9, 29.8,
29.5, 29.44, 29.42, 29.4, 29.3, 29.2, 27.3, 27.2, 26.2, 25.8, 25.4, 25.2,
22.7, 22.3, 20.0, 19.7, 18.6, 18.0, 14.3, 14.2, 14.1, 11.4, 10.5, 1.1, 1.0, 0.15,
ꢂ4.5, ꢂ4.7; nmax/cmꢂ1: 2924, 2854, 1733, 1465, 1375, 1254, 1215,
Please cite this article in press as: Taher, S. G.; et al., Tetrahedron (2016), http://dx.doi.org/10.1016/j.tet.2016.05.004

12
S.G. Taher et al. / Tetrahedron xxx (2016) 1e14
836, 760. The second fraction was glucopyranoside 27 (0.114 g,
32%), a2D1þ28 (c 0.50, CHCl3) [MALDI-Found (MþNa)þ: 2160.5;
mixture was stirred at 43 ꢀC for 17 h, then diluted with THF (5 mL).
The excess of the HF was neutralized with triethylamine (2 mL), and
the solvent was evaporated under high vacuum; column chroma-
tography eluting with CHCl3/MeOH 10:1 gave the glucopyranoside
29 (0.095 g, 58%), a2D1þ31 (c 0.50, CHCl3) [MALDI-Found (MþNa)þ:
2863.6340; C182H350NaO19 requires 2863.6314]; dH (400 MHz,
CDCl3þfew drops of CD3OD): 4.98 (2H, d, J 3.4 Hz), 4.87 (2H, sextet, J
6.4 Hz), 4.68 (2H, br d, J 10.8 Hz), 4.24 (2H, t, J 9.3 Hz), 3.93 (2H, dd, J
3.8, 10.9 Hz), 3.73 (2H, t, J 9.2 Hz), 3.66e3.62 (2H, m), 3.49 (2H, dd, J
3.7, 9.8 Hz), 3.19 (2H, t, J 9.5 Hz), 2.40 (2H, br m), 2.23 (4H, t, J 7.4 Hz),
1.58e1.53 (8H, m), 1.52e1.22 (284 H, m), 1.16 (6H, d, J 6.2 Hz), 0.86
(6H, d, J 6.8 Hz), 0.86e0.82 (12H, t, including two t with J 6.9 Hz),
0.66e0.59 (2H, m), 0.43e0.37 (2H, m), 0.18e0.04 (6H, m); dC
(101 MHz, CDCl3þfew drops of CD3OD): 175.7, 173.6, 95.2, 72.5, 71.1,
70.9, 70.7, 69.8, 64.7, 52.1, 38.0, 37.3, 34.7, 34.4, 33.8, 32.0, 29.7, 29.6,
29.5, 29.42, 29.4, 29.3, 29.2, 29.0, 27.2, 26.0, 25.3, 25.0, 22.7, 22.6,
19.9, 19.5, 18.5, 14.0, 10.4; nmax/cmꢂ1: 3432, 2918, 2850, 1721, 1712,
1470.
