Synthesis of truncated analogues of the iNKT cell agonist, α-galactosyl ceramide (KRN7000), and their biological evaluation

Article abstract of DOI:10.1016/j.bmc.2010.11.032

Stimulation of iNKT cells by α-galactosyl ceramide (α-GalCer), also known as KRN7000, and its truncated analogue OCH induces both Th1- and Th2-cytokines, with OCH inducing a Th2-cytokine bias. Skewing of the iNKT cells' response towards either a Th1- or Th2-cytokine profile offers potential therapeutic benefits. The length of both the acyl and the sphingosine chains in α-galactosyl ceramides is known to influence the cytokine release profile. We have synthesized analogues of α-GalCer with truncated sphingosine chains for biological evaluation, with particular emphasis on the Th1/Th2 distribution. Starting from a common precursor, d-lyxose, the sphingosine derivatives were synthesised via a straightforward Wittig condensation.

Full text of DOI:10.1016/j.bmc.2010.11.032

Bioorganic & Medicinal Chemistry 19 (2011) 221–228
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of truncated analogues of the iNKT cell agonist, a-galactosyl
ceramide (KRN7000), and their biological evaluation
Natacha Veerapen a, Faye Reddington a, Mariolina Salio b, Vincenzo Cerundolo b, Gurdyal S. Besra a,
⇑
a School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
b The Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Stimulation of iNKT cells by
analogue OCH induces both Th1- and Th2-cytokines, with OCH inducing a Th2-cytokine bias. Skewing of
the iNKT cells’ response towards either a Th1- or Th2-cytokine profile offers potential therapeutic bene-
fits. The length of both the acyl and the sphingosine chains in
ence the cytokine release profile. We have synthesized analogues of
chains for biological evaluation, with particular emphasis on the Th1/Th2 distribution. Starting from a
a
-galactosyl ceramide (
a
-GalCer), also known as KRN7000, and its truncated
Received 7 October 2010
Revised 9 November 2010
Accepted 10 November 2010
Available online 19 November 2010
a-galactosyl ceramides is known to influ-
a-GalCer with truncated sphingosine
Keywords:
common precursor,
condensation.
D-lyxose, the sphingosine derivatives were synthesised via a straightforward Wittig
a-Galactosyl ceramide
OCH
Th1
Ó 2010 Elsevier Ltd. All rights reserved.
Th2
Sphingosine
1. Introduction
Crystal structures of both mouse12 and human CD1d13 have iden-
tified the antigen-binding site as consisting of two channels or pock-
The distinctive class of T cells, known as invariant Natural Killer T
(iNKT) cells, display characteristics of both T cells and NK cells and
play a crucial role in diverse immune responses and other pathologic
ets; A0 and F0, lined with hydrophobic residues. While the A0 channel
can accommodate an alkyl chain consisting of up to 26 carbons (the
acyl chain in
chain of 18 carbons (the sphingosine chain in a
-GalCer).13,14 The sta-
a
-GalCer), the F0 channel can accommodate an alkyl
conditions.1 The synthetic glycolipid
a
-galactosyl ceramide
(a
-GalCer),2 known as KRN7000 (1) (Fig. 1), is a powerful agonist,
bility of the bound glycolipid/CD1d complex and in turn the binding
affinity of the latter to T cell receptors (TCR) are believed to largely
influence the immunological response.6,14 The apparent skewing to-
wards a Th2 response in the case of OCH (5) is allegedly due to a less
stable bound CD1d complex resulting in a less prolonged TCR stim-
which when presented by CD1d, activates iNKT cells to release
diverse cytokines, including both Th1- and Th2-cytokines.3 Once
stimulated, iNKT cells also activate other cells such as dendritic cells,
T cells and B cells.4 It is believed that the release of proinflammatory
Th1 cytokines such as interferon-
c
(INF-c
) may contribute to antitu-
ulation.6,14,15
a-Galactosyl ceramides with variations in the acyl
mour and antimicrobial functions while that of immunomodulatory
Th2-cytokines such as interleukin 4 (IL-4) may help alleviate auto-
immune diseases5 such as multiple sclerosis6 (MS) and arthritis.7
Maintaining the right balance between Th1- and Th2-cytokines is
of utmost importance as over activation of Th1 cells or suppression
of Th2 ones can lead to autoimmune diseases.8
chain have been extensively studied, and have also exhibited similar
effects on the cytokine release profile.9 However, the properties of
analogues of
a-GalCer with variations in the sphingosine chain, such
as OCH (5) have yet to be fully explored.
Moreover, skewing of the cytokine release profile towards a Th2
one can help in the treatment of autoimmune and inflammatory
conditions.9,10 For example, compound 5, commonly referred to
as OCH (Fig. 1), has been shown to protect mice against experimen-
tal encephalomyelitis (an animal model for MS) by favouring the
release of the Th2-cytokine IL-4 and suppressing the myelin
antigen-specific Th1 responses.6,11
OH
OH
HO
HO
HO
HO
O
C25H51
O
C23H47
O
O
NH OH
NH OH
HO
HO
O
O
CnH2n+1
C5H11
OH
OH
5
1 n =14
2 n = 5
3 n = 8
4 n = 11
⇑
Corresponding author. Tel.: +44 1214158123.
E-mail address: g.besra@bham.ac.uk (G.S. Besra).
Figure 1. CD1d agonist KRN7000, OCH, and analogues.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.11.032

222
N. Veerapen et al. / Bioorg. Med. Chem. 19 (2011) 221–228
a
-GalCer and its counterparts have proved to be and remain
of the double bond after the Wittig condensation via catalytic hydro-
genation using palladium hydroxide (Pd(OH)2). However, in our
hands, after several attempts the hydrogenation failed to go to com-
pletion after prolonged reaction periods. In an attempt to drive the
reaction to completion, the catalyst was filtered off and replaced
with fresh one. Although the reaction was eventually completed
after 48 h, the trityl protecting group was also cleaved, thereby leav-
ing the primary alcohol unprotected. An alternative strategy where
the protecting groups would be removed prior to hydrogenation of
the double bond was therefore envisaged. Hence, the remaining free
secondary hydroxyl group was mesylated by reaction with meth-
anesulfonyl chloride in dichloromethane. The mesyl group acts both
as a temporary protecting group and a good leaving group for the fol-
lowing inversion of stereochemistry at this position. Removal of the
trityl and acetonide protecting groups by acid treatment then
yielded compounds 13, 14 and 15 in quantitative yields. The reduc-
tion of the double bond by hydrogenation, catalysed by 5% palla-
dium on barium sulphate proceeded smoothly to give compounds
16, 17 and 18. We substituted the Pd(OH)2 with palladium on bar-
ium sulphate because the latter is less active and is more suitable
for use in the presence of the mesylate group. Finally, the amine
functional group was introduced into the molecule by an SN2 dis-
placement of the mesylate with sodium azide in DMF. Sphingosine
analogues 19, 20 and 21 were thus obtained in reasonable yields
for further use as glycosyl acceptors.
invaluable tools in understanding the functioning of CD1d and
NKT cells in a wide range of immune responses. Specifically,
compounds such as OCH (5), that are able to alter the polarisation
of Th1 or Th2, have potential therapeutic values for certain dis-
eases. As such, we have synthesised compounds 2, 3 and 4
(Fig. 1), with varying phytosphingosine chain lengths consisting
of 9–15 carbons, to investigate their effect on the Th1/Th2 balance
as well as to study their overall biological properties.
2. Results and discussion
Various methods, including dihydroxylation reactions16 and
Sharpless asymmetric epoxidation17 have been described in the lit-
erature for the synthesis of sphingolipids.18 Yet, the preparation of
such compounds remains nontrivial. In contrast to many reported
syntheses, the strategy employed by Lin et al.19 is quite concise and
affords a relatively high yield of the final phytosphingosine from
the readily available
tive as -lyxose already possesses the required stereogenic centres
D-lyxose. Their method is particularly attrac-
D
and an SN2 displacement of a suitable leaving group at C-4 allows
the introduction of the amine functionality in the molecule. It is
noteworthy that Plettenburg et al.20 also used a similar strategy,
involving conversion of their substrate to phytosphingosine via a
Wittig condensation, in their reported synthesis.
