Novel synthesis of α-galactosyl-ceramides and confirmation of their powerful NKT cell agonist activity

Article abstract of DOI:10.1016/j.carres.2006.09.006

α-Galactosyl-ceramide (1) has been identified as a powerful modulator of immunological processes through its capacity to bind CD1d molecules and specifically activate invariant natural killer (NK)-like T cells (iNKT cells). This paper describes the synthe

Full text of DOI:10.1016/j.carres.2006.09.006

Carbohydrate Research 341 (2006) 2785–2798
Novel synthesis of a-galactosyl-ceramides and confirmation
of their powerful NKT cell agonist activity
Adrianne Lee,a Kathryn J. Farrand,b Nina Dickgreber,b Colin M. Hayman,a
Stefan Jurs,a,c Ian F. Hermansb and Gavin F. Paintera,*
¨
aCarbohydrate Chemistry, Industrial Research Limited, PO Box 31-310, Lower Hutt, New Zealand
bThe Malaghan Institute of Medical Research, PO Box 7060, Wellington, New Zealand
cInstitut fu¨r Organische Chemie der Universita¨t Hamburg, Martin-Luther-King-Platz 6, 20146 Hamburg, Germany
Received 7 August 2006; received in revised form 5 September 2006; accepted 7 September 2006
Available online 2 October 2006
Abstract—a-Galactosyl-ceramide (1) has been identified as a powerful modulator of immunological processes through its capacity
to bind CD1d molecules and specifically activate invariant natural killer (NK)-like T cells (iNKT cells). This paper describes the
synthesis of 1, the analogous a-galactosyl-ceramide 3, and its short chain analogue ‘OCH’ (2), by use of the 4,6-di-O-tert-butyl-
silylene (DTBS) protecting group to produce a powerful a-galactosylating agent. In vivo experiments confirmed these compounds
to be potent and selective activators of iNKT cells in a CD1d-dependent manner, each inducing a unique profile of cytokine release.
This synthesis strategy will permit the generation of novel derivatives for use in the study of the mechanism of iNKT cell activation.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Keywords: a-Galactosyl-ceramide; iNKT cells; Lipid antigen presentation; CD1d; Agelasphins; KRN 7000
1. Introduction
may attenuate autoimmune diseases such as multiple
sclerosis10 and arthritis.11 The best known class of ago-
nist ligands for iNKT cells12 are the agelasphins, first
isolated from a marine sponge for their antitumour
attributes.13,14 Later structure activity relationships
identified the compound (2S,3S,4R)-1-(a-D-galactopyr-
anosyloxy)-2-(N-hexacosanoylamino)octadecane-3,4-
diol (1),15 commonly referred to as a-galactosyl-cera-
mide (a-GalCer), as a candidate for clinical trials. a-Gal-
Cer specifically, and potently, stimulates iNKT cells to
release Th1 and Th2 cytokines in vitro and in vivo. A
short chain derivative of a-GalCer, known as ‘OCH’
(2), was shown to drive the release of a cytokine profile
with a Th2 bias, thereby indicating a potential applica-
tion in the treatment of autoimmune conditions.10,12
While there has been considerable interest in using a-
GalCer to stimulate iNKT cells in a therapeutic manner,
it has recently been shown that repeated administration
of a-GalCer induces long-term iNKT unresponsiveness
in mice,16 suggesting that there are limitations to the
use of a-GalCer as a therapeutic option.
In contrast to most T cells that recognize specific peptide
fragments bound to major histocompatibility complex
(MHC) molecules, invariant natural killer (NK)-like T
cells (iNKT cells) recognize glycolipids bound by the
MHC-like molecule CD1d. After stimulation, iNKT
cells can modulate the function of a number of immune
cells including T cells,1 B cells,2 NK cells3 and den-
dritic cells (DC),4–6 primarily through the release of a
spectrum of cytokines, including ‘Th1’ cytokines such
as IFN-c, and ‘Th2’ cytokines such as IL-4 and IL-13.
Controlling the profile of cytokine release may be key
to exploiting iNKT cell function for the treatment of
various diseases. For example, the release of Th1 cyto-
kines is likely to contribute to antitumour7,8 and anti-
microbial functions,9 whereas the release of Th2 cytokines
*
Corresponding author. Tel.: +64 4 931 3103; fax: +64 4 931 3055;
e-mail: g.painter@irl.cri.nz
0008-6215/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2006.09.006

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A. Lee et al. / Carbohydrate Research 341 (2006) 2785–2798
Stimulation of iNKT cells in vivo by injection of a-
anomerization, has also been used successfully in the
synthesis of a-galactosyl-ceramides in a selective man-
ner. However, these reactions generally require extended
GalCer has been shown to provide a powerful stimulus
to vaccine-induced immune responses.4–6 However, a-
GalCer may not be the best candidate for this adjuvant
activity if multiple rounds of vaccination are required. It
has been suggested that less potent stimulation of iNKT
cells may avoid the induction of long-term unrespon-
siveness.17 Here we report a novel method for the gen-
eral synthesis of a-galactosyl-ceramides that can be
used to generate different analogues of a-GalCer. We
confirm that this synthesis strategy can be used to gener-
ate known structures a-GalCer (1) and OCH (2), and
show that a third structure (3) has the capacity to stim-
ulate iNKT cells in a CD1d-dependent manner with less
potency than a-GalCer (1), but without the stronger Th2
bias of OCH (2).
reaction times22 and are often not high yielding.
27
´
Figueroa-Perez and Schmidt have demonstrated
that use of a 4,6-O-benzylidene acetal group within a
glycosyl trichloroacetimidate donor can be used to
afford a-galactosyl-ceramides in high yield and with good
anomeric selectivity. Similarly, the 4,6-O-di-tert-butylsil-
ylene (DTBS) group can function as a powerful a-direct-
ing group in galactosylation donors and, significantly,
under circumstances in which the corresponding 4,6-O-
benzylidene donors can give only b-products.28 We
therefore reasoned that it should be useful within donors
in the synthesis of a-galactosyl-ceramides. With this in
mind, we prepared the galactosyl donor 8 from allyl a-
D-galactopyranoside (4)29 via compounds 5–7 (Scheme
1). The 4,6-O-DTBS group was installed by reaction
with di-tert-butylsilyl bis(trifluoromethanesulfonate) in
the presence of DMAP. Benzylation of diol 5 with
NaH/BnBr needed to be monitored carefully as excess
reagent and/or extended reaction times led to cleavage
of the DTBS group. Nevertheless, the fully protected 6
was made in 66% yield. Deprotection of the anomeric
hemi-acetal and installation of the a-imidate function
proceeded without incident.
OH
HO
O
1 R = C11H23, R1 = C25H51
2 R = C2H5, R1 = C23H47
3 R = C13H27, R1 = C23H47
NHCOR1
OH
HO
HO
O
R
OH
2. Results and discussion
There are a number of elegant syntheses of the cera-
mide moiety or precursors thereof. In this study, we
chose as a convenient glycosyl acceptor the sphingosine
derivative 13, which was prepared via compounds 10–12
from the 2-deoxy-D-lyxo-hexose derivative 9 by applica-
tion of the existing protocols (Scheme 2).19
2.1. Synthesis of a-galactosyl-ceramides 1, 2 and 3
A number of the reported syntheses of a-galactosyl-
ceramides depend on chemical glycosylation of a sphin-
gosine derivative in the key step.18–26 Because the
glycosidic bond forming reactions are not normally
stereospecific, most of these methods require tedious
separations of anomeric products. Furthermore, those
reactions that are highly a-selective are often not high
yielding.25 A method for facilitating the isolation of
the a-anomers of the mixtures is based on preparing
them by use of 3,4,6-tri-O-acetyl-2-O-benzyl-a-D-galac-
tosyl bromide. Release of the C-2 hydroxyl group of
the products aids in the chromatographic removal of
the b-anomers.21 The application of 2,3,4,6-tetra-O-benz-
yl-a-D-galactosyl bromide in the presence of tetrabutyl-
ammonium bromide, which induces its in situ
The glycosylation with imidate 8 of acceptor 13 pro-
ceeded smoothly to afford the desired a-glycoside 14 in
good yield with no detectable b-anomer (Scheme 2).
The stereochemistry of the newly formed glycosidic link-
age was established as a from the C-1 heteronuclear one-
bond 13C–1H coupling constant of 168.6 Hz.30,31 In a
comparative experiment, to confirm the stereo-directing
effect of the DTBS group, glycosylation of 13 with
donor 15 afforded the expected glycoside together with
the b-anomer (70% combined yield, a:b ratio 2.5:1, esti-
1
mated from H NMR) that proved difficult to separate.
In a similar experiment reported by Wong and co-work-
Si
O
Si
O
OH
HO
HO
O
O
iii
i
O
O
O
RO
BnO
HO
O
BnO
OR
RO
O
5 R = H
6 R = Bn
7 R = H
8 R = C(=NH)CCl3
4
ii
iv
Scheme 1. Reagents, conditions and yields: (i) tBu2Si(OTf)2, DMAP, Py (65%); (ii) NaH, BnBr, DMF (66%); (iii) Ph2P2ðCODÞIrþPF ꢀ, THF, then
6
AcCl, MeOH–CH2Cl2; (iv) CCl3CN, DBU, CH2Cl2 (steps iii and iv) 40% in total.