C
121H248O15Si7Na requires 2160.6]; dH (400 MHz, CDCl3): 4.92 (2H,
d, J 3 Hz), 4.91 (1H, sextet, J 6.2 Hz), 4.85 (1H, d, J 2.9 Hz), 4.36 (1H,
dd, J 2, 11.6 Hz), 4.08 (1H, dd, J 4.0, 11.7 Hz), 4.04e3.98 (4H, m), 3.85
(1H, dt, J 3.2, 9.5 Hz), 3.69 (2H, m), 3.5 (2H, dt, J 4.6, 9.1 Hz), 3.41 (1H,
dd, J 3.1, 9.3 Hz), 3.38 (1H, dd, J 2.8, 9.1 Hz), 2.55 (1H, ddd, J 3.4, 5.4,
10.3 Hz), 2.27 (2H, t, J 7.4 Hz), 1.73 (1H, br m), 1.68e1.21 (141H, m),
1.20 (3H, d, J 6.2 Hz), 0.88 (9H, s), 0.90 (3H, d, J 6.8 Hz), 0.91e0.87
(6H, t, including two t with J 6.9 Hz), 0.70e0.40 (1H, m), 0.48e0.41
(1H, m), 0.22e0.06 (3H, m), 0.17 (18H, s), 0.15 (18H, s), 0.14 (18H, s),
0.060 (3H, s), 0.052 (3H, s); dC (101 MHz, CDCl3): 174.5, 173.6, 94.5,
94.4, 73.4, 73.3, 72.9, 72.8, 72.7, 72.0, 71.4, 70.7, 62.4, 61.7, 51.8, 38.1,
37.4, 36.0, 34.8, 34.4, 34.1, 33.4, 31.9, 30.1, 29.72, 29.7, 29.68, 29.65,
29.63, 29.6, 29.5, 29.45, 29.4, 28.1, 27.3, 26.3, 26.1, 25.8, 25.4, 25.1,
22.62, 22.6, 22.3, 20.0, 19.7, 18.7, 18.0, 14.1, 14.0, 10.5, 1.1, 1.05, 1.004,
1.0, 0.9, 0.8, 0.2, 0.03, ꢂ4.5, ꢂ4.7; nmax/cm-1: 2924, 2853, 1743,
1464.9, 1251.6, 1163, 1099, 872, 839.
(ii) Tetrabutylammonium fluoride (0.334 mL, 0.334 mmol, 1M)
was added to a stirred solution of glucopyranoside 26 (0.305 g,
0.087 mmol) in dry THF (25 mL) at 5 ꢀC under nitrogen. The mixture
was allowed to reach rt and stirred for 30 min, then the solvent was
evaporated and the residue was purified by column chromatogra-
phy eluting with CHCl3/MeOH (10:1) to give glucopyranoside 28 as
a viscous oil (0.19 g, 71%), a2D1þ26 (c 0.13, CHCl3) [MALDI-Found
(MþNa)þ: 3091.8; C194H378O19Si2Na requires 3091.8]; dH
(400 MHz, CDCl3þfew drops of CD3OD): 5.05 (2H, d, J 3.2 Hz), 4.92
(2H, sextet, J 6.3 Hz), 4.37 (2H, br, dd, J 3.5, 11.3 Hz), 4.21 (2H, br d, J
11.1 Hz), 3.93e3.85 (4H, m), 3.77 (2H, t, J 9 Hz), 3.51 (2H, dd, J 3.4,
9.4 Hz), 3.36 (2H, m), 3.30 (2H, t, J 9.4 Hz), 2.55 (2H, br m), 2.22 (4H,
t, J 7.4 Hz), 1.61e1.24 (288H, m), 1.18 (6H, d, J 6.3 Hz), 0.88 (18H, s),
0.90 (6H, d, J 6.8 Hz), 0.89e0.87 (12H, t, including two t with J
6.9 Hz), 0.68e0.58 (2H, m), 0.49e0.38 (2H, m), 0.20e0.06 (6H, m),
0.02 (6H, s), 0.007 (6H, s); dC (101 MHz, CDCl3þfew drops of
CD3OD): 175.2, 173.8, 93.5, 73.2, 72.8, 70.8, 70.2, 70.1, 67.8, 62.9,
51.6, 50.2, 49.7, 49.5, 49.3, 49.1, 38.0, 37.3, 34.7, 34.4, 33.8, 32.0, 29.7,
29.6, 29.5, 29.42, 29.4, 29.3, 29.2, 26.3, 25.8, 25.4, 25.1, 22.7, 22.4,
20.0, 19.7, 18.6, 18.2, 14.0, 10.4, ꢂ4.6, ꢂ5.0; nmax/cmꢂ1: 3421, 2922,
2853, 1732, 1728, 1465, 1375, 1253, 836, 721.