Hence, following the standard conditions described previ-
ously21 and summarised in Scheme 2, the sphingosine acceptors
22–24 were synthesized. Consistent with our previous work, ben-
zoate esters, rather than benzyl ethers, were adopted as protecting
groups to circumvent the hydrogenolysis reaction.
We have therefore adapted both these methods to synthesise our
analogous sphingosines. We first embarked on the synthesis of the
appropriate phosphonium salts required for the chain extending
Wittig olefination reaction. This was easily achieved by refluxing tri-
phenylphoshine with the corresponding alkyl halides in toluene
overnight. For the C-9, C-12, and C-15 phytosphingosine chain
lengths, 1-iodobutane, 1-bromooctane and 1-bromodecane were
used, respectively. Once isolated, they were resuspended in THF
and treated with n-BuLi at ꢀ78 °C to generate the Wittig ylids, as de-
scribed by Plettenburg et al.20 Subsequently, the protected lyxose
derivative 6, which was synthesised as previously described,19 was
condensed with the ylids to afford the desired olefins 7, 8 and 9 as
a mixture of cis and trans isomers (Scheme 1). In their preparation
of the C-18 phytosphingosine, Lin et al.19 proceeded to the reduction
With respect to the glycosyl donor, we have previously success-
fully employed the bulky 4, 6-O-di-tert-butylsilylene (DTBS) group
as
sive formation of an
a
-directing in galactosylation donors.22 DTBS ensures the exclu-
a-glycosidic linkage, irrespective of the nature
of the acceptor, and remains the protecting group of choice in chal-
lenging glycosylation reactions. However with our glycosyl accep-
tors, we were inclined to adopt Gervay-Hague’s rather simplified
glycosylation strategy employing glycosyl iodides and promoted
by tetrabutyl ammonium iodide (TBAI).23 Indeed, the latter’s group
recently reported both excellent stereoselectivity and yields for the
synthesis of
a
-GalCer and other similar compounds.24,25 It is
hypothesised that the
isomerization of the
strategy represents a straightforward approach to the synthesis
of our target compounds given that the donor can be easily ob-
tained in large quantities in contrast to the multi-step preparation
a
a
-selectivity is due to the TBAI-catalysed
-glycosyliodide to the b-anomer.26 This
TrO
OH
CnH2n+1
O
OH
b
TrO
a
O
O
O
O
of other galactosyl donors. The per-O-tetramethylsilyl-
pyranosyl iodide 25 was therefore generated by the reaction of the
a-D-galacto-
7 n= 3
8 n= 6
9 n= 9
6
per-O-pentamethylsilyl-a-D-galactose with 1 equiv of iodotrimeth-
ylsilane26 and then added to the respective phytosphingosine
OMs
O
CnH2n+1
OMs OH
acceptors 22–24 which were premixed with diisopropylethyl-
TrO
d
c
HO
O
CnH2n+1
OH
N3 OH
OH
OH
N3
Ph
Si
a
b
13 n= 3
14 n= 6
15 n= 9
10 n= 3
11 n= 6
12 n= 9
HO
O
CnH2n+1
CnH2n+1
Ph
OH
N3 OH
OMs OH
e
CnH2n+1
HO
HO
OBz
N3
CnH2n+1
N3 OBz
Ph
c
HO
OH
O
Si
OH
CnH2n+1
CnH2n+1
Ph
19 n = 3
20 n = 6
21 n = 9
16 n= 3
17 n= 6
18 n= 9
OBz
OBz
22 n = 3
23 n = 6
24 n = 9
Scheme 1. Reagents: (a) n-BuLi, THF, phosphonium salts (C4H9PPh3+Iꢀ,
C7H15PPh3+Brꢀ, C10H21PPh3+Brꢀ; (b) MsCl, pyridine, CH2Cl2, quant; (c) HCl, MeOH/
CH2Cl2; (d) H2, Pd–BaSO4, THF; (e) NaN3, DMF.
Scheme 2. Reagents: (a) TBDPSCl, pyridine, quant; (b) BzCl, Pyr, 86%; (c) TBAF, THF,
80%.

N. Veerapen et al. / Bioorg. Med. Chem. 19 (2011) 221–228
223
OTMS
TMSO
TMSO
A
180
150
120
90
OTMS
O
TMSO
TMSO
a
O
α-GalCer 1
OCH9 2
TMSO
TMSO
I
OTMS
OCH12 3
OCH15 4
25
OH
O
OH
HO
b, c
26 n= 3
27 n= 6
28 n= 9
60
N3 OBz
OBz
HO
O
30
CnH2n+1
0
OH
HO
HO
0
50
100
O
d
GSL concentration (ng/mL)
29 n= 3
30 n= 6
31 n= 9
N3 OH
OH
HO
O
CnH2n+1
18
15
B
OH
HO
HO
C25H51
O
e, f
O
12
9
α-GalCer 1
OCH9 2
OCH12 3
OCH15 4
2 n= 3
3 n= 6
4 n= 9
NH OH
HO
O
CnH2n+1
OH
6
Scheme 3. Reagents and conditions: (a)TMSI, CH2Cl2, 0 °C, quant; (b) TBAI, DIPEA,
22, 23 and 24, benzene, rt; (c) Dowex 50WX8-200, MeOH, rt, 62% over two-steps;
(d) NaOMe/MeOH, quantitative; (d) H2, Pd, MeOH, 80%; (e) C25H51COCl, THF/NaOAc
(1:1), 71%.
3
0
0
50
100
GSL concentration (ng/mL)
amine (DIPEA) and TBAI (Scheme 3). After two days at room
temperature, the solvent was evaporated and the TMS protecting
groups were removed by treatment with an acidic resin in MeOH.
Figure 2. CIR-hCD1d cells were pulsed with GSL and used to stimulate iNKT cells.
Supernatant was assayed for IL-4 (A) and IFN- (B) by ELISA.
c
The glycosylated compounds 26–28 were obtained as the
mer exclusively in good overall yields of over 60%. The formation
of the desired -linkage in compounds 26–28 was confirmed by
a-ano-
a
the H-1 and C-1 signals in 1H and 13C NMR, respectively. Finally,
Zemplen’s deprotection of the benzoate protecting groups, fol-
lowed by hydrogenation of the azido group in methanol provided
the amines which were acylated with the fully saturated fatty acid,
hexacosanoic acid. This was accomplished by reaction of the corre-
sponding acid chloride with the free amine in a 1:1 mixture of THF
and saturated sodium acetate solution. Glycosphingolipds (GSL) 2
(OCH9), 3 (OCH12) and 4 (OCH15) were obtained as white solids
after concentration of the organic phase and purification of the res-
idue by flash chromatography.
To evaluate the biological activity of compounds OCH9 (2),
OCH12 (3) and OCH15 (4), human B cells expressing CD1d (C1R
CD1d) were pulsed with different concentrations of lipids and
incubated with a human iNKT cell line. iNKT cell activation (as deter-
mined by both IL-4 and IFN-
elicited by OCH15 (4) was comparable to that obtained with
-GalCer 1 (Fig. 2). On the other hand, compounds OCH9 (2) and
OCH12 (3) were found to be weaker stimulants of iNKT cells as less
IL-4 and IFN- were observed. These data clearly indicate that the
c secretion in the culture supernatant)
Figure 3. A (upper and lower pannel) IL4/IFN
cells), B (upper and lower pannel) IL4/IFN and IFN
mice per group were injected with 1 g of lipid ip and sera was assayed at 2 h (IL4)
or 24 h (IFN ) by ELISA.