A. Lee et al. / Carbohydrate Research 341 (2006) 2785–2798
2787
OTIPS
O
HO
HO
OH OBn
OBn
OH OBn
OBn
i
ii
TIPSO
TIPSO
C2H5
O
STol
10
11
9
Si
O
O
N3 OBn
OBn
O
RO
C2H5
BnO
N3 OBn
OBn
iii
BnO
O
C2H5
12 R = TIPS
13 R = H
v
iv
14
Scheme 2. Reagents, conditions and yields: (i) Ref. 19; (ii) Ph3P+PrÆBrꢀ, n-BuLi, THF (88%); (iii) Ph3P, DEAD, HN3, THF (91%); (iv) TBAF, THF
(85%); (v) 8, TMSOTf, CH2Cl2 (66%).
ers,19 glycosylation of the related acceptor 29 (vide infra)
with donor 15 afforded the corresponding a-glycoside in
50% yield with a similar a:b ratio. Thioglycoside 16
was also prepared, but standard reaction conditions
including activation with NIS/TFOH,32 DMTSF33 and
MeOTf34 only gave, at best, minor amounts of the
desired galactoside 14.
give saturated amine 17 (Scheme 3). Somewhat surpris-
ingly, the four benzyl ethers appeared to be completely
resistant to hydrogenolysis under the conditions used.
However, simultaneous hydrogenolysis of the benzyl
groups, reduction of the azide and hydrogenation of
the alkene to give compound 18 were achieved by hydro-
gen transfer reaction with ammonium formate.35 This
strategy should allow the introduction of unsaturated
fatty acid moieties within the amido alkyl chain, which
is currently under investigation. In an alternative proce-
dure, as adopted by others,19 the azido group of com-
pound 14 was reduced with PMe3 to afford amine 19
that was subsequently coupled with tetracosanoic acid
to give amide 20 (Scheme 3). The silyl protecting group
was removed with HF/Py to afford diol 21, and the final
deprotective-hydrogenation/hydrogenolysis with Pd-
black afforded target compound 2.
OBn
Si
O
BnO
BnO
O
O
O
SPh
BnO
BnO
OC(NH)CCl3
OBn
15
16
Reduction of the azide functional group and hydro-
genation of the double bond of glycoside 14 were accom-
plished in one step by hydrogenation over Pd/C to
The above methodology, used to prepare the short
chain a-galactosyl-ceramide derivative 2, was then applied
Si
O
O
O
RO
NH2
OR
RO
i
O
C2H5
17 R = Bn
18 R = H
14
ii
OR
iii
Si
O
O
OH
HO
R1O
O
O
v
BnO
NHR OR1
OR1
NHR OBn
OBn
R1O
BnO
R2
O
O
C2H5
19 R = H
20 R = COC23H47
21 R1 = Bn, R2 = CH=CHC2H5, R = COC23H47
R1 = H, R2 = CH2CH2C2H5, R = COC23H47
iv
vi
2
Scheme 3. Reagents, conditions and yields: (i) Pd–C, H2 (96%); (ii) HCO2NH4, Pd–C, MeOH (40%); (iii) Me3P, THF (99%); (iv) C23H47CO2H,
CH2Cl2, DMF, then DIPEA (70%); (v) HFÆPy, THF (81%); (vi) Pd-black, H2, CHCl3/MeOH (50%).

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A. Lee et al. / Carbohydrate Research 341 (2006) 2785–2798
with acceptor 24 and donor 8 to give a-galactosyl-
ceramide 3 via intermediates 25–28. Acceptor 24 was
prepared from aldehyde 10, by the method used for
alcohol 13, via intermediates 22 and 23 (Scheme 4).
In a similar way, a-galactosyl-ceramide 1 was pre-
pared from acceptor 2919 and donor 8. As before, the
glycosylation reaction afforded the a product 30 in good
yield (82%) with no detectable amounts of the b anomer
(Scheme 5). This result compares favourably with the
2.2. The stimulation effects of a-galactosyl-ceramides
on iNKT cells in vivo
A hallmark of iNKT cell activation is the release of an
array of different cytokines into the tissues. Included
within this ‘cytokine storm’ are the cytokines IFN-c,
IL-4 and IL-12, which can be derived from the iNKT
cells themselves, or from other immune cells that have
been stimulated in turn by the activated iNKT cells.
Thus, activated iNKT cells can interact with DC, driv-
ing ‘maturation’ characterized by the release of signifi-
cant quantities of bioactive IL-12 (IL-12p70) into the
serum. Maturation of DC is also associated with an in-
crease in the expression of molecules involved in T cell
stimulation on the DC surface.4–6 We used these two
attributes of iNKT cell-induced DC maturation to
examine the biological activity of the synthetic glyco-
literature. Figueroa-Perez and Schmidt27 observed glyco-
´
sylation of a triol acceptor with the 4,6-benzylidene-pro-
tected trichloroacetimidate affording the a-glycoside in
73% yield. Later Wong19 demonstrated that glycosyla-
tion of 29 with the same 4,6-benzylidene-protected
donor afforded the corresponding a-glycoside in 68%.
The spectroscopic data collected for 1 were in good
agreement with that already reported.15,23
R2
OBn
OH
OBn
R1O
iii
C13H27
i
ii
TIPSO
C13H27
10
OBn
22
OBn
23 R1 = TIPS, R2 = N3
24 R1 = H, R2 = N3
OR1
OH
R1O
HO
O
O
HO
BnO
R2
NHR OH
OBn
HO
BnO
O
C13H27
O
C13H27
OH
OBn
25 R1 = tBu2Si, R2 = N3
iv
3 R = COC23H47
24
v
vi
26 R1 = tBu2Si, R2 = NH2
27 R1 = tBu2Si, R2 = NHCOC23H47
28 R1 = H, R2 = NHCOC23H47
vii
viii
Scheme 4. Reagents, conditions and yields: (i) Ph3P+C14H29ÆBrꢀ, n-BuLi, THF (84%); (ii) Ph3P, DEAD, HN3, THF (77%); (iii) TBAF, THF (94%);
(iv) 8, TMSOTf, CH2Cl2 (63%); (v) Me3P, THF (94%); (vi) EDCl, HOBt, C23H47CO2H, DIPEA, CH2Cl2/DMF (62%); (vii) HFÆPy, THF (63%); (viii)
Pd-black, H2, THF/MeOH/H2O (35:10:1) (79%).
OR1
R1O
OBn
N3
O
BnO
R2
i
HO
OBn
C
11H23
BnO
O
C11H23
OBn
29
OBn
30 R1 = tBu2Si, R2 = N3
31 R1 = tBu2Si, R2 = NH2
ii
OH
iii
iv
HO
HO
32 R1 = tBu2Si, R2 = NHCOC25H51
33 R1 = H, R2 = NHCOC25H51
O
NHR OH
HO
O
C11H23
v
OH
1 R = COC25H51
Scheme 5. Reagents, conditions and yields: (i) 8, TMSOTf, CH2Cl2 (82%); (ii) Me3P, THF; (iii) EDCl, HOBt, C25H51CO2H, DIPEA, CH2Cl2/DMF
(86%); (iv) HFÆPy, THF (79%); (v) Pd-black, H2, THF/MeOH/H2O (35:10:1) (85%).

A. Lee et al. / Carbohydrate Research 341 (2006) 2785–2798
C57BL/6
CD1d-/-
2789
500
450
400
350
300
250
200
150
100
50
C57BL/6
CD1d-/-
A
B
100
80
60
40
20
100
80
60
40
20
100
80
60
40
20
100
80
60
40
20
100
80
60
40
20
100
80
60
40
20
80
60
40
20
100
80
60
40
20
80
60
IL-12p70 at 6 h
compound 1
40
Wild-type
CD1d-/-
20
1000
1
2
3
1
2
3
10
0
1
2
3
04
4
1
2
3
10
10
10
10
10
10
10
10
10
10
10
10
80
60
compound 2
compound 3
40
20
100
0
100
1
10
2
10
3
10
1
10
2
10
3
10
1
2
3
1
2
10
3
10
10
10
10
10
10
80
60
40
20
80
60
40
20
0
1
2
3
4
1
2
3
4
10
0
10 10
10 10
1
2
3
4
1
2
3
4
10
10
10 10
10
10
10
10 10
10 10
10 10
CD86
CD40
0
100
80
60
40
20
100
80
60
40
20
100
80
60
40
20
compound 1
compound 2
compound 3
80
60
40
compound 1
compound 2
compound 3
20
untreated
1 µg glycolipid
100
1
2
3
4
1
2
3
10
10
10
10
10
10
10
80
60
40
20
0
3
1
2
1
2
3
10
10
10
10
10
10
80
60
40
20
0
1
2
3
4
1
2
3
4
10
10
10
10 10
10 10
10 10
CD80
C
600
500
400
300
200
100
0
compound 1
compound 2
compound 3
1
10
100
1000
10000
Amount of glycolipid injected (ng)
Figure 1. Injection of synthetic glycolipids induces activation of iNKT cells and subsequent maturation of dendritic cells. C57BL/6 (wild-type) and
CD1dꢀ/ꢀ mice (n = 3) were injected intravenously with 1 lg of glycolipid. (A) Serum was collected 6 h after the injection for the analysis of IL-12p70
levels. Mean cytokine levels SE are presented. (B) Spleens were removed 20 h after injection for antibody labelling and flow cytometry. The
expression of CD40, CD80 and CD86 was assessed on CD11c+ dendritic cells. Representative FACS profiles are shown. (C) Titrated doses of
glycolipids were injected intravenously into groups of C57BL/6 mice (n = 3) and spleens were removed 20 h later for the analysis of CD86 expression
on CD11c+ dendritic cells. Mean fluorescence index SE are presented.
lipids. Injection of 1 lg of the synthetic compounds 1, 2 or
3 into wild-type mice induced the release of IL-12p70 into
the serum (Fig. 1A). In contrast, no IL-12p70 was re-
leased when the compounds were injected into CD1d-
deficient mice, which are completely devoid of iNKT
cells. This readout of iNKT cell activity showed com-
pound 1 (a-GalCer) to have the most potent activity,
and compound 2 (OCH) the least. The new derivative
of a-GalCer, compound 3, had intermediate activity.
Using antibody labelling and flow cytometry, expression
of the co-stimulatory molecules CD80 and CD86, and
CD40 was shown to be increased on splenic DC (defined
as CD11c+ cells) in wild-type animals following injec-
tion with 1 lg of 1, 2 or 3 (Fig. 1B). No increase in the
expression of these molecules was observed in CD1d-defi-
cient animals. To assess the potency of the different
compounds to induce iNKT cell-mediated maturation
of DC, the mice were injected with titrated doses of
the glycolipids. This analysis, shown in Figure 1C, again
showed compound 1 (a-GalCer) to have the most potent
activity, compound 2 (OCH) the least and compound 3
to have intermediate activity.
The ability of the different compounds to induce
different profiles of cytokine release was also assessed
(Fig. 2). In agreement with the published data,10,36 injec-
tion of 1 lg of a-GalCer (1) led to the release of consid-
erable quantities of both IL-4 and IFN-c into the serum,
whereas injection of 1 lg of analogue OCH (2) induced
a weaker response, with considerably less IFN-c, indi-
cating a Th2 bias. The injection of 1 lg of compound
3 induced a similar release of IL-4 to OCH (2), but the
intermediate levels of IFN-c indicated less Th2 bias.