(v) A dry polyethylene vial equipped with an acid-proof rubber
septum was charged with glucopyranoside 30 (0.062 gm,
0.038 mmol) and pyridine (0.06 mL) in dry THF (10 mL) and stirred
at rt under nitrogen. The mixture was cooled to 10 ꢀC, and then HF-
pyridine complex (w70%, 0.5 mL) was added dropwise. The mix-
ture was stirred at 43 ꢀC for 17 h then worked up and purified as
above, eluting with CHCl3/MeOH (5:1) to give the title glucopyr-
anoside 31 as a white solid (0.035 mg, 58%), a2D1þ26 (c 1.12, CHCl3),
mp 116e117 ꢀC [MALDI-Found (MþNa)þ: 1614.3698; C97H187NaO15
requires 1614.3684]; dH (400 MHz, CDCl3þfew drops of CD3OD):
5.08 (1H, d, J 2.8 Hz), 5.02 (1H, d, J 3.0 Hz), 4.88 (1H, sextet, J 6.3 Hz),
4.69 (1H, d, J 11.3 Hz), 4.22 (1H, t, J 9.1 Hz), 3.99e3.90 (2H, m),
3.88e3.78 (3H, m), 3.63e3.54 (3H, m), 3.5 (1H, dd, J 2.9, 10 Hz), 3.28
(1H, t, J 9.4 Hz), 3.21 (1H, t, J 9.4 Hz), 2.38 (1H, m), 2.23 (2H, t, J
7.5 Hz),1.58e1.50 (4H, m), 1.49e1.21 (147H, m), 1.17 (3H, d, J 6.2 Hz),
0.86 (3H, d, J 6.8 Hz), 0.86e0.83 (6H, t, including two t with J 6.9 Hz),
0.66e0.59 (1H, m), 0.44e0.36 (1H, m), 0.18e0.04 (3H, m); dC
(101 MHz, CDCl3þfew drops of CD3OD): 175.4, 173.9, 94.32, 94.3,
74.4, 73.3, 72.5, 71.4, 71.3, 71.2, 70.7, 64.0, 62.9, 52.4, 38.0, 37.2, 35.7,
34.5, 34.3, 31.7, 30.1, 29.9, 29.5, 29.3, 29.2, 29.1, 28.1, 27.3, 25.3, 25.1,
24.9, 22.6, 19.8, 19.5, 18.7, 14.0, 10.3; nmax/cmꢂ1: 3357, 2919, 2851,
1730, 1467, 1375, 759, 721.
(iii) Tetrabutylammonium fluoride (0.144 mL, 0.144 mmol, 1M)
was added to a stirred solution of glucopyranoside 27 (0.103 g,
0.0480 mmol) in dry THF (13 mL) at 5 ꢀC under nitrogen. The
mixture was allowed to reach rt and stirred for 20 min, then the
solvent was evaporated and the residue was purified by column
chromatography eluting with CHCl3/MeOH (5:1) to give glucopyr-
anoside 30 as a semi-solid (0.073 g, 89%), a2D1þ17 (c 0.78, CHCl3)
[MALDI-Found (MþNa)þ: 1728.3; C103H200O15SiNa requires
1728.4]; dH (400 MHz, CDCl3þfew drops of CD3OD): 5.05 (2H, d, J
3.4 Hz), 4.85 (1H, sextet, J 6.2 Hz), 4.32e4.22 (2H, m), 3.92 (1H, br d,
J 9.6 Hz), 3.86e3.78 (4H, m), 3.67 (1H, m), 3.5 (2H, dd, J 3.4, 9.7 Hz),
3.37e3.25 (3H, m), 2.52 (1H, ddd, J 3.4, 5.4, 10.3 Hz), 2.22 (2H, t, J
7.5 Hz),1.58e1.50 (4H, m),1.49e1.21 (145H, m),1.16 (3H, d, J 6.3 Hz),
0.88 (9H, s), 0.7 (3H, d, J 6.8 Hz), 0.91e0.87 (6H, t, including two t
with J 6.9 Hz), 0.66e0.57 (1H, m), 0.44e0.36 (1H, m), 0.10e0.03
(3H, m), 0.007 (3H, s), 0.015 (3H, s); dC (101 MHz, CDCl3þfew drops
of CD3OD): 175.1, 173.9, 93.5, 93.4, 73.2, 73.0, 72.6, 72.1, 71.5, 70.9,
70.7, 70.2, 69.9, 67.9, 62.4, 61.7, 51.6, 38.0, 37.3, 35.8, 34.7, 33.5, 31.8,
30.0, 29.72, 29.7, 29.6, 29.5, 29.4, 29.39, 29.3, 29.2, 29.0, 27.6, 26.9,
26.1, 25.6, 25.3, 25.0, 24.2, 22.62, 22.6, 19.8, 19.6, 18.5, 17.8, 14.6, 14.0,
10.4, ꢂ4.6, ꢂ5.0; nmax/cmꢂ1: 3428, 2924, 2852, 1732, 1465, 1375,
1251, 835, 759.