c
and IFN
c/IL4 ratio (human iNKT
c
c
/IL4 ratio (mice iNKT cells); two
a
l
c
c
iNKT cells’ activation by the GSLs OCH9 (2), OCH12 (3) and OCH15
(4) correlate with the length of their sphingosine chains, with the
longest chain inducing the greatest cytokine response. However, un-
like what was previously observed with mouse iNKT cells,27 the
cytokine profile of human iNKT cells stimulated by OCH9 (2) and
OCH12 (3) was not as strongly biased towards a Th2 response
binding affinity.14 We reported that shortening the phytosphingo-
sine chain increased the rate of dissociation of the GSLs from
hCD1d molecules and led to a decrease of the binding affinity of
the iNKT TCR for hCD1d–GSL complexes.14 The increase in the Kd
value as the phytosphingosine chain got shorter was attributed
to both a decrease in kon and an increase in koff. Furthermore, our
results indicated an important role of the phytosphingosine chain
in controlling the formation of the immunological synapse and
iNKT cell activation.14
(Fig. 2A).14 Increasing amounts of IFN-
cwere observed with increas-
ing concentrations of OCH9 (2) and OCH12 (3), as opposed to what
was observed with mouse iNKT cells (Fig. 3).27
We previously performed kinetic and affinity experiments to
investigate the effect of the phytosphingosine chain length on
the stability of the bound glycolipid/CD1d complex and on TCR
As a control,
a-GalCer, OCH9, OCH12 and OCH15 were com-
pared with C20:2 (Fig. 4), a compound known to induce a Th2

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N. Veerapen et al. / Bioorg. Med. Chem. 19 (2011) 221–228
OH
allowed to cool and then filtered. The precipitate was washed with
cold toluene and dried under vacuo to give the phosphonium salts
which were used in the next step without further purification.
HO
HO
O
(CH2)8
C4H9
O
NH OH
HO
O
C14H29
OH
4.2. General procedure for the synthesis of Wittig salts (ylids)
and subsequent olefination (b)
C20:2
Figure 4. Analogue of a-GalCer inducing a Th2 skewing.
The phosphonium salt was resuspended in THF and cooled
to ꢀ10 °C. nBuLi (2.5 M solution in hexanes, 2.8 equiv) was added
dropwise and the reaction mixture was stirred for 30 min, before
a solution of 6 (1 equiv) in dry THF was added. The resulting solu-
tion was stirred overnight at room temperature. Upon completion
of the reaction, the reaction was quenched with MeOH, followed by
80% MeOH in H2O, and the mixture was extracted with heptane
(4 ꢁ 20 mL). The combined organic layers were then washed with
brine (30 mL), dried over anhydrous Na2SO4 and concentrated in
vacuo. The residue was purified by purified by flash chromatogra-
phy using hexanes/EtOAc (10:1) to give the desired products.
skewing in mice.9,10, While no apparent change in IL4/IFN
was observed between -GalCer and the various GSLs with human
c ratio
a
iNKT cells (Fig. 3A, upper panel), a net increase was observed with
mice iNKT cells (Fig. 3B, upper pannel). In mice C20:2 and the trun-
cated GSLs OCH9, OCH12 and OCH15 favour the release of the Th2-
cytokine IL4. Similarly, the IFN
with mice iNKT cells (Fig. 3B, lower pannel) demonstrates that
the latter favours the release of the Th1 cytokine IFN , confirming
c/IL4 ratio observed with a-GalCer
c
that in mice, compounds C20:2, OCH9, OCH12 and OCH15 induce
varying extents of Th2 skewing.
3. Conclusions
4.3. (2R,3S,4R,5E/Z)-3,4-O-Isopropylidene-1-O-trityloxy-non-5-
en-2-ol 7
In conclusion we have developed a synthetic strategy for the
successful syntheses of three truncated analogues of
a-GalCer,
Prepared following general procedures (a) and (b) using triphen-
ylphosphine (18.36 g, 70.0 mmol) and iodobutane (4.29 g,
adapted from examples present in the literature. ELISA assays of
these compounds with hCD1d and human iNKT cells showed dif-
ferent results than those observed with mice iNKT cells. The trun-
cated analogues OCH9 (2), OCH12 (3) and OCH15 (4) were found to
23.3 mmol), n-BuLi (26.8 mL, 65.2 mmol) and
6
(10.10 g,
23.3 mmol) to afford compound 7 as a white solid in 82% yield as a
mixture of cis and trans isomer in a 1:2.3 ratio (9.03 g, 19.1 mmol).
1H NMR (CDCl3): d 7.19–7.49 (15H, m, Ar–H), 5.53–5.57 (2H, m,
H-5, H-6), 4.90–4.94 (0.7 H, m, H-4trans), 4.45 (0.3H, dd,
J4,5 = J3,4 = 7.4 Hz, H-4cis), 4.28–4.30 (0.3H, m, H-3cis), 4.22–4.25
(0.7H, m, H-3trans), 3.77–3.81 (0.7H, m, H-2trans), 3.69–3.73 (0.3H,
m, H-2cis), 3.25 (0.3H, dd, J1a,2 = 5.1, J1a,1b = 9.5 Hz, H-1acis),
3.18–3.20 (0.7H, m, H-1atrans), 3.07–3.15 (1H, m, H-1btrans, H-1bcis),
1.75–2.06 (2H, m, H-7acis, H7bcis, H-7atrans, H-7btrans), 1.58, 1.52,
1.39, 1.37 (6H, 4s, 4 ꢁ C(CH3)2), 1.22–1.32 (2H, m, CH2), 0.87 (3H, t,
J = 7.3 Hz, CH3); 13C NMR (75 MHz, CDCl3): d 144.1 (O2C(CH3)2),
135.8 (C-5), 127.3, 128.0 (CAr), 125.5 (C-6), 79.5 (C-2), 73.3 (C-3),
69.4 (C-4), 65.2 (C-1), 30.6, 30.1 (C-7, C-8), 28.5, 25.7 (2 ꢁ C(CH3)3),
14.9 (C-9); HRMS calcd for C31H36O4 [M+Na]+: 495.2511, found
495.2511.
be generally less potent than a-GalCer (1). Also there was no Th2
skewing observed in this present study with the shorter phyto-
sphingosine chain lengths. Furthermore, control comparison
experiments showed that GSLs OCH9 (2), OCH12 (3), OCH15 (4)
and C20:2 cause a Th2 skewing in mice iNKT cells, thereby con-
firming the viability of our assays.
4. Experimental
1H NMR spectra were recorded at 400 MHz or 300 MHz, using
Bruker AMX 400, Bruker AV 400, Bruker AV 300 and Bruker AC
300 spectrometers. 13C NMR spectra were recorded at 100 MHz
or 75 MHz, respectively, using Bruker AMX 400, Bruker AV 400,
Bruker AV 300 and Bruker AC 300 spectrometers. Chemical shifts
are reported as d values (ppm) referenced to the following solvent
signals: CHCl3, dH 7.26; CDCl3, dC 77.0; CH3OH, dH 3.34; CD3OD, dC
49.9. Quaternary carbons were reported only when observed. Opti-
cal rotations were measured at 23 °C and reported in
degdmꢀ1 gꢀ1 cm3. HRMS were recorded on a Micromass LCT spec-
trometer using a lock mass incorporated into the mobile phase. All
reagents were obtained from commercial sources, and were used
without further purification unless stated otherwise. Anhydrous
solvents were purchased from Sigma–Aldrich, UK, stored over 4 Å
molecular sieves and under an Ar atmosphere. Analytical thin layer
chromatography (TLC) was performed on aluminium plates pre-
coated with Merck silica gel 60A F-254 as adsorbent. The devel-
oped plates were air dried, visualised by UV detection (at
254 nm) and/or stained with 5% phosphomolybdic acid in EtOH
(MPA spray). Compounds were purified by flash column chroma-
tography on Merck silica gel (particle size 40–63 lm mesh) or Fluka
60 (40–60 lm mesh) silica gel.