2790
A. Lee et al. / Carbohydrate Research 341 (2006) 2785–2798
1800
1600
9000
8000
7000
6000
5000
4000
3000
2000
1000
IFN-γ at 6 h
2 h
20 h
IFN-γ
1400
1200
1000
800
600
400
200
0
0
800
700
600
500
400
300
200
100
0
1
10
100
1000
10000
Amount of glycolipid injected (ng)
IL-4
compound 1
compound 2
compound 3
compound 1
compound 2
compound 3
Figure 2. Kinetics of cytokine release into serum following the injection of glycolipids. C57BL/6 mice were injected intravenously with 1 lg of
glycolipid (left panels, n = 8), or titrated doses of glycolipid (right panel) and then the serum was collected at the indicated times for analysis of IL-4
and IFN-c levels.
This hierarchy of IFN-c responses was shown again
when titrated doses of glycolipids were injected (Fig. 2,
right panel), with compound 3 inducing the release of
significantly higher levels of IFN-c than OCH (2) when
more glycolipid was injected, but never exceeding the
levels induced by a-GalCer (1).
the mechanism of iNKT cell activation, and ways in
which this cell type can be exploited therapeutically.
4. Experimental
4.1. General methods
Melting points were measured on a Gallenkamp
capillary melting point apparatus and are uncorrected.
Specific optical rotations, given in 10ꢀ1 deg cm2 gꢀ1, were
measured at ambient temperature using either a Jasco
DIP-370 or DIP-1000 polarimeter with a cell of path
length 1.0 dm. 1H NMR spectra were obtained at either
300 or 500 MHz and referenced to the residual solvent
peak. Chemical shifts are reported using the d scale,
and coupling constants (J) and separations are reported
in hertz. 13C NMR spectra were recorded at 75 or
125 MHz and chemical shifts are reported using the d
scale. FAB mass spectra were recorded on a Kratos
MSORF or a VG70-250S double focusing, magnetic
sector mass spectrometer under chemical ionization con-
ditions using isobutene, ammonia or xenon as the ioniz-
ing gas, or under high-resolution FAB conditions in a
glycerol or nitrobenzyl alcohol matrix. Electro-spray
ionization (ESI) mass spectra were recorded on a
PerSeptive Biosystems Mariner time of flight mass spec-
trometer. Elemental analyses were carried out by Dr. R.
G. Cunninghame and associates at the Campbell Micro-
3. Conclusions
We have reported a new synthetic protocol for the
synthesis of a-galactosyl-ceramides that should have
general applicability. The selectivity of the key
glycosylation reaction was controlled by incorporation
of the powerful a-directing 4,6-O-DTBS group into the
galactosylating donor. The synthetic compounds pre-
pared by this methodology were shown to be powerful
iNKT cell agonists with the capacity to induce cytokine
release and DC maturation in a CD1d-dependent man-
ner. One of these structures (3), has the capacity to stim-
ulate iNKT with less potency than a-GalCer (1), but
without the stronger Th2 bias of OCH (2). Further
study on the adjuvant activity of compound 3 is now
being carried out, with specific emphasis on the ability
of this compound to be utilized in repeated vaccination
procedures. The new a-galactosyl-ceramide synthesis
protocol described here may permit the investigation
of new derivatives37 in an attempt to further understand

A. Lee et al. / Carbohydrate Research 341 (2006) 2785–2798
2791
analytical Laboratory, University of Otago, Dunedin,
New Zealand. Thin layer chromatography (TLC) was
performed on Merck silica gel DC Alurolle Kieselgel
60F254 plates and were visualized under UV light and/
or with a spray (5% w/v dodecamolybdophosphoric acid
in EtOH) with subsequent heating. Flash column chro-
matography was carried out using Merck Kieselgel 60
(230–400 mesh). All chromatographic solvents were of
reagent grade. THF was distilled from sodium-benzo-
phenone ketyl under nitrogen and dichloromethane
was distilled from phosphorus pentoxide. All other
solvents and reagents were purified using the methods
described by Perrin and Armarego.38 Organic solutions
were dried over MgSO4.
tography on silica gel. Elution with EtOAc/light petro-
leum (1:4 to 3:7) afforded the title compound 6 as an oil
22
1
(1.67 g, 3.09 mmol, 62%); ½aꢁD +66 (c 0.6, CHCl3); H
NMR (300 MHz, CDCl3) d 7.44–7.21 (m, 10H), 5.96–
5.83 (m, 1H), 5.34–5.25 (m, 1H), 5.20–5.13 (m, 1H),
4.86 (d, J = 12.0 Hz, 1H), 4.78 (d, J = 3.7 Hz, 1H), 4.73
(s, 2H), 4.67 (d, J = 12.0 Hz, 1H), 4.49 (d, J = 2.6 Hz,
1H), 4.22–3.97 (m, 5H), 3.85 (dd, J = 10.0, 3.0 Hz, 1H),
3.62 (s, 1H), 1.06 (s, 9H), 0.99 (s, 9H); 13C NMR
(75 MHz, CDCl3) d 139.1, 138.6, 134.1, 128.2, 127.5,
127.4, 117.5, 97.1, 77.7, 74.2, 73.6, 71.2, 71.1, 68.2, 67.3,
67.2, 27.6, 27.3, 23.4, 20.6; HRMS-ESI [M+Na]+ calcd
for C31H44O6NaSi: 563.2805. Found 563.2813.
4.4. 2,3-Di-O-benzyl-4,6-O-di-tert-butylsilylene-D-galac-
topyranose (7)
4.2. Allyl 4,6-O-di-tert-butylsilylene-D-galactopyranoside
(5)
1,5-Cyclooctadiene)bis(methyldiphenylphosphine)irid-
ium(I) hexafluorophosphate (390 mg, 0.63 mmol) was
added to a stirred solution of allyl glycoside 7 (1.75 g,
3.24 mmol) in THF (50 mL) under argon. This atmo-
sphere was replaced with hydrogen for ca. 1 min and
the hydrogen was replaced with argon. The mixture
was stirred at 20 ꢁC for 70 min, the solvent was removed
in vacuo and the residue dissolved with stirring in
CH2Cl2/MeOH (2:1, 30 mL). Acetyl chloride (1.0 mL,
12.9 mmol) was added to this solution and stirring con-
tinued for a further 90 min when solid NaHCO3 (2.00 g,
23.8 mmol) was added. The mixture was stirred for an
additional 5 min when H2O (50 mL) was added. This
mixture was extracted with ether (2 · 100 mL) and after
drying and filtration the solvent was removed and the
residue purified by column chromatography on silica
gel. Elution with EtOAc/light petroleum (1:4 to 35:65)
afforded the title compound 7 as an oil (843 mg,
1.68 mmol, 52%); 13C NMR (75 MHz, CDCl3), a-ano-
mer only, d 138.8, 138.2, 129.1, 128.3, 127.8, 127.4,
92.2, 77.5, 77.4, 74.8, 73.9, 70.9, 67.5, 67.4, 27.7, 26.9,
23.4, 20.7; HRMS-ESI [M+Na]+ calcd for C28H40Si-
NaO6: 523.2492. Found 523.2493.
Di-tert-butylsilyl bis(trifluoromethanesulfonate) (3.40 mL,
10.6 mmol) was added drop-wise to a stirred solution
of allyl a-D-galactopyranoside (1.94 g, 8.80 mmol)
and DMAP (537 mg, 4.40 mmol) in pyridine (15 mL)
cooled to ꢀ10 ꢁC. After being stirred at 0 ꢁC for 1 h
and rt for 3 h, the reaction solution was diluted with
H2O (100 mL) and extracted with ether (2 · 100 mL).
The combined organic extracts were washed with HCl
(1 M, 150 mL) and H2O (150 mL). After drying and fil-
tration, the solvent was removed and the residue puri-
fied by column chromatography on silica gel. Elution
with EtOAc/light petroleum (3:7 to 35:65) afforded the
title compound 5 as an oil (1.85 g, 5.13 mmol, 58%);
½aꢁD +127 (c 0.9, CHCl3); H NMR (300 MHz, CDCl3)
d 5.97–5.83 (m, 1H), 5.35–5.25 (m, 1H), 5.23–5.16 (m,
1H), 4.99 (d, J = 3.7 Hz, 1H), 4.41 (d, J = 3.2 Hz, 1H),
4.28 (dd, J = 12.5, 2.2 Hz, 1H), 4.22–4.12 (m, 2H),
4.09–4.01 (m, 1H), 3.90–3.70 (m, 3H), 2.67 (d,
J = 9.6 Hz, 1H), 2.37 (d, J = 10.1 Hz, 1H), 1.06 (s,
9H), 1.03 (s, 9H); 13C NMR (75 MHz, CDCl3) d
133.7, 117.6, 98.2, 73.3, 71.3, 69.8, 68.6, 67.5, 66.9,
27.6, 27.2, 23.4, 20.7; HRMS-ESI [M+H]+ calcd for
C17H33O6Si: 361.2046. Found 361.2050.
20
1
4.5. O-(2,3-Di-O-benzyl-4,6-O-di-tert-butylsilylene-a-D-
galactopyranosyl) trichloroacetimidate (a:b, 9:1) (8)
4.3. Allyl 2,3-di-O-benzyl-4,6-O-di-tert-butylsilylene-a-D-
galactopyranoside (6)
DBU (24 lL, 0.17 mmol) was added drop-wise to a stir-
red solution of hemi-acetal 7 (825 mg, 1.65 mmol) and
trichloroacetonitrile (0.830 mL, 8.25 mmol) in CH2Cl2
(20 mL) cooled to 0 ꢁC. After 1 h, the solvent was
removed and the residue purified by column chromato-
graphy on silica gel. Elution with EtOAc/light petroleum
(15:85) afforded the title a-compound 8 as an oil
(840 mg, 1.30 mmol, 79%); 1H NMR (300 MHz,
CDCl3), a anomer, d 8.61 (s, 1H), 7.42–7.23 (m, 10H),
6.47 (d, J = 3.5 Hz, 1H), 4.89–4.67 (m, 4H), 4.59 (d,
J = 2.5 Hz, 1H), 4.25–4.10 (m, 3H), 3.91 (dd, J = 9.9,
2.9 Hz, 1H), 3.83 (br s, 1H), 1.06 (s, 9H), 1.01 (s, 9H);
Sodium hydride (60% dispersion in mineral oil, 439 mg,
11.0 mmol) was added to a stirred solution of diol 5
(1.80 g, 4.99 mmol) and benzyl bromide (1.44 mL,
12.5 mmol) in DMF (30 mL) cooled to ꢀ20 ꢁC. After
3 h, the cold bath was removed and the reaction mixture
was stirred at rt for a further 12 h when NH4Cl solution
(100 mL, satd aq soln) and H2O (100 mL) were added.
The mixture was extracted with ether (2 · 150 mL) and
the combined ethereal extracts were washed with H2O
(2 · 100 mL). After drying and filtration, the solvent
was removed and the residue purified by column chroma-