5.17. 6-O-[(R)-2-((R)-1-Hydroxy-17-((1S,2R)-2-((S)-19-((S)-
eicosan-2-yloxy)-21-oxo-nonadecan-2-yl)-cyclopropyl)non-
adecyl)tetracosanoate]-a-D
-glucopyranosyl-(1-10)-60-O-[(R)-2-
((R)-1-hydroxy-17-((1S,2R)-2-((S)-19-((S)-eicosan-2-yloxy)-21-
oxononadecan-2-yl)cyclopropyl)nonadecyl)tetracosanoate]-
a-
D
-glucopyranoside (32) and 6-O-[(R)-2-((R)-1-hydroxy-17-
((1S,2R)-2-((S)-19-((S)-eicosan-2-yloxy)-21-oxononadecan-2-
yl)cyclo-propyl)nonadecyl)tetracosanoate]- -glucopyran-
osyl-(1-10)-
-gluco-pyranoside (33)
a-D
a-D
Compound 32, a waxy colourless solid, was prepared as de-
scribed in detail in the supplementary section. It showed a2D1þ34.2
(c 1.32, CHCl3) [MALDI-Found (MþNa)þ: 2863.6353, C182H350NaO19
requires: 2863.6314]; dH (CDCl3þfew drops of CD3OD): 4.98 (2H, d, J
3.4 Hz), 4.87 (2H, sext, J 6.2 Hz), 4.68 (2H, br d, J 10.9 Hz), 4.24 (2H, t,
J 8.8 Hz), 3.93 (2H, m), 3.73 (2H, t, J 9.2 Hz), 3.66e3.62 (2H, m), 3.49
(2H, dd, J 3.4, 9.7 Hz), 3.19 (2H, t, J 9.5 Hz), 2.40 (2H, br m), 2.23 (4H,
t, J 7.5 Hz), 1.58e1.53 (8H, m), 1.52e1.22 (284H, m), 1.16 (6H, d, J
6.2 Hz), 0.86 (6H, d, J 6.8 Hz), 0.86e0.82 (12H, br t, J 6.9 Hz),
0.66e0.59 (2H, m), 0.43e0.38 (2H, m), 0.18e0.05 (6H, m); dC
(CDCl3þfew drops of CD3OD): 175.5,174.1, 94.9, 72.5, 71.8, 71.3, 71.0,
70.6, 69.8, 64.7, 52.1, 38.0, 37.3, 34.7, 34.4, 33.8, 31.9, 29.7, 29.6, 29.5,
29.42, 29.4, 29.3, 29.2, 29.0, 27.3, 26.0, 25.3, 25.0, 22.7, 22.6, 19.8,
19.5, 18.5, 14.0, 10.5; nmax/cmꢂ1: 3371, 2918, 2850, 1732, 1467, 758,
723.