4.4. (2R,3S,4R,5E/Z)-3,4-O-Isopropylidene-1-O-trityloxy-dodec-
5-en-2-ol 8
Prepared following general procedures (a) and (b) using tri-
phenylphosphine (18.00 g, 68.7 mmol) and 1-bromoheptane
(12.24 g, 22.9 mmol), n-BuLi (26.4 mL, 64.2 mmol) and 6 (9.90 g,
22.9 mmol) to afford compound 8 as a colourless syrup in 63% yield
as a mixture of cis and trans isomer in a 1:2.3 ratio (5.88 g,
13.68 mmol). 1H NMR (CDCl3): d 7.19–7.48 (15H, m, Ar–H),
5.47–5.58 (2H, m, H-5, H-6), 4.91–4.93 (0.7 H, m, H-4trans),
4.43–4.46 (0.3H, m, H-4cis), 4.25 (0.3H, dd, J2,3 = J3,4 = 4.6 Hz,
H-3cis), 4.21 (0.7H, dd, J2,3 = J3,4 = 4.4 Hz, H-3trans), 3.68–3.81
(0.7H, m, H-2trans), 3.51–3.56 (0.3H, m, H-2cis), 3.22 (0.3H, dd,
J1a,2 = 5.1, J1a,1b = 5.3 Hz, H-1acis), 3.18 (0.7H, dd, J1a,2 = 9.5,
J1a,1b = 5.3 Hz, H-1atrans), 3.07–3.17 (1H, m, H-1btrans, H-1bcis),
1.91–2.09 (2H, m, H-7acis, H7bcis, H-7atrans, H-7btrans), 1.54, 1.49,
1.39, 1.38 (6H, 4s, 4 ꢁ C(CH3)2), 1.19–1.39 (8H, m, CH2), 0.88 (3H,
t, J = 5.7 Hz, CH3); 13C NMR (75 MHz, CDCl3):
d
145.9,
4.1. Generalprocedure for the synthesis of phosphonium salts (a)
(O2C(CH3)2), 137.3 (C-5), 129.1, 129.9, 130.8 (CAr), 127.1 (C-6),
79.5 (C-2), 75.1 (C-3), 71.3 (C-4), 67.1 (C-1), 34.2, 31.3, 31.1, 29.7,
25.0 (C-7–C-12), 29.8, 27.1 (2 ꢁ C(CH3)3), 17.1 (C-12); HRMS calcd
for C34H42O4 [M+Na]+: 537.2980, found 537.2982.
A mixture of triphenyl phosphine (3 equiv) and the alkyl halide,
1-iodobutane, 1-bromoheptane and 1-bromodecane, respectively,
(1 equiv) in toluene was refluxed overnight. The mixture was then

N. Veerapen et al. / Bioorg. Med. Chem. 19 (2011) 221–228
225
4.5. (2R,3S,4R,5E/Z)-3,4-O-Isopropylidene-1-O-trityloxy-
pentadec-5-en-2-ol 9
2.99, 3.18 (3H, 2s, OSO2CH3), 1.57–1.82 (2H, m, H-7acis, H7bcis
,
H-7atrans, H-7btrans), 1.48, 1.41, 1.39, 1.38 (6H, 4s, 4 ꢁ C(CH3)2),
1.10–1.33 (8H, m, CH2), 0.85 (3H, t, J = 6.7 Hz, CH3); 13C NMR
(75 MHz, CDCl3): d 144.8 (O2C(CH3)2), 137.8 (C-5), 130.2, 129.4,
128.7 (CAr), 125.1 (C-6), 82.5 (C-2), 77.6 (C-3), 73.7 (C-4), 64.6
(C-1), 39.9 (COSO2CH3), 35.1, 30.9, 30.2 (C-7, C-8, C-9), 29.5, 28.1
(2 ꢁ C(CH3)3), 29.0 (C-10), 24.5 (C-11), 16.8 (C-9); HRMS calcd for
Prepared following general procedures (a) and (b) using triphen-
ylphosphine (54. 6 g, 0.20 mol, 3 equiv) and 1-bromodecane (15.4 g,
69.5 mmol), n-BuLi (74.0 mL, 0.18 mol) and 6 (10.00 g, 23.2 mmol)
to afford compound 9 as a colourless syrup in 55% yield as a mixture
of cis and trans isomer in a 1:2.3 ratio (7.12 g, 12.8 mmol). 1H NMR
(CDCl3): d 7.19–7.48 (15H, m, Ar–H), 5.47–5.58 (2H, m, H-5, H-6),
4.91–4.93 (0.7 H, m, H-4trans), 4.40–4.46 (0.3H, m, H-4cis), 4.25
C
35H44SO6 [M+Na]+: 615.2757, found 615.2560.
4.9. (2R,3S,4R,5E/Z)-3,4-O-Isopropylidene-2-
methanesulfonyloxy-1-O-trityloxy-pentadec-5-enol 12
(0.3H, dd, J2,3 = J3,4 = 4.6 Hz, H-3cis), 4.21 (0.7H, dd, J2,3 = J3,4
=
4.4 Hz, H-3trans), 3.72–3.79 (0.7H, m, H-2trans), 3.68–3.70 (0.3H, m,
H-2cis), 3.22 (0.3H, dd, J1a,2 = 5.1, J1a,1b = 5.3 Hz, H-1acis), 3.15 (0.7H,
Prepared following general procedure (c) using 9 (5.39 g,
dd, J1a,2 = 9.5, J1a,1b = 5.3 Hz, H-1atrans), 3.08–3.13 (1H, m, H-1btrans
,
9.67 mmol) and methanesulfonyl chloride (1.3 mL, 16.1 mmol) in
a mixture of CH2Cl2 (45 mL) and pyridine (15 mL) to afford
compound 12 (5.52 g, 8.70 mmol) as an opaque oil in 90% yield as
a mixture of cis and trans isomer in a 1:2.3 ratio. 1H NMR (CDCl3):
H-1bcis), 1.71–2.03 (2H, m, H-7acis, H7bcis, H-7atrans, H-7btrans),
1.56, 1.47, 1.39, 1.38 (6H, 4s, 4 ꢁ C(CH3)2), 1.11–1.35 (14H, m,
CH2), 0.87 (3H, t, J = 6.7 Hz, CH3); 13C NMR (75 MHz, CDCl3):
d 145.7, (O2C(CH3)2), 137.2 (C-5), 130.6, 129.7, 128.9 (CAr), 126.8
(C-6), 79.5 (C-2), 74.9 (C-3), 70.8 (C-4), 66.5 (C-1), 34.0, 31.3, 31.2,
30.0 (C-7–C-14), 29.9, 27.0 (2 ꢁ C(CH3)3), 16.0 (C-15); HRMS calcd
for C37H48O4 [M+Na]+: 579.3450, found 579.3454.
d
7.19–7.48 (15H, m, Ar–H), 5.40–5.48 (0.7H, m, H-6trans),
5.28–5.37 (0.7H, m, H-5trans), 5.20–5.25 (0.3H, m, H-6cis),
4.97–5.06 (0.3H, M, H-5cis), 4.79–4.85 (0.7H, m, H-2trans), 4.72–
4.75 (0.7H, m, H-4trans), 4.62–4.66 (0.3H, m, H-2cis), 4.57–
4.61(0.3H, m, H-4cis), 4.49 (0.7H, dd, J3,4 = 8.7, J2,3 = 5.6 Hz, H-3trans),
4.20 (0.3H, dd, J3,4 = 9.4, J3,2 = 4.2 Hz, H-3cis), 3.45–3.56 (2H, m, H-1a,
4.6. General procedure for mesylation reaction (c)
H-1b), 3.04, 3.10 (3H, 2s, OSO2CH3), 1.85–2.12 (2H, m, H-7acis, H7bcis
,
Compounds 7, 8 and 9 (1 equiv) were, respectively, dissolved in
a mixture of anhydrous CH2Cl2 and dry pyridine (3:1) and cooled to
0 °C. Methanesulfonylchloride (1.6 equiv) was then added drop-
wise and the reaction mixture stirred for 4 h at room temperature.
Upon completion of the reaction, the reaction was quenched with
sodium bicarbonate solution, diluted with CH2Cl2 (20 mL) and
washed successively with water (20 mL) and brine (20 mL). The or-
ganic layer was dried over anhydrous Na2SO4 and evaporated un-
der vacuo to give the mesylated compounds 10, 11 and 12 as
thick syrups in quantitative yields.