2792
A. Lee et al. / Carbohydrate Research 341 (2006) 2785–2798
13C NMR (75 MHz, CDCl3), a anomer, d 161.4, 138.7,
138.4, 128.3, 128.1, 127.8, 127.6, 95.8, 77.0, 73.8, 73.3,
71.1, 70.9, 70.1, 66.9, 27.7, 27.3, 23.4, 20.8.
4.8. (2S,3S,4R)-2-Azido-3,4-di(benzyloxy)-non-6-en-1-ol
(E:Z, 4:1) (13)
A
solution of TBAF in THF (1.0 M, 3.83 mL,
4.6. (2R,3S,4R)-3,4-Di(benzyloxy)-1-triisopropylsilyloxy-
non-6-en-2-ol (E:Z, 4:1) (11)
3.83 mmol) was added to a stirred solution of silyl ether
12 (1.41 g, 2.56 mmol) in THF (30 mL) cooled to 5 ꢁC.
After the addition the cold bath was removed and the
reaction mixture stirred at ambient temperature for
90 min The solvent was removed in vacuo and the resi-
due purified by column chromatography on silica gel.
Elution with EtOAc/light petroleum (1:9) afforded the
title compound 13 as an oil (860 mg, 2.17 mmol, 85%);
1H NMR (300 MHz, CDCl3) d 7.35–7.21 (m, 10H),
5.55–5.38 (m, 2H), 4.64–4.45 (m, 4H), 3.88–3.62 (m,
5H), 2.49–2.38 (m, 3H), 2.11–1.97 (m, 2H), 0.98–0.93
(m, 3H); 13C NMR (75 MHz, CDCl3), major isomer, d
138.0, 137.7, 134.4, 128.5, 128.4, 128.1, 128.0, 127.9,
124.1, 80.5, 78.8, 73.7, 72.4, 63.3, 62.4, 28.2, 20.9, 14.1;
HRMS-ESI [M+HꢀN2]+ calcd for C23H30NO3:
368.2226. Found 368.2216.
n-BuLi (1.44 M in hexanes, 5.60 mL, 8.10 mmol) was
added drop-wise to a stirred solution of propyl tri-
phenylphosphonium bromide (3.42 g, 8.88 mol) in THF
(30 mL) cooled to ꢀ60 ꢁC. After being stirred for
45 min, a solution of 3,4-di-O-benzyl-2-deoxy-6-O-tri-
isopropylsilyl-D-galactopyranose (10) (1.69 g, 3.38 mmol)
in THF (15 mL) was added drop-wise over 30 min to the
reaction mixture. After being stirred for a further 12 h,
the mixture was quenched with the addition of NH4Cl
solution (100 mL, satd aq soln) and H2O (100 mL).
The mixture was extracted with ether (2 · 150 mL) and
the combined ethereal extracts were washed with brine
(100 mL) and H2O (100 mL). After drying and filtration
the solvent, was removed in vacuo and the residue puri-
fied by column chromatography on silica gel. Elution
with EtOAc/light petroleum (5:95) afforded the title
4.9. (2S,3S,4R)-2-Azido-3,4-di(benzyloxy)-1-(2,3-di-O-
benzyl-4,6-O-di-tert-butylsilylene-a-D-galactopyranosyl-
oxy)-non-6-ene (E:Z, 4:1) (14)
1
compound 11 as an oil (1.56 g, 2.96 mmol, 88%); H
NMR (300 MHz, CDCl3) d 7.32–7.21 (m, 10H), 5.55–
5.37 (m, 2H), 4.70–4.59 (m, 4H), 3.93–3.85 (m, 1H),
3.79–3.67 (m, 4H), 3.05 (d, J = 6.0 Hz, 1H), 2.45–2.38
(m, 2H), 2.05–1.97 (m, 2H), 1.10–0.85 (m, 24H); 13C
NMR (75 MHz, CDCl3), major isomer, d 138.4, 133.9,
128.4, 128.3, 128.0, 127.8, 127.7, 127.6, 124.7, 80.2,
78.3, 73.8, 72.8, 71.4, 64.0, 29.0, 20.8, 18.0, 14.1, 12.0;
HRMS-ESI [M+H]+ calcd for C32H51O4Si: 527.3557.
Found 527.3550.
TMSOTf (46.0 lL, 0.251 mmol) was added drop-wise to
a stirred mixture of donor (8) (809 mg, 1.25 mmol),
acceptor (13) (378 mg, 0.956 mmol) and 4 A molecular
˚
sieves (220 mg) in CH2Cl2 (30 mL) at ꢀ40 ꢁC. The reac-
tion mixture was allowed to warm to 0 ꢁC over 2 h when
Et3N (2.5 mL) was added. The mixture was filtered
through Celite and the filter cake washed with further
CH2Cl2 (2 · 100 mL). The solvent was removed and
the residue purified by column chromatography on silica
gel. Elution with EtOAc/light petroleum (1:19) afforded
the title compound 14 (554 mg, 0.631 mmol, 66%) as an
4.7. (2S,3S,4R)-2-Azido-3,4-di(benzyloxy)-1-triisopropyl-
silyloxy-non-6-ene (E:Z, 4:1) (12)
1
oil; H NMR (300 MHz, CDCl3) d 7.45–7.39 (m, 2H),
Diethyl azodicarboxylate (1.80 mL, 11.4 mmol) and
hydrazoic acid (0.7 M in toluene, 16.3 mL, 11.4 mmol)
were added together to a stirred solution of triphenyl-
phosphine (2.99 g, 11.4 mmol) in THF (25 mL) cooled
to ꢀ78 ꢁC. After 1 min, a solution of alcohol 11
(1.50 g, 2.85 mmol) was cannulated into the reaction
mixture. After stirring for 10 h, the solvent was removed
in vacuo and the residue purified by column chromato-
graphy on silica gel. Elution with EtOAc/light petroleum
(5:95) afforded the title compound 12 as an oil (1.43 g,
7.35–7.20 (m, 18H), 5.55–5.35 (m, 2H), 4.87–4.45 (m,
10H), 4.15–3.60 (m, 9H), 3.55 (br s, 1H), 2.47–2.34 (m,
2H), 2.09–1.95 (m, 2H), 1.05 (s, 9H), 0.99 (s, 9H),
0.92–0.85 (m, 3H); 13C NMR (75 MHz, CDCl3), major
isomer, d 139.1, 138.8, 138.3, 138.2, 134.1, 128.4,
128.3, 128.1, 127.8, 127.8, 127.7, 127.5, 127.5, 124.5,
99.1, 79.3, 79.3, 77.6, 74.3, 73.8, 73.6, 72.1, 71.2, 71.0,
68.2, 67.7, 67.1, 62.0, 28.1, 27.7, 27.4, 23.5, 20.9, 20.7,
14.2; Gated decoupled 13C NMR (75 MHz, CDCl3)
1
0
0
selected data, d 99.1, JC1 –H1 168.6 Hz; HRMS-ESI
[M+HꢀN2]+ calcd for C51H68NO8Si: 850.4714. Found
850.4706.
1
2.58 mmol, 91%); H NMR (300 MHz, CDCl3) d 7.33–
7.21 (m, 10H), 5.45–5.37 (m, 2H), 4.62–4.43 (m, 4H),
3.97–3.90 (m, 1H), 3.75–3.65 (m, 1H), 3.63–3.56 (m,
2H), 3.54–3.48 (m, 1H), 2.40–2.32 (m, 2H), 2.00–1.90
(m, 2H), 1.11–0.86 (m, 24H); 13C NMR (75 MHz,
CDCl3), major isomer, d 138.4, 138.2, 133.9, 128.4,
128.0, 127.7, 127.6, 124.8, 79.6, 79.1, 73.7, 72.2, 64.7,
64.6, 28.0, 20.8, 18.0, 14.1, 12.0; HRMS-ESI [M+H]+
calcd for C32H50N3O3Si: 552.3616. Found 552.3590.
4.10. (2S,3S,4R)-2-Amino-3,4-di(benzyloxy)-1-(2,3-di-O-
benzyl-4,6-O-di-tert-butylsilylene-a-D-galactopyranosyl-
oxy)-nonane (17)
A mixture of Pd on carbon (20%, 70 mg), and alkene 14
(355 mg, 0.404 mmol) in MeOH (30 mL) was stirred