(iv) A dry polyethylene vial equipped with an acid-proof rubber
septum was charged with glucopyranoside 28 (0.180 gm,
0.058 mmol) and pyridine (0.07 mL) in dry THF (20 mL) and stirred
at rt under nitrogen. The mixture was cooled to 10 ꢀC, and then HF-
pyridine complex (w70%, 1.35 mL) was added dropwise. The
Please cite this article in press as: Taher, S. G.; et al., Tetrahedron (2016), http://dx.doi.org/10.1016/j.tet.2016.05.004

S.G. Taher et al. / Tetrahedron xxx (2016) 1e14
13
Compound 33, a colourless waxy solid, showed a2D1þ33 (c 1.3,
CHCl3), mp 115e117 ꢀC [MALDI-Found (MþNa)þ: 1614.3674,
5. Barry, C. E.; Lee, R. E.; Mdluli, K.; Sampson, A. E.; Schroeder, B. G.; Slayden, R. A.;
Yuan, Y. Prog. Lipid Res. 1998, 37, 143e179.
6. Minnikin, D. E.; Kremer, L.; Dover, L. G.; Besra, G. S. Chem. Biol. 2002, 9,
C
97H187NaO15 requires: 1614.3684]; dH (CDCl3þfew drops of
545e553.
7. Verschoor, J. A.; Baird, M. S.; Grooten, J. Prog. Lipid Res. 2002, 51, 325e339.
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272, 10041e10049.
CD3OD): 5.07 (1H, d, J 3.1 Hz), 5.02 (1H, d, J 3.2 Hz), 4.88 (1H, sext, J
6.2 Hz), 4.69 (1H, d, J 11.3 Hz), 4.22 (1H, t, J 9.1 Hz), 3.99e3.90 (2H,
m), 3.88e3.78 (3H, m), 3.63e3.54 (3H, m), 3.5 (1H, dd, J 2.9, 10 Hz),
3.28 (1H, t, J 9.4 Hz), 3.21 (1H, t, J 9.3 Hz), 2.38 (1H, m), 2.23 (2H, t, J
7.5 Hz), 1.58e1.50 (4H, m), 1.49e1.21 (147H, m),1.17 (3H, d, J 6.2 Hz),
0.86 (3H, d, J 6.8 Hz), 0.86e0.83 (6H, br t, J 6.9 Hz), 0.67e0.59 (1H,
m), 0.44e0.37 (1H, m), 0.18e0.04 (3H, m); dC (CDCl3þfew drops of
CD3OD): 175.5, 173.9, 94.3, 94.2, 74.5, 73.3, 72.5, 71.5, 71.3, 71.2,
70.6, 63.9, 62.9, 52.4, 38.0, 37.2, 35.7, 34.5, 34.4, 33.4, 31.7, 30.0, 29.9,
29.6, 29.3, 29.2, 29.1, 28.1, 27.3, 25.9, 25.2, 25.0, 24.9, 22.5, 19.8, 18.8,
18.4, 14.0, 10.3; nmax/cmꢂ1: 3369, 2922, 2852, 1732, 1466, 1373, 759.
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L
Chua, G. H.; Bratschi, M. W.; Rahim, S. Z. Z.; Michelle, A. L. T.; Hwang, S.-H.; Lee,
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J.-S.; Eum, S.-Y.; Kwak, H.-K.; Daffe, M.; Dartois, V.; Michel, G.; Barry, C. E.;
and non-essential amino acids (Thermo Fisher Scientific) for 7 days.
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according to a previously described protocol.73 Briefly, glycolipids
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injected subcutaneously in both hind footpads with 25
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m
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Acknowledgements
SGT wished to thank the Ministry of Higher Education in Kur-
distan Region-Iraq and HMA the Government of Iraq for the award
of PhD studentships. We wish to thank Dr. Paul Gates of Bristol
University for carrying out accurate mass MALDI determinations.
HGT holds a FRIA PhD fellowship. This research was partially fun-
ded by the Interuniversity Attraction Pole Programme IAP 7/32 and
by TBVAC2020 under grant agreement no. H2020-PHC-643381.
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Supplementary data
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Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.tet.2016.05.004.
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Please cite this article in press as: Taher, S. G.; et al., Tetrahedron (2016), http://dx.doi.org/10.1016/j.tet.2016.05.004