H-7atrans, H-7btrans), 1.59–1.82 (2H, m, H-7), 1.48, 1.47, 1.38, 1.36
(6H, 4s, 4 ꢁ C(CH3)2), 1.08–1.32 (14H, m, CH2), 0.88 (3H, t,
J = 6.5 Hz, CH3); 13C NMR (75 MHz, CDCl3): d 144.3 (O2C(CH3)2),
137.3 (C-5), 129.7, 128.9, 128.2 (CAr), 124.6 (C-6), 82.0 (C-2), 77.1
(C-3), 73.2 (C-4), 64.1 (C-1), 40.1 (COSO2CH3), 33.1, 32.4, 30.5,
30.3, 30.2 (C-7, C-14), 26.8, 26.6 (2 ꢁ C(CH3)3), 16.9 (C-15); HRMS
calcd for C38H50SO6 [M+Na]+: 657.3226, found 657.3223.
4.10. General procedure for deprotection and reduction of
double bond (d)
4.7. (2R,3S,R,5E/Z)-3,4-O-Isopropylidene-2-methanesulfonyl-
oxy-1-O-trityloxy-non-5-enol 10
The mesylated compounds 10, 11 and 12 were, respectively,
dissolved in a mixture of dry CH2Cl2 and MeOH (2:1) (20 mL) and
concentrated hydrochloric acid (3 mL) was added dropwise and
the mixture stirred at room temperature for 2 h, after which time
TLC analysis indicated that the reaction was complete. Solid sodium
bicarbonate was then added to quench the reaction until the
solution was neutral. The mixture was then filtered and the filtrate
concentrated. The residue was dissolved again in EtOAc and the or-
ganic solution washed consecutively with water (2 ꢁ 20 mL), brine
(20 mL), dried over anhydrous Na2SO4 and concentrated in vacuo.
The residues were purified by flash chromatography (gradient from
to hexanes/EtOAc (4:1) to neat EtOAc) and dissolved in THF (10 mL).
Fivepercentage of Pd–Ba(SO4)2 (0.1 equiv)was added to thesolution
and the mixture was stirred under H2 overnight, after which time it
was filtered through a pad of Celite, which was subsequently
washed with CHCl3/MeOH (1:1). The combined filtrates were
concentrated to yield compounds 13, 14 and 15.
Prepared following general procedure (c) using 7 (6.46 g,
13.7 mmol) and methanesulfonyl chloride (1.8 mL, 22.7 mmol) in
a mixture of CH2Cl2 (45 mL) and pyridine (20 mL) to afford com-
pound 10 (6.13 g, 11.2 mmol) as an off-white solid in 82% yield
as a mixture of cis and trans isomer in a 1:2.3 ratio. 1H NMR
(CDCl3): d 7.21–7.49 (15H, m, Ar–H), 5.89–5.48 (2H, m, H-5, H-6),
4.78–5.07 (1H, m, H-2), 4.34–4.50 (2H, m, H-3, H-4), 3.92–4.26
(2H, m, H-1a, H-1b), 3.14, 3.12 (3H, 2s, OSO2CH3), 1.85–2.12 (2H,
m, H-7acis, H7bcis, H-7atrans, H-7btrans), 1.58, 1.52, 1.39, 1.37 (6H,
4s, 4 ꢁ C(CH3)2), 1.22–1.31 (2H, m, CH2), 0.85 (3H, t, J = 7.2 Hz,
CH3); 13C NMR (75 MHz, CDCl3): d 141.4 (O2C(CH3)2), 135.3
(C-5), 126.9, 126.8, 126.1 (CAr), 121.9 (C-6), 79.2 (C-2), 74.3 (C-3),
70.2 (C-4), 61.3 (C-1), 37.2 (COSO2CH3), 34.6, 28.7 (C-7, C-8),
28.5, 25.7 (2 ꢁ C(CH3)3), 14.9 (C-9); HRMS calcd for C32H38SO6
[M+Na]+: 573.2287, found 573.2290.
4.11. (2R,3S,4R)-2-Methanesulfonyloxy-nonane-1,3,4-triol 16
Prepared following general procedure (d) using 10 (5.06 g,
4.8. (2R,3S,4R,5E/Z)-3,4-O-Isopropylidene-2-
methanesulfonyloxy-1-O-trityloxy-dodec-5-enol 11
9.18 mmol) to give 16 as an off-white wax (1.91 g, 7.07 mmol) in
23
Prepared following general procedure (c) using 8 (2.59 g,
5.0 mmol) and methanesulfonyl chloride (0.62 mL, 8.06 mmol) in
a mixture of CH2Cl2 (25 mL) and pyridine (8 mL) to afford compound
11 (2.80 g, 4.7 mmol) as a colourless oil in 94% yield as a mixture of
cis and trans isomer in a 1:2.3 ratio. 1H NMR (CDCl3): d 7.17–7.52
(15H, m, Ar–H), 5.03–5.52 (2H, m, H-5, H-6), 4.79–4.82 (1H, m,
H-2), 4.58–4.75 (2H, m, H-3, H-4), 4.48 (0.7H, dd, J1a,2 = 5.9,
J1a,1b = 8.4 Hz, H-1atrans), 4.24 (0.3H, dd, J1a,2 = 6.9, J1a,1b = 14.0 Hz,
H-1acis), 3.54–3.57 (0.3H, m, H-1btrans), 3.45 (0.7H, dd, H-1bcis),
77% yield.
[a
]
D = ꢀ80.0 (c 1.00, CH3OH). 1H NMR (CD3OD): d
4.84–4.88 (1H, m, H-2), 3.78–3.88 (2H, m, H1a, H-1b), 3.49–3.55
(1H, m, H-4), 3.43–3.47 (1H, dd, J2,3 = 8.3, J3,4 = 2.2 Hz, H-3), 3.15
(3H, s, OSO2CH3), 2.76 (3H, br s, OH), 1.67–1.75 (1H, m, H-5a),
1.46–1.53 (1H, m, H-7a), 1.19–1.38 (6H, m, H-5b, H-6a, H-6b, H-
8a, H-8b), 0.88 (3H, t, J = 6.8 Hz, CH3); 13C NMR (75 MHz, CD3OD):
d 82.9 (C-2), 73.6 (C-3), 70.5 (C-4), 62.3 (C-1), 38.4 (COSO2CH3),
33.1, 32.0, 25.2, 22.8 (C-5–C-8), 14.0 (C-9); HRMS calcd for
C
10H22SO6 [M+Na]+: 293.1035, found 293.1026.

226
N. Veerapen et al. / Bioorg. Med. Chem. 19 (2011) 221–228
4.12. (2R,3S,4R)-2-Methanesulfonyloxy-dodecane-1,3,4-triol 17
Prepared following general procedure (d) using 11 (2.46 g,
4.17. (2S,3S,4R)-2-Azido-pentadecane-1,3,4-triol 21
Prepared following general procedure (e) using 18 (2.64 g,
4.15 mmol) to give 17 as a colourless syrup (0.946 g, 3.03 mmol)
7.45 mmol), sodium azide (0.97 g, 14.91 mmol) to afford a colour-
23
in 73% yield.
[
a]
D = ꢀ32.2 (c 1.00, CH3OH). 1H NMR (CD3OD): d
less oil (1.61 g, 5.36 mmol, 72%).23 D = +31.4 (c 1.00, CH3OH). 1H
[a]
4.74–4.78 (1H, m, H-2), 3.62–3.78 (2H, m, H1a, H-1b), 3.31–3.47
(1H, m, H-4), 3.18–3.21 (1H, m, H-3), 3.06 (3H, s, OSO2CH3),
1.48–1.56 (2H, m, H-5a, H-7a), 1.22–1.43 (12H, m, CH2), 0.73
(3H, t, J = 6.5 Hz, CH3); 13C NMR (75 MHz, CD3OD): d 83.9 (C-2),
74.7 (C-3), 71.5 (C-4), 63.3 (C-1), 40.1 (COSO2CH3), 34.2, 33.1,
30.9, 30.8 30.5, 26.5, 23.8 (C-5–C-11), 15.1 (C-12); HRMS calcd
for C13H28SO6 [M+Na]+: 335.1504, found 335.1511.