A. Lee et al. / Carbohydrate Research 341 (2006) 2785–2798
2793
under a hydrogen atmosphere for 12 h. The mixture was
filtered through Celite and the cake washed with MeOH
(100 mL). The solvent was removed in vacuo to afford
the title compound 17 (332 mg, 0.389 mmol, 96%) as
an oil; 1H NMR (300 MHz, CDCl3) d 7.44–7.19 (m,
20H), 4.89–4.45 (m, 9H), 4.15–3.90 (m, 4H), 3.82 (dd,
J = 10.1, 2.8 Hz, 1H), 3.75–3.65 (m, 2H), 3.59–3.50
(m, 2H), 3.40–3.30 (m, 1H), 3.20–3.10 (m, 1H), 1.95–
1.75 (m, 2H), 1.38–1.15 (m, 6H), 1.05 (s, 9H), 0.97 (s,
9H), 0.92–0.85 (m, 3H); 13C NMR (75 MHz, CDCl3) d
139.0, 138.7, 138.6, 128.4, 128.1, 127.8, 127.6, 127.5,
127.0, 99.2, 82.1, 80.0, 77.9, 74.7, 73.7, 73.6, 72.1, 71.5,
71.0, 70.8, 67.4, 67.3, 52.9, 32.1, 30.6, 27.7, 27.4, 25.5,
23.5, 22.7, 20.7, 14.1; HRMS-ESI [M+H]+ calcd for
C51H72NO8Si: 854.5027. Found 854.5019.
1H), 3.17–3.08 (m, 1H), 2.41–2.29 (m, 2H), 2.04–1.90
(m, 2H), 1.05 (s, 9H), 0.99 (s, 9H), 0.92–0.85 (m, 3H);
13C NMR (75 MHz, CDCl3), major isomer, d 139.0,
138.6, 133.7, 128.4, 128.1, 127.8, 127.7, 127.6, 127.6,
127.5, 125.1, 99.2, 82.1, 79.9, 77.9, 74.7, 73.7, 72.0,
71.5, 71.1, 70.8, 67.4, 67.3, 52.9, 30.4, 28.4, 27.7, 27.4,
23.5, 20.9, 20.8, 14.2; HRMS-ESI [M+H]+ calcd for
C51H70NO8Si: 852.4871. Found 852.4873.
4.13. (2S,3S,4R)-3,4-Di(benzyloxy)-1-(2,3-di-O-benzyl-
4,6-O-di-tert-butylsilylene-a-D-galactopyranosyloxy)-2-
tetracosanoylamino-non-6-en (E:Z, 4:1) (20)
1-(3-Dimethylaminopropyl)-3-ethyl-carbodiimide hydro-
chloride (38 mg, 0.197 mmol) and 1-hydroxybenzo-
triazole (27 mg, 0.197 mg) were added to a stirred
suspension of tetracosanoic acid (60 mg, 0.164 mmol) in
a CH2Cl2 (10 mL), DMF (4 mL) solvent mixture cooled
to 0 ꢁC. After 2 min, a solution of amine 19 (140 mg,
0.164 mmol) and DIPEA (70 lL, 0.394 mmol) in
CH2Cl2 (10 mL) was added. The reaction mixture was
stirred at ambient temperature for 12 h when it was
diluted with the addition of EtOAc/Et2O (4:1, 200 mL).
The organic phase was washed with a satd aq soln of
NaHCO3 (100 mL), HCl (1 M, 100 mL) and brine
(150 mL). After drying and filtration, the solvent was
removed and the residue purified by column chromato-
graphy on silica gel. Elution with EtOAc/light petroleum
(0:1 to 1:9) afforded the title compound 20 (139 mg,
0.115 mmol, 70%) as an oil; 1H NMR (300 MHz,
CDCl3) d 7.44–7.39 (m, 2H), 7.36–7.21 (m, 18H), 5.85
(d, J = 8.5 Hz, 1H), 5.55–5.37 (m, 2H), 4.86–4.45 (m,
10H), 4.36–4.25 (m, 1H), 4.15–3.93 (m, 3H), 3.82–3.70
(m, 4H), 3.59–3.51 (m, 2H), 2.54–2.35 (m, 2H), 2.10–
1.97 (m, 2H), 1.92–1.83 (m, 2H), 1.53–1.40 (m, 2H),
1.28–1.20 (m, 40H), 1.06 (s, 9H), 0.99 (s, 9H), 0.91–
0.84 (m, 6H); 13C NMR (75 MHz, CDCl3), major iso-
mer, d 172.9, 138.9, 138.5, 133.8, 128.4, 128.2, 127.9,
127.9, 127.8, 127.7, 127.6, 125.0, 99.5, 79.7, 79.4, 77.8,
74.6, 73.9, 73.4, 71.7, 70.9, 70.7, 68.1, 67.7, 67.3, 50.2,
36.8, 32.0, 29.8, 29.5, 29.4, 27.9, 27.7, 27.4, 25.8, 23.9,
22.7, 20.8, 20.8, 14.2; HRMS-ESI [M+H]+ calcd for
C75H115NO9Si: 1202.8419. Found 1202.8389.
4.11. (2S,3S,4R)-2-Amino-1-(4,6-O-di-tert-butylsilylene-
a-D-galactopyranosyloxy)-nonane-3,4-diol (18)
A mixture of Pd on carbon (20%, 73 mg), ammonium
formate (76 mg, 0.15 mmol) and tetrabenzyl ether 17
(30 mg, 0.03 mmol) in MeOH (5 mL) was heated under
reflux for 3 h. The mixture was cooled, filtered through
Celite and the cake washed with MeOH (3 · 30 mL).
The solvent was removed in vacuo and the residue puri-
fied by column chromatography on silica gel. Elution
with MeOH/CHCl3 (0:1 to 1:9) afforded the title com-
1
pound 18 (6 mg, 0.012 mmol, 40%) as an oil; H NMR
(300 MHz, CD3OD) d 4.42–4.33 (m, 1H), 4.20 (dd,
J = 12.5, 1.9 Hz, 1H), 4.01 (dd, J = 12.5, 1.4 Hz, 1H),
3.88–3.76 (m, 2H), 3.73–3.62 (m, 2H), 3.57–3.27 (m,
5H), 3.06–2.95 (m, 1H), 1.75–1.60 (m, 1H), 1.55–1.40
(m, 1H), 1.35–1.15 (m, 6H), 1.03 (s, 9H), 0.93 (s, 9H),
0.88–0.78 (m, 3H); 13C NMR (75 MHz, CD3OD) d
101.5, 76.9, 75.4, 74.2, 71.8, 70.9, 70.1, 69.0, 68.2, 54.1,
35.0, 33.2, 28.2, 27.9, 26.2, 24.2, 23.7, 21.6, 14.4;
HRMS-ESI [M+H]+ calcd for C23H48NO8Si: 494.3149.
Found 494.3155.
4.12. (2S,3S,4R)-2-Amino-3,4-di(benzyloxy)-1-(2,3-di-O-
benzyl-4,6-O-di-tert-butylsilylene-a-D-galactopyranosyl-
oxy)-non-6-ene (E:Z, 4:1) (19)
Trimethylphosphine (1 M in THF, 0.820 mL, 0.880
mmol) was added to a stirred solution of azide 14
(145 mg, 0.165 mmol) in THF (15 mL) cooled to 0 ꢁC
and the mixture was stirred for 45 min. After stirring
at ambient temperature for a further 1.5 h, NaOH (aq,
1 M, 50 mL, 1.50 mmol) was added. After a further
2 h, the mixture was diluted with EtOAc (150 mL) and
washed with H2O (2 · 100 mL), brine (100 mL) dried
and filtered. The solvent was removed to give the title
4.14. (2S,3S,4R)-3,4-Di(benzyloxy)-1-(2,3-di-O-benzyl-a-
D-galactopyranosyloxy)-2-tetracosanoylamino-non-6-ene
(E:Z, 4:1) (21)
A solution of HFÆpy (40 M, 0.350 mL, 14.0 mmol) was
added to a stirred solution of amide 20 (135 mg,
0.112 mmol) in THF (10 mL) cooled to 0 ꢁC. After
1 h, the mixture was diluted with EtOAc (100 mL) and
washed with a satd aq soln of NaHCO3 (50 mL). After
drying and filtration, the solvent was removed and the
residue purified by column chromatography on silica
gel. Elution with EtOAc/light petroleum (1:1 to 3:1)
1
compound 19 (140 mg, 0.164 mmol, 99%) as an oil; H
NMR (300 MHz, CDCl3) d 7.37–7.30 (m, 2H), 7.28–
7.14 (m, 18H), 5.52–5.30 (m, 2H), 4.78–4.40 (m, 11H),
4.07–3.60 (m, 7H), 3.50–3.42 (m, 2H), 3.32–3.23 (m

2794
A. Lee et al. / Carbohydrate Research 341 (2006) 2785–2798
1
afforded the title compound 21 (96 mg, 0.09 mmol, 81%)
as an oil; H NMR (300 MHz, CDCl3) d 7.36–7.22 (m,
pound 22 as an oil (1.99 g, 2.92 mmol, 84%); H NMR
1
(300 MHz, CDCl3) d 7.37–7.26 (m, 10H), 5.57–5.31
(m, 2H), 4.73–4.59 (m, 4H), 4.00–3.90 (m, 1H), 3.80–
3.69 (m, 4H), 3.09–3.01 (m, 1H), 2.53–2.35 (m, 2H),
2.07–1.93 (m, 2H), 1.33 (m, 22H), 1.08–1.02 (m, 21H),
0.91–0.82 (m, 3H); 13C NMR (75 MHz, CDCl3), major
isomer, d 138.5, 133.9, 133.7, 132.4, 128.4, 128.4,
128.3, 128.2, 128.1, 127.9, 127.8, 127.6, 125.2, 80.2,
78.4, 73.8, 72.8, 71.5, 64.1, 32.0, 29.8, 29.7, 29.4, 29.1,
27.7, 22.8, 18.1, 14.2, 12.0; HRMS-ESI [M+H]+ calcd
for C43H73O4Si: 681.5278. Found 681.5265.
20H), 5.78 (d, J = 8.2 Hz, 1H), 5.60–5.38 (m, 2H), 4.84
(d, J = 2.9 Hz, 1H), 4.79–4.39 (m, 9H), 4.02 (br s, 1H),
3.92–3.54 (m, 9H), 2.80–2.35 (m, 4H), 2.11–1.97 (m,
3H), 1.94–1.75 (m, 3H), 1.55–1.37 (m, 2H), 1.28–1.20
(m, 40H), 0.99–0.82 (m, 6H); 13C NMR (75 MHz,
CDCl3), major isomer, d 173.1, 138.5, 138.3, 138.1,
133.9, 128.6, 128.4, 128.0, 127.9, 127.8, 127.7, 124.7,
99.5, 80.3, 79.1, 77.6, 75.9, 73.5, 73.2, 72.6, 71.7, 69.9,
69.4, 68.5, 62.8, 50.5, 36.8, 32.0, 29.7, 29.6 29.5, 29.4,
28.0, 25.7, 22.7, 20.8, 14.2, 14.1; HRMS-ESI [M+H]+
calcd for C67H100NO9: 1062.7398. Found 1062.7408.
4.17. (2S,3S,4R)-2-Azido-3,4-di(benzyloxy)-1-triisoprop-
ylsilyloxy-icos-6-ene (E:Z, 4:1) (23)
4.15. (2S,3S,4R)-1-(a-D-Galactopyranosyloxy)-2-tetra-
cosanoylamino-nonane-3,4-diol (2)
Diethyl azodicarboxylate (1.80 mL, 11.4 mmol) and
hydrazoic acid (0.7 M in toluene, 16.4 mL, 11.5 mmol)
were added together to a stirred solution of triphenyl-
phosphine (3.00 g, 11.5 mmol) in THF (40 mL) cooled
to ꢀ78 ꢁC. After 1 min a solution of alcohol 22
(1.95 g, 2.86 mmol) was cannulated onto the reaction
mixture. After stirring for 10 h MeOH (1 mL) was
added and the solvent was removed in vacuo. The resi-
due was purified by column chromatography on silica
gel. Elution with EtOAc/light petroleum (0:1 to 5:95)
afforded the title compound 23 as an oil (1.56 g,
A mixture of Pd-black (9 mg) and 21 (90 mg, 0.085
mmol) in CHCl3/MeOH (15 mL, 1:2) was stirred under
a hydrogen atmosphere for 12 h. The mixture was
filtered through Celite and the cake washed with
CHCl3/MeOH/H2O (70:20:2, 200 mL). The solvent
was removed in vacuo and the residue purified by
column chromatography on silica gel. Elution with
MeOH/chloroform (1:49 to 1:9) afforded the title com-
pound 2 (30 mg, 0.043 mmol, 50%) as a white hygro-
scopic solid; 1H NMR (300 MHz, CDCl3/CD3OD,
3:1) d 4.83 (d, J = 3.7 Hz, 1H), 4.15–4.08 (m 1H),
3.88–3.76 (m, 2H), 3.75–3.55 (m, 7H), 3.49–3.42 (m,
2H), 2.16–2.07 (m, 2H), 1.65–1.41 (m, 2H), 1.27–1.10
(m, 51H), 0.85–0.76 (m, 6H); 13C NMR (150 MHz,
CD3OD) d 174.3, 99.6, 74.7, 71.8, 70.6, 70.1, 69.6,
68.8, 67.3, 61.7, 50.2, 36.3, 32.4, 31.7, 29.5, 29.2, 25.6,
25.2, 22.4, 13.7; HRMS-ESI [M+H]+ calcd for C39H78-
NO9: 704.5677. Found 704.5694; Anal. (C39H77NO9Æ
0.2CHCl3) C, H, N.
1
2.21 mmol, 77%); H NMR (300 MHz, CDCl3) d 7.35–
7.26 (m, 10H), 5.55–5.39 (m, 2H), 4.73–4.50 (m, 4H),
4.08–4.00 (m, 1H), 3.87–3.78 (m, 1H), 3.75–3.64 (m,
2H), 3.63–3.58 (m, 1H), 2.50–2.37 (m, 2H), 2.08–1.95
(m, 2H), 1.35–1.20 (m, 22H), 1.08–1.02 (m, 21H),
0.91–0.83 (m, 3H); 13C NMR (75 MHz, CDCl3), major
isomer, d 138.5, 138.3, 132.4, 128.4, 128.0, 127.9,
127.8, 127.6, 125.3, 79.7, 79.1, 73.7, 72.2, 64.8, 64.7,
32.0, 29.8, 29.5, 29.4, 28.1, 27.7, 22.8, 18.0, 14.2, 12.0;
HRMS-ESI [M+HꢀN2]+ calcd for C43H72NO3Si:
678.5281. Found 678.5281.
4.16. (2R,3S,4R)-3,4-Di(benzyloxy)-1-triisopropylsilyl-
oxy-icos-6-en-2-ol (E:Z, 4:1) (22)
4.18. (2S,3S,4R)-2-Azido-3,4-di(benzyloxy)-icos-6-en-1-
ol (E:Z, 4:1) (24)
n-BuLi (1.44 M in hexanes, 6.75 mL, 9.74 mmol) was
added drop-wise to a stirred solution of tetradecyl tri-
phenylphosphonium bromide (5.60 g, 10.5 mmol) in
THF (40 mL) cooled to ꢀ60 ꢁC. After stirring for 1 h
a solution of 3,4-di-O-benzyl-2-deoxy-6-O-triisopropyl-
silyl-D-galactopyranose (10) (1.74 g, 3.48 mmol) in
THF (20 mL) was added drop-wise over 15 min to the
reaction mixture. After being stirred for a further 12 h,
the mixture was quenched with the addition of NH4Cl
solution (100 mL, satd aq soln) and H2O (100 mL).
The mixture was extracted with ether (2 · 200 mL) and
the combined ethereal extracts were washed with brine
(100 mL) and H2O (100 mL). After drying and filtration,
the solvent was removed in vacuo and the residue puri-
fied by column chromatography on silica gel. Elution
with EtOAc/light petroleum (1:9) afforded the title com-
A
solution of TBAF in THF (1.0 M, 2.90 mL,
2.90 mmol) was added to a stirred solution of silyl ether
23 (1.40 g, 1.98 mmol) in THF (20 mL) cooled to 0 ꢁC.
After the addition, the cold bath was removed and the
reaction mixture stirred at ambient temperature for
1 h. The solvent was removed in vacuo and the residue
purified by column chromatography on silica gel. Elu-
tion with EtOAc/light petroleum (1:9) afforded the title
1
compound 24 as an oil (1.02 g, 1.86 mmol, 94%); H
NMR (300 MHz, CDCl3) d 7.38–7.27 (m, 10H), 5.59–
5.35 (m, 2H), 4.68–4.52 (m, 4H), 3.90–3.62 (m, 5H),
2.50–2.38 (m, 3H), 2.07–1.95 (m, 2H), 1.33–1.21 (m,
22H), 0.91–0.82 (m, 3H); 13C NMR (75 MHz, CDCl3),
major isomer, d 138.0, 137.7, 132.8, 128.5, 128.4,
128.0, 128.0, 127.8, 124.6, 80.5, 78.8, 73.7, 72.4, 63.3,