NMR (CD3OD): d 3.92–3.98 (1H, dd, J1a,1b = 11.4, J1a,2 = 3.3 Hz,
H-1a), 3.74–3.88 (1H, m, H-1b), 3.58–3.66 (1H, m, H-4),
3.41–3.58 (1H, m, H-3), 3.32–3.36 (1H, m, H-2), 1.51–1.76(3H, m,
CH2), 1.18–1.47 (17H, m, CH2), 0.91 (3H, t, J = 6.6 Hz, CH3); 13C
NMR (75 MHz, CD3OD): d 73.9 (C-3), 70.8 (C-4), 64.6 (C-2), 60.4
(C-1), 31.8, 31.0, 28.7, 28.4, 24.7, 21.7 (C-5–C-14), 13.6 (C-15);
HRMS calcd for C12H25N3O3[M+Na]+: 324.2263, found 324.2275.
4.18. Preparation of glycosyl acceptors 22, 23 and 24
4.13. (2R,3S,4R)-2-Methanesulfonyloxy-pentadecane-1,3,4-triol
18
Compounds 19, 20 and 21 were subjected to the same standard
reaction conditions described by Akimoto et al.21 and depicted in
Scheme 2 for the preparation of (2R, 3S, 4R)-2-azido-3, 4-di-O-(ben-
zoyloxy)-octadecan-1-ol.
Prepared following general procedure (d) using 12 (6.93 g,
10.9 mmol) to give 18 as a white solid (2.79 g, 7.87 mmol) in
72% yield.
23
[
a]
D = ꢀ180.0 (c 1.00, CH3OH). 1H NMR (CD3OD):
d 4.99–5.05 (1H, m, H-2), 3.98–4.04 (2H, m, H1a, H-1b), 3.55–3.62
(1H, m, H-4), 3.45–3.49 (1H, m, H-3), 3.15 (3H, s, OSO2CH3),
1.48–1.86 (2H, m, H-5a, H-7a), 1.22–1.43 (18H, m, CH2), 0.89 (3H,
t, J = 6.8 Hz, CH3); 13C NMR (75 MHz, CD3OD): d 85.4 (C-2), 75.6 (C-
3), 72.8 (C-4), 64.1 (C-1), 40.1 (COSO2CH3), 34.3, 33.1, 32.0, 30.8,
30.4, 29.2, 28.7, 26.2, 25.2, 22.8 (C-5–C-14), 16.2 (C-15); HRMS calcd
for C16H34SO6 [M+Na]+: 377.1974, found 377.1980.
4.19. (2S,3S,4R)-2-Azido-3,4-di-O-(benzoyloxy)-nonan-1-ol 22
23
[a]
D = +81.4 (c 1.00, CHCl3). 1H NMR (CDCl3): d 7.48–8.07
(10H, m, Ar–H), 5.49–5.46 (2H, m, H-3, H-4), 3.98–4.01 (1H, m,
H-1a), 3.75–3.82 (2H, m, H-1b, H-2), 1.82–1.99 (2H, m, H-5a,
H-5b), 1.18–1.51 (6H, m, CH2), 0.89 (3H, t, J = 6.8 Hz, CH3); 13C
NMR (75 MHz, CDCl3): d 166.1, 165.7 (C@O), 133.7–129.7 (CAr),
129.3, 129.1, 128.6, 128.4 (CAr), 73.4 (C-3), 72.9 (C-4), 63.1 (C-2),
62.1 (C-1), 31.5 (C-7), 29.7 (C-5), 25.2 (C-6), 22.4 (C-8), 13.9
4.14. General procedure for displacement of mesylate group
with sodium azide (e)
(C-9); HRMS calcd for
448.1829.
C
23H27N3O5[M+Na]+: 448.1848, found
The mesylated compounds were dissolved in DMF (10 mL) and
sodium azide (2 equiv) was added. The reaction was stirred over-
night at 60 °C, after which, it was taken up in water (50 mL) and
extracted with EtOAc (3 ꢁ 15 mL). The combined organic layers
were then washed with brine (30 mL), dried over anhydrous
Na2SO4 and evaporated. The residue was then purified by flash
chromatography using hexanes/EtOAc (4:1).
4.20. (2S,3S,4R)-2-Azido-3,4-di-O-(benzoyloxy)-dodecan-1-ol 23
23
[a]
D = +8.8 (c 1.00, CHCl3). 1H NMR (CDCl3): d 7.26–7.89 (10H,
m, Ar–H), 5.32–5.39 (2H, m, H-3, H-4), 3.65–3.82 (1H, m, H-1a),
3.54–3.60 (2H, m, H-1b, H-2), 1.62–1.82 (2H, m, H-5a, H-5b),
1.06–1.30 (12H, m, CH2), 0.68 (3H, t, J = 6.7 Hz, CH3); 13C NMR
(75 MHz, CDCl3): d 136.4.7–131.1 (CAr), 76.1 (C-3), 75.4 (C-4),
66.2 (C-2), 64.8 (C-1), 34.5, 33.0, 30.7, 30.3, 26.6, 23.6 25.3
(C-5–C11), 16.7 (C-12); HRMS calcd for
490.2318, found 490.2316.
C
26H33N3O5[M+Na]+:
4.15. (2S,3S,4R)-2-Azido-nonane-1,3,4-triol 19
Prepared following general procedure (e) using 16 (0.74 g,
2.75 mmol), sodium azide (0.36 g, 5.50 mmol) to afford a white solid
(0.38 g, 1.73 mmol, 63%).
4.21. (2S,3S,4R)-2-Azido-3,4-di-O-(benzoyloxy)-pentadecan-1-ol
24
23
[a]
D = +144.0 (c 1.00, CH3OH). 1H NMR
(CD3OD): d 3.82–3.86 (1H, dd, J1a,1b = 11.5, J1a,2 = 4.9 Hz, H-1a),
3.71–3.77 (1H, m, H-1b), 3.58–3.64 (2H, m, H-3, H-4), 3.44–3.47
(1H, ddd, J1b,2 = 9.9, J2,3 = 4.5 Hz), 1.45–1.56 (2H, m, H-5a, H-7a),
1.38 (1H, m, H-5b), 1.27–1.14 (5H, m, CH2), 0.79 (3H, t, J = 6.1 Hz,
CH3); 13C NMR (75 MHz, CD3OD): d 74.0 (C-3), 71.9 (C-4), 62.7
(C-2), 61.0 (C-1), 31.5 (C-5), 31.3 (C-6), 24.9 (C-7), 22.1 (C-8), 13.5
(C-9); HRMS calcd for C9H19N3O3[M+Na]+: 240.1324, found
240.1315.
23
[a]
D = +157.2 (c 1.00, CHCl3). 1H NMR (CDCl3): d 7.25–8.11
(10H, m, Ar–H), 5.48–5.5.4 (2H, m, H-3, H-4), 4.82 (1H, dd,
J1a,2 = 7.3, J1a,1b = 14.6 Hz, H-1a), 3.67–4.03 (2H, m, H-1b, H-2),
177–1.97 (2H, m, H-5a, H-5b), 1.09–1.46 (18H, m, CH2), 0.85 (3H,
t, J = 6.6 Hz, CH3); 13C NMR (75 MHz, CDCl3): d 162.5 (C@O),
135.8–130.6 (CAr), 75.4 (C-3), 75.1 (C-4), 65.3 (C-2), 64.3 (C-1),
34.0, 32.6, 32.4, 31.5, 29.7, 25.2, 23.8, 23.2, 22.8 22.4 (C-5–C14),
16.2 (C-15); HRMS calcd for C29H39N3O5[M+Na]+: 532.6370, found
532.6373.
4.16. (2S,3S,4R)-2-Azido-dodecane-1,3,4-triol 20
Prepared following general procedure (e) using 17 (0.95 g,
4.22. General procedure for glycosylation reaction (f)
3.03 mmol), sodium azide (0.39 g, 6.06 mmol) to afford a colour-
less syrup (0.69 g, 2.64 mmol, 87%). 23
[a]D = +45.0 (c 1.00, CH3OH).