A. Lee et al. / Carbohydrate Research 341 (2006) 2785–2798
2795
62.3, 32.0, 29.7, 29.7, 29.6, 29.4, 28.3, 27.6, 22.7, 17.7,
14.1, 12.4; HRMS-ESI [M+HꢀN2]+ calcd for
C34H52NO3: 522.3947. Found 522.3947.
127.5, 125.6, 99.2, 82.1, 79.8, 77.9, 74.7, 73.7, 72.0,
71.5, 71.0, 70.8, 67.4, 67.3, 52.9, 32.0, 30.4, 29.7, 29.5,
29.4, 28.5, 27.7, 27.4, 23.5, 22.7, 20.7, 14.2; HRMS-
ESI [M+H]+ calcd for C62H92NO8Si: 1006.6592. Found
1006.6567.
4.19. (2S,3S,4R)-2-Azido-3,4-di(benzyloxy)-1-(2,3-di-O-
benzyl-4,6-O-di-tert-butylsilylene-a-D-galactopyranosyl-
oxy)-icos-6-ene (E:Z, 4:1) (25)
4.21. (2S,3S,4R)-3,4-Di(benzyloxy)-1-(2,3-di-O-benzyl-
4,6-O-di-tert-butylsilylene-a-D-galactopyranosyloxy)-2-
tetracosanoylamino-icos-6-ene (E:Z, 4:1) (27)
TMSOTf (19 lL, 0.108 mmol) was added drop-wise to a
stirred mixture of donor 8 (350 mg, 0.542 mmol), accep-
˚
tor 24 (265 mg, 0.482 mmol) and 4 A molecular sieves
1-(3-Dimethylaminopropyl)-3-ethyl-carbodiimide hydro-
chloride (52 mg, 0.269 mmol) and 1-hydroxybenzo-
triazole (36 mg, 0.269 mmol) were added to a stirred
suspension of tetracosanoic acid (83 mg, 0.224 mmol)
in a CH2Cl2 (10 mL), DMF (4 mL) solvent mixture
cooled to 0 ꢁC. After 30 min a solution of amine 26
(226 mg, 0.224 mmol) and DIPEA (90 lL, 0.539 mmol)
in CH2Cl2 (10 mL) was added. The reaction mixture
was stirred at ambient temperature for 12 h when it
was diluted with the addition of EtOAc/Et2O (4:1,
200 mL). The organic phase was washed with a satd
aq soln of NaHCO3 (100 mL), HCl (1 M, 100 mL) and
brine (150 mL). After drying and filtration, the solvent
was removed and the residue purified by column chro-
matography on silica gel. Elution with EtOAc/light
petroleum (1:19 to 3:17) afforded the title compound
27 (188 mg, 0.138 mmol, 62%) as an oil; 1H NMR
(300 MHz, CDCl3) d 7.44–7.39 (m, 2H), 7.36–7.21 (m,
18H), 5.85 (d, J = 8.5 Hz, 1H), 5.55–5.39 (m, 2H),
4.86–4.45 (m, 10H), 4.36–4.25 (m, 1H), 4.15–3.94 (m,
3H), 3.82–3.70 (m, 4H), 3.60–3.50 (m, 2H), 2.54–2.35
(m, 2H), 2.08–1.95 (m, 2H), 1.91–1.83 (m, 2H), 1.52–
1.40 (m, 2H), 1.27–1.21 (m, 62H), 1.04 (s, 9H), 1.00 (s,
9H), 0.91–0.84 (m, 6H); 13C NMR (75 MHz, CDCl3),
major isomer, d 172.8, 138.9, 138.5, 132.2, 128.4,
128.2, 127.9, 127.8, 127.7, 127.7, 127.6, 125.4, 99.5,
79.7, 79.3, 77.8, 74.6, 73.8, 73.4, 71.7, 70.9, 70.7, 68.0,
67.6, 67.2, 50.2, 36.8, 32.0, 29.7, 29.5, 29.4, 28.0, 27.7,
27.6, 27.4, 25.8, 23.4, 22.7, 20.7, 14.2; HRMS-ESI
[M+H]+ calcd for C86H138NO9Si: 1357.0141. Found
1357.0155.
(120 mg) in CH2Cl2 (20 mL) at ꢀ40 ꢁC. The reaction
mixture was allowed to warm to 0 ꢁC over 2 h when
Et3N (2.5 mL) was added. The mixture was filtered
through Celite and the filter cake was further washed
with CH2Cl2 (2 · 100 mL). The solvent was removed
and the residue purified by column chromatography
on silica gel. Elution with EtOAc/light petroleum (0:1
to 1:19) afforded the title compound 25 (312 mg,
0.302 mmol, 63%) as an oil; 1H NMR (300 MHz,
CDCl3) d 7.44–7.38 (m, 2H), 7.35–7.20 (m, 18H),
5.53–5.37 (m, 2H), 4.84 (d, J = 11.8 Hz, 1H), 4.79 (d,
J = 3.6 Hz, 1H), 4.75–4.45 (m, 8H), 4.11–3.62 (m, 9H),
3.55 (s, 1H), 2.48–2.35 (m, 2H), 2.06–1.94 (m, 2H),
1.37–1.22 (m, 22H), 1.05 (s, 9H), 0.99 (s, 9H), 0.92–
0.85 (m, 3H); 13C NMR (75 MHz, CDCl3), major iso-
mer, d 139.1, 138.8, 138.4, 138.2, 132.5, 128.4, 128.3,
128.1, 127.8, 127.8, 127.7, 127.5, 127.5, 125.0, 99.1,
79.3, 79.2, 77.6, 74.3, 73.9, 73.6, 72.1, 71.2, 71.0, 68.2,
67.7, 67.1, 62.0, 32.0, 29.8, 29.7, 29.5, 29.4, 28.1, 27.7,
27.4, 23.5, 22.7, 20.7, 14.2; Gated decoupled 13C NMR
1
0
0
(75 MHz, CDCl3) selected data, d 99.1, JC1 –H1 168.3
Hz. HRMS-ESI [M+HꢀN2]+ calcd for C62H90NO8Si:
1004.6436. Found 1004.6425.
4.20. (2S,3S,4R)-2-Amino-3,4-di(benzyloxy)-1-(2,3-di-O-
benzyl-4,6-O-di-tert-butylsilylene-a-D-galactopyranosyl-
oxy)-icos-6-ene (E:Z, 4:1) (26)
Trimethylphosphine (1 M in THF, 1.20 mL, 1.20 mmol)
was added to a stirred solution of azide 25 (246 mg,
0.238 mmol) in THF (20 mL) cooled to 0 ꢁC and stirred
for 45 min. After stirring at ambient temperature for a
further 1 h, NaOH (aq, 1 M, 2.00 mL, 2.00 mmol) was
added. After a further 2 h, the mixture was diluted with
EtOAc (150 mL) and washed with H2O (2 · 100 mL),
brine (100 mL) dried and filtered. The solvent was
removed to give the title compound 26 (226 mg,
0.225 mmol, 94%); 1H NMR (300 MHz, CDCl3) d
7.45–7.37 (m, 2H), 7.35–7.19 (m, 18H), 5.55–5.40 (m,
2H), 4.87–4.43 (m, 10H), 4.13–3.65 (m, 8H), 3.58–3.49
(m, 2H), 3.40–3.31 (m, 1H), 3.25–3.16 (m, 1H), 2.49–
2.37 (m, 2H), 2.10–1.92 (m, 2H), 1.29–1.21 (m, 22H),
1.06 (s, 9H), 1.02 (s, 9H), 0.91–0.85 (m, 3H); 13C
NMR (75 MHz, CDCl3), major isomer, d 139.0, 138.7,
138.6, 132.2, 128.3, 128.1, 128.0, 127.8, 127.7, 127.6,
4.22. (2S,3S,4R)-1-(2,3-Di-O-benzyl-a-D-galactopyranos-
yloxy)-3,4-di(benzyloxy)-2-tetracosanoylamino-icos-6-
ene (E:Z, 4:1) (28)
A solution of HFÆpy (40 M, 0.600 mL, 24.0 mmol) was
added to a stirred solution of 27 (164 mg, 0.121 mmol)
in THF (10 mL) cooled to 0 ꢁC. After 1 h the mixture
was diluted with EtOAc (100 mL) and washed with a satd
aq soln of NaHCO3 (50 mL). After drying and filtration,
the solvent was removed and the residue purified by
column chromatography on silica gel. Elution with
EtOAc/light petroleum (1:1 to 4:1) afforded the title
1
compound 28 (93 mg, 0.076 mmol, 63%) as an oil; H
NMR (300 MHz, CDCl3) d 7.36–7.24 (m, 20H), 5.75