To a solution of the persilylated galactose (3 equiv) in anhy-
drous CH2Cl2 (20 mL), iodotrimethylsilane (3 equiv) was added
and the reaction mixture stirred at room temperature under argon
for 30 min. The mixture was then concentrated and azeotroped
twice with dry benzene (5 mL). The yellow residue was dissolved
in dry benzene and kept under argon. Meanwhile, activated 4 Å
molecular sieves, tetrabutyl ammonium iodide (6 equiv), respec-
tive glycosyl acceptors 22, 23 and 24 (1 equiv) and diisopropyleth-
1H NMR (CD3OD): d 3.88–3.95 (1H, dd, J1a,1b = 11.1, J1a,2 = 3.3 Hz,
H-1a), 3.71–3.80 (1H, m, H-1b), 3.54–3.63 (1H, m, H-4),
3.48–3.53 (1H, m, H-3), 3.28–3.32 (1H, m, H-2), 1.20–1.45 (11H,
m, CH2), 0.89 (3H, t, J = 6.2 Hz, CH3); 13C NMR (75 MHz, CD3OD):
d 75.9 (C-3), 72.8 (C-4), 66.2 (C-2), 62.4 (C-1), 33.8, 32.9, 30.7,
30.3, 26.6, 23.6 (C-5–C-11), 13.8 (C-12); HRMS calcd for
C
12H25N3O3[M+Na]+: 282.1794, found 282.1794.

N. Veerapen et al. / Bioorg. Med. Chem. 19 (2011) 221–228
227
ylamine (4.5 equiv) were added to a two-necked flask fitted with a
condenser. Benzene (10 mL) was added and the solution stirred at
70 °C for 20 min. The solution of the glycosyl iodide 25 in benzene
was then cannulated into the two-necked flask and stirred at room
temperature for two days. Upon completion of the reaction, the
mixture was filtered through Celite and the Celite pad was washed
with CH2Cl2 (10 mL). The filtrate was washed with saturated so-
dium thiosulfate (10 mL), brine (10 mL), dried over anhydrous
Na2SO4 and concentrated in vacuo. The residue was dissolved in
MeOH and Dowex 50WX8-200 ion exchange resin was added
and the mixture stirred at room temperature overnight. The latter
was then filtered and the filtrate concentrated to give a residue
which was purified by flash chromatography using EtOAc/hexanes
(7:1) to give the glycosylated product.
m, Ar–H), 5.62–5.66 (1H, m, H-3Cer), 5.48–5.55 (1H, m, H-4Cer),
4.80 (1H, d, J1,2 = 2.4 Hz H-1), 4.12–4.16 (1H, m, H-6a), 3.90–3.95
(2H, m, H-2Cer, H-4), 3.83–3.85 (1H, m, H-3), 3.71–3.74 (4H, m,
H-2, H-5, H-1aCer, H-1bCer), 3.63–3.69 (1H, m, H-6b), 1.82–1.92
(2H, m, H-5aCer, H-5bCer), 1.15–1.30 (16H, m, CH2), 0.82 (3H, t,
J = 6.9 Hz, CH3); 13C NMR (75 MHz, CDCl3): d 135.1–139.5 (CAr),
106.5 (C-1), 79.5 (C-4Cer), 78.1 (C-3Cer), 71.4 (C-3), 70.8 (C-5),
70.0 (C-4), 69.5 (C-2), 67.7 (C-6), 63.4 (C-1Cer), 62.6 (C-2Cer),
22.9–36.8 (11 ꢁ CH2Cer), 19.5 (CH3Cer); HRMS calcd for
C
35H49N3O10[M+Na]+: 694.7686, found 694.7683.
4.26. General procedure for the removal of benzoate esters,
hydrogenation of the azido group and subsequent N-acylation
(g)
4.23. (2S,3S,4R)-2-Azido-3,4-di-O-(benzoyloxy)-1-(
pyranosyl)-nonane 26
a
-
D
-galacto-
Compounds 26, 27 and 28 were, respectively, dissolved in MeOH
and a 1 M solution of NaOMe (5 mL) was added. The mixture was
stirred at room temperature for 2 h, after which time TLC analysis
indicated that the reaction was complete. The reaction was neutra-
lised by the addition of Dowex 50WX8-200 resin. The latter was then
filtered and the filtrate concentrated to give a residue which was
purified by flash chromatography using EtOAc/MeOH (7:1) to give
compounds 29, 30 and 31 in quantitative yields. Compounds 29,
30 and 31 were, respectively, dissolved in MeOH (10 mL) and
stirred with Pd–C (5 mg) under H2 overnight, after which time the
mixture was filtered through a pad of Celite, which was subse-
quently washed with MeOH. The combined filtrates were concen-
trated to give the respective amines as white solids. The crude
amine was then dissolved in a 1:1 mixture of THF/NaOAC (saturated
solution) (5 mL) and the freshly prepared acid chloride of hexacosa-
noic acid was added. The reaction was allowed to stir vigorously
overnight at room temperature after which the organic phase was
removed. The aqueous phase was further extracted with THF
(2 ꢁ 1 mL), and the combined organic phases were concentrated.
The residue was finally purified by flash chromatography (gradient
from CHCl3 to 15% MeOH in CHCl3) to give target compounds 2, 3
and 4.
Prepared following general procedure (f) using per-O-pentam-
ethylsilyl- -galactose (0.27 g, 0.49 mmol), iodotrimethylsilane
a
-D
(0.09 g, 0.07 mL, 0.49 mmol), TBAI (3.65 mg, 0.99 mmol), DIPEA
(97.5 mg, 0.13 mL, 0.74 mmol) and 22 (70 mg, 0.16 mmol) to afford
26 as
a pale yellow oil (43 mg, 0.07 mmol) in 46% yield.
23
[a]
D = +36.1 (c 1.00, CHCl3). 1H NMR (CDCl3): d7.38–7.96 (10H,
m, Ar–H), 5.59–5.61 (1H, m, H-3Cer), 5.40–5.50 (1H, m, H-4Cer),
4.80 (1H, s, H-1), 4.10–4.13 (1H, dd, J6a, 6b = 7.9, J5,6a = 3.0 Hz,
H-6a), 3.91–3.94 (2H, m, H-2Cer
, H-4), 3.80–3.84 (1H, dd,
J2,3 = J3,4 = 6.0 Hz, H-3), 3.70–3.75 (4H, m, H-2, H-5, H-1aCer, H-1bCer),
3.62–3.67 (1H, m, H-6b), 1.81–1.91 (2H, m, H-5aCer, H-5bCer),
1.15–1.41 (6H, m, CH2), 0.83 (3H, t, J = 6.4 Hz, CH3); 13C NMR
(75 MHz, CDCl3): d 167.1, 167.0 (C@O), 127.4–133.4 (CAr), 101.4
(C-1), 73.2 (C-4Cer), 72.1 (C-3Cer), 70.9 (C-3), 70.6 (C-5), 70.2 (C-4),
69.7 (C-2), 68.1 (C-6), 62.4 (C-1Cer), 61.0 (C-2Cer), 21.9–33.3
(4 ꢁ CH2Cer), 13.9 (CH3Cer); HRMS calcd for C29H37N3O10[M+Na]+:
610.6183, found 610.6188.
4.24. (2S,3S,4R)-2-Azido-3,4-di-O-(benzoyloxy)-1-(a-D-galacto-
pyranosyl)-dodecane 27
4.27. (2S,3S,4R)-1-(
a-D-Galactopyranosyl)-2-hexacosanoylami-
Prepared following general procedure (f) using per-O-pentam-
ethylsilyl- -galactose (0.85 g, 1.58 mmol), iodotrimethylsilane
no-3,4-nonanediol 2
a
-D
(0.32 g, 0.23 mL, 1.58 mmol), TBAI (1.17 g, 3.16 mmol), DIPEA
Prepared following general procedure (g) using 26 (50 mg,
(1.05 g, 1.42 mL, 8.16 mmol) and 23 (0.25 g, 0.53 mmol) to afford
0.12 mmol) and hexacosanoic acid (71 mg, 0.18 mmol, 1.5 equiv)
27 as an off-white solid (110 mg, 0.17 mmol) in 33% yield..
to give 2 as a white solid (31 mg, 0.04 mmol) in 35% yield..