2796
A. Lee et al. / Carbohydrate Research 341 (2006) 2785–2798
(d, J = 8.9 Hz, 1H), 5.55–5.41 (m, 2H), 4.84 (d,
J = 3.2 Hz, 1H), 4.79–4.40 (m, 9H), 4.02 (br s, 1H),
3.89–3.55 (m, 9H), 2.75–2.36 (m, 4H), 2.09–1.95 (m,
2H), 1.92–1.83 (m, 2H), 1.53–1.40 (m, 2H), 1.27–1.22
(m, 62H), 0.91–0.84 (m, 6H); 13C NMR (75 MHz,
CDCl3), major isomer, d 173.1, 138.5, 138.4, 138.1,
132.4, 128.6, 128.5, 128.0, 127.9, 127.8, 127.7, 125.1,
99.5, 80.4, 79.0, 77.6, 76.0, 73.5, 73.2, 72.7, 71.7, 69.9,
69.5, 68.6, 62.9, 50.5, 36.8, 32.0, 29.8, 29.5, 29.4, 28.1,
27.6, 25.8, 22.7, 14.2; HRMS-ESI [M+H]+ calcd for
C78H122NO9: 1216.9120. Found 1216.9157.
1.39–1.18 (m, 18H), 1.05 (s, 9H), 1.00 (s, 9H), 0.92–
0.82 (m, 3H); 13C NMR (75 MHz, CDCl3), major iso-
mer, d 139.1, 138.8, 138.4, 138.2, 132.5, 128.40, 128.35,
128.3, 128.1, 127.81, 127.76, 127.7, 127.6, 127.54,
127.47, 125.0, 99.1, 79.33, 79.26, 77.6, 74.3, 73.9, 73.6,
72.1, 71.2, 71.0, 68.2, 67.7, 67.1, 62.0, 32.0, 29.74,
29.70, 29.65, 29.54, 29.47, 29.40, 28.1, 27.71, 27.65,
27.4, 23.4, 22.7, 20.7, 14.2; Gated decoupled 13C NMR
1
0
0
(75 MHz, CDCl3) selected data,
d
99.1, JC1 –H1
168.2 Hz. HRMS-ESI [M+Na]+ calcd for C60H85N3O8-
NaSi: 1026.6004. Found 1026.6007.
4.23. (2S,3S,4R)-1-(a-D-Galactopyranosyloxy)-2-tetra-
cosanoylamino-icosane-3,4-diol (3)
4.25. (2S,3S,4R)-2-Amino-3,4-di(benzyloxy)-1-(2,3-di-O-
benzyl-4,6-O-di-tert-butylsilylene-a-D-galactopyranosyl-
oxy)-octadeca-6-ene (E:Z, 4:1) (31)
A
mixture of Pd-black (9 mg) and 28 (90 mg,
0.074 mmol) in THF/MeOH/H2O (7, 2 and 0.2 mL)
was stirred under a hydrogen atmosphere for 5 h. The
mixture was filtered through Celite and the cake washed
with THF/MeOH/H2O (70:20:2, 200 mL). The solvent
was removed in vacuo and the residue purified by
column chromatography on silica gel. Elution with
MeOH/chloroform (1:9) afforded the title compound 3
(50 mg, 0.058 mmol, 79%) as a white hygroscopic solid;
1H NMR (600 MHz, CDCl3/CD3OD, 3:1) d 4.91 (d,
J = 3.8 Hz, 1H), 4.19 (dd, J = 9.5 and 4.6 Hz, 1H),
4.07 (s, 1H), 3.93 (d, J = 2.8 Hz, 1H), 3.88 (dd,
J = 10.7 and 4.7 Hz, 1H), 3.82–3.67 (m, 6H), 3.57–3.52
(m, 2H), 2.21 (t, J = 7.5 Hz, 2H), 1.70–1.52 (m, 4H),
1.35–1.24 (m, 68H), 0.91–0.87 (m, 6H); 13C NMR
(150 MHz, CDCl3/CD3OD, 3:1), referenced to CD3OD,
d 172.9, 99.5, 74.4, 71.7, 70.5, 70.0, 69.5, 68.7, 67.1, 61.6,
50.2, 36.2, 32.2, 31.6, 29.4, 29.0, 25.5, 22.3, 13.6; HRMS-
ESI [M+H]+ calcd for C50H100NO9: 858.7398. Found
858.7417.
Trimethylphosphine (1 M in THF, 4.0 mL, 4.0 mmol)
was added to a stirred solution of azide 30 (779 mg,
0.776 mmol) in THF (40 mL) cooled to 0 ꢁC. After
45 min the cooling bath was removed and the reaction
mixture was stirred at ambient temperature for a further
2 h. Sodium hydroxide (aq, 1 M, 7.5 mL, 7.50 mmol)
was added and after a further 2 h the mixture was
diluted with EtOAc (200 mL) and washed with H2O
(3 · 100 mL), brine (50 mL), dried (MgSO4) and filtered.
The solvent was removed to give the title compound 31
(836 mg), which was used for the next step without
further purification; 1H NMR (300 MHz, CDCl3) d 7.46–
7.38 (m, 2H), 7.38–7.13 (m, 18H), 5.58–5.32 (m, 2H),
4.87–4.41 (m, 10H), 4.16–3.67 (m, 6H), 3.59–3.48 (m,
2H), 3.41–3.30 (m, 1H), 3.25–3.15 (m, 1H), 2.53–2.34
(m, 2H), 2.09–1.91 (m, 2H), 1.39–1.16 (m, 18H), 1.05
(s, 9H), 1.00 (s, 9H), 0.92–0.82 (m, 3H); 13C NMR
(75 MHz, CDCl3), major isomer, d 139.0, 138.7, 138.6,
132.2, 128.4, 128.1, 128.0, 127.8, 127.7, 127.6, 127.54,
127.50, 125.6, 99.2, 82.1, 79.9, 77.9, 74.7, 73.7, 71.9,
71.5, 71.0, 70.8, 67.4, 67.3, 52.9, 32.0, 30.4, 29.7, 29.6,
29.5, 28.5, 27.7, 27.4, 23.5, 22.7, 20.7, 14.2; HRMS-
ESI [M+H]+ calcd for C60H88NO8Si: 978.6279. Found
978.6264.
4.24. (2S,3S,4R)-2-Azido-3,4-di(benzyloxy)-1-(2,3-di-O-
benzyl-4,6-O-di-tert-butylsilylene-a-D-galactopyranosyl-
oxy)-octadeca-6-ene (E:Z, 4:1) (30)
TMSOTf (19 lL, 0.105 mmol) was added drop-wise to a
stirred mixture of donor 8 (884 mg, 1.37 mmol), accep-
tor 29 (550 mg, 1.054 mmol) and 4 A molecular sieves
4.26. (2S,3S,4R)-3,4-Di(benzyloxy)-1-(2,3-di-O-benzyl-
4,6-O-di-tert-butylsilylene-a-D-galactopyranosyloxy)-2-
hexacosanoylamino-octadeca-6-ene (E:Z, 4:1) (32)
˚
(1.5 g) in CH2Cl2 (30 mL) at ꢀ20 ꢁC. The reaction mix-
ture was allowed to warm to 10 ꢁC over 2.25 h when
Et3N (1 mL) was added. The mixture was filtered
through Celite and the filter cake was further washed
with CH2Cl2 (2 · 100 mL). The solvent was removed
and the residue purified by column chromatography
on silica gel. Elution with EtOAc/light petroleum (1:39
to 1:19) afforded the title compound 30 (863 mg,
0.859 mmol, 82%) as an oil; 1H NMR (300 MHz,
CDCl3) d 7.45–7.38 (m, 2H), 7.38–7.12 (m, 18H),
5.56–5.33 (m, 2H), 4.84 (d, J = 11.9 Hz, 1H), 4.79 (d,
J = 3.5 Hz, 1H), 4.75–4.42 (m, 8H), 4.12–3.58 (m, 9H),
3.55 (br s, 1H), 2.49–2.32 (m, 2H), 2.10–1.92 (m, 2H),
1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydro-
chloride (80 mg, 0.418 mmol) and 1-hydroxybenzotri-
azole (57 mg, 0.418 mmol) were added to a stirred
suspension of hexacosanoic acid (138 mg, 0.349 mmol)
in a CH2Cl2 (20 mL), DMF (8 mL) solvent mixture
cooled to 0 ꢁC. After 30 min a solution of amine 31
(341 mg, 0.349 mmol) and DIPEA (146 lL, 0.836 mmol)
in CH2Cl2 (20 mL) was added. The reaction mixture was
stirred at ambient temperature for 18 h when it was di-
luted with the addition of CH2Cl2 (150 mL) and washed
with water (3 · 100 mL). The organic phase was dried

A. Lee et al. / Carbohydrate Research 341 (2006) 2785–2798
2797
(MgSO4), concentrated and taken up again in EtOAc
(200 mL) and washed further with water (3 · 100 mL).
After drying and filtration, the solvent was removed
and the residue purified by column chromatography
on silica gel. Elution with EtOAc/light petroleum (1:19
to 3:17) afforded the title compound 32 (406 mg,
0.299 mmol, 86%) as an oil; 1H NMR (300 MHz,
CDCl3) d 7.44–7.39 (m, 2H), 7.45–7.16 (m, 18H), 5.80
(d, J = 8.5 Hz, 1H), 5.56–5.32 (m, 2H), 4.83 (d,
J = 11.7 Hz, 1H), 4.78–4.42 (m, 9H), 4.36–4.25 (m,
1H), 4.16–3.93 (m, 3H), 3.84–3.68 (m, 4H), 3.61–3.46
(m, 2H), 2.56–2.30 (m, 2H), 2.11–1.91 (m, 2H), 1.91–
1.80 (m, 2H), 1.56–1.40 (m, 2H), 1.39–1.12 (m, 62H),
1.05 (s, 9H), 0.99 (s, 9H), 0.93–0.81 (m, 6H); 13C
NMR (75 MHz, CDCl3), major isomer, d 172.8, 139.0,
138.6, 138.5, 132.2, 128.5, 128.4, 128.2, 127.9, 127.75,
127.72, 127.63, 127.57, 127.5, 125.4, 99.5, 79.8, 79.4,
77.8, 74.6, 73.8, 73.5, 71.7, 71.0, 70.8, 68.1, 67.7, 67.2,
50.3, 36.8, 32.0, 29.8, 29.7, 29.53, 29.50, 29.46, 28.0,
27.7, 27.6, 27.4, 25.8, 23.5, 22.8, 20.8, 14.2; HRMS-
ESI [M+Na]+ calcd for C86H137NO9NaSi: 1378.9960.
Found 1379.0016.
which time the atmosphere was swapped for Ar. The
reaction mixture was diluted with THF/MeOH/H2O
(35:10:1, 30 mL) and stirred for 30 min before filtering
through Celiteꢂ. The filter cake was rinsed with further
THF/MeOH/H2O (35:10:1, approx 100 mL). The sol-
vent was removed in vacuo and the residue purified by
flash chromatography on reversed phase (C18) silica
(eluant neat MeOH to 1:1 MeOH/CHCl3) to provide
the title compound 1 (67 mg, 85%) as a white powder.
[a]D +42.4 (c 0.4, pyridine); lit.23 +42.2 (c 0.54, pyri-
dine), lit.15 +43.6 (c 1.0, pyridine