23
23
[a
]
D = +92.4 (c 1.00, CHCl3). 1H NMR (CDCl3): d7.38–7.98 (10H,
[a]
D = +12.6 (c 1.00, CHCl3/CH3OH 2:1). 1H NMR (CDCl3/CD3OD
m, Ar–H), 5.61–5.64 (1H, m, H-3Cer), 5.48–5.52 (1H, m, H-4Cer),
4.79 (1H, s, H-1), 4.11–4.13 (1H, dd, J6a, 6b = 8.0, J5.6a = 2.9 Hz,
H-6a), 3.90–3.93 (2H, m, H-2Cer, H-4), 3.82–3.85 (1H, m, H-3),
3.71–3.75 (4H, m, H-2, H-5, H-1aCer, H-1bCer), 3.63–3.67 (1H, dd,
J5,6b = 6.0 Hz, H-6b), 1.81–1.94 (2H, m, H-5aCer, H-5bCer), 1.18–1.42
(12H, m, CH2), 0.79 (3H, t, J = 6.7 Hz, CH3); 13C NMR (75 MHz, CDCl3):
d 166.8, 166.3 (C@O), 128.7–134.2 (CAr), 100.2 (C-1), 73.7 (C-4Cer),
72.6 (C-3Cer), 71.4 (C-3), 70.6 (C-5), 70.2 (C-4), 69.5 (C-2), 67.9
(C-6), 62.1 (C-1Cer), 61.2 (C-2Cer), 22.9–32.1 (6 ꢁ CH2Cer), 14.2
(CH3Cer); HRMS calcd for C32H43N3O10[M+Na]+: 652.2846, found
610.2845.
2:1): d 4.85 (1H, d, J1,2 = 3.4 Hz, H-1), 4.13–4.16 (1H, m, H-2Cer),
3.86–3.89 (1H, m, H-3), 3.81–3.86 (1H, dd, J1a,2 = 4.6, J1a,1b = 10.5 Hz,
H-1aCer), 3.72–3.78 (2H, m, H-2, H-5), 3.66–3.72 (3H, m, H-4, H-6a,
H-6b), 3.60–3.66 (1H, m, H-1bCer), 3.47–3.55 (2H, m, H-3Cer
,
H-4Cer), 2.13–2.18 (2H, m, NHCOCH2C24H49), 1.53–1.67 (3H, m,
CH2), 0.94–1.46 (49H, m, CH2), 0.79 (6H, t, J = 6.7 Hz, CH3); 13C
NMR (75 MHz, (CDCl3/CD3OD 2:1)): d 103.6 (C-1), 77.7 (C-3Cer),
75.4 (C-4Cer), 73.7 (C-5), 73.2 (C-4), 72.6 (C-3), 71.7 (C-2), 70.7
(C-1Cer), 65.2 (C-6), 64.7 (C-2Cer), 23.8–33.4 (CH2), 15.9 (CH3, CH3Cer);
HRMS calcd for C41H81NO9 [M+Na]+: 754.5809, found 754.5812.
4.28. (2S,3S,4R)-1-(
a-D-Galactopyranosyl)-2-hexacosanoylami-
4.25. (2S,3S,4R)-2-Azido-3,4-di-O-(benzoyloxy)-1-(a-D-galacto-
no-3,4-dodecanediol 3
pyranosyl)-pentadecane 28
Prepared following general procedure (g) using 27 (100 mg,
Prepared following general procedure (f) using per-O-pentam-
ethylsilyl- -galactose (1.61 g, 2.97 mmol), iodotrimethylsilane
(0.59 g, 0.43 mL, 2.97 mmol), TBAI (2.19 g, 5.94 mmol), DIPEA
0.16 mmol) and hexacosanoic acid (95 mg, 0.24 mmol, 1.5 equiv)
a
-D
to give 3 as a white solid (45 mg, 0.06 mmol) in 37% yield.
23
[a]
D = +15.6 (c 1.00, CHCl3/CH3OH 2:1). 1H NMR (CDCl3/CD3OD
(0.58 g, 0.78 mL, 4.45 mmol) and 24 (0.50 g, 0.99 mmol) to afford
2:1): d 4.85 (1H, d, J1,2 = 3.8 Hz, H-1), 4.12–4.16 (1H, m, H-2Cer),
3.87–3.89 (1H, m, H-3), 3.81–3.85 (1H, dd, J1a,2 = 4.8,
J1a,1b = 10.5 Hz, H-1aCer), 3.72–3.77 (2H, m, H-2, H-5), 3.66–3.72
28 as an off-white solid (175 mg, 0.26 mmol) in 26% yield..
23
[a]
D = +81.4 (c 1.00, CHCl3). 1H NMR (CDCl3): d7.37–8.02 (10H,

228
N. Veerapen et al. / Bioorg. Med. Chem. 19 (2011) 221–228
(3H, m, H-4, H-6a, H-6b), 3.61–3.66 (1H, m, H-1bCer), 3.47–3.51
(2H, m, H-3Cer, H-4Cer), 2.14–2.18 (2H, m, NHCOCH2C24H49), 1.46–
1.65 (3H, m, CH2), 1.16–1.34 (57H, m, CH2), 0.82 (6H, t, J = 6.6 Hz,
CH3); 13C NMR (75 MHz, (CDCl3/CD3OD 2:1)): d 101.8 (C-1), 76.9
(C-3Cer), 74.2 (C-4Cer), 73.2 (C-5), 73.0 (C-4), 72.6 (C-3), 71.8 (C-
2), 70.1 (C-1Cer), 65.5 (C-6), 64.4 (C-2Cer), 24.8–36.0 (CH2), 17.0
(CH3, CH3Cer); HRMS calcd for C44H87NO9 [M+Na]+: 797.6289,
found 797.6291.
References and notes
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4.29. (2S,3S,4R)-1-(a-D-Galactopyranosyl)-2-hexacosanoylami-
no-3,4-pentadecanediol 4
Prepared following general procedure (g) using 28 (100 mg,
0.15 mmol) and hexacosanoic acid (90 mg, 0.22 mmol, 1.5 equiv)
to give 4 as a white solid (44 mg, 0.05 mmol) in 36% yield.
23
[a]
D = +26.7 (c 1.00, CHCl3/CH3OH 2:1). 1H NMR (CDCl3/CD3OD
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3.87–3.89 (1H, m, H-3), 3.81–3.86 (1H, dd, J1a,2 = 4.8, J1a,1b = 11.0 Hz,
H-1aCer), 3.72–3.77 (2H, m, H-2, H-5), 3.66–3.71 (3H, m, H-4, H-6a,
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Porcelli, S. A. J. Immunol. 2007, 178, 1415.
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H-6b), 3.61–3.66 (1H, m, H-1bCer), 3.46–3.51 (2H, m, H-3Cer
,
H-4Cer), 2.13–2.18 (2H, m, NHCOCH2C24H49), 1.46–1.65 (3H, m,
CH2), 1.11–1.35 (70H, m, CH2), 0.83 (6H, t, J = 6.8 Hz, CH3); 13C
NMR (75 MHz, (CDCl3/CD3OD 2:1)): d 102.5 (C-1), 76.8 (C-3Cer),
74.9 (C-4Cer), 73.7 (C-5), 73.1 (C-4), 72.9 (C-3), 71.9 (C-2), 70.7
(C-1Cer), 65.0 (C-6), 64.5 (C-2Cer), 25.6–35.1 (CH2), 16.9 (CH3, CH3Cer);
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4.30. Elisa
C1R-hCD1d cells were pulsed for overnight with GSLs. 5 ꢁ 104
cell-pulsed targets were incubated at 37 °C with 2 ꢁ 104 iNKT cells
in a final volume of 200 lL. After 36 h, the supernatants were har-
vested, and the concentrations of IFN-c and IL-4 were determined
by commercial ELISA (BD Pharmingen).
Acknowledgments
GSB acknowledges support in the form of a Personal Research
Chair from Mr. James Badrick, Royal Society Wolfson Research
Merit Award, as a former Lister Institute-Jenner Research Fellow,
the Medical Council and The Wellcome Trust (084923/B/08/7).
V.C. is supported by Cancer Research UK grant C399/A2291. The
Bruker spectrometers used in this research was obtained, through
Birmingham Science City: Innovative Uses for Advanced Materials
in the Modern World (West Midlands Centre for Advanced Materi-
als Project 2), with support from Advantage West Midlands (AWM)
and part funded by the European Regional Development Fund
(ERDF).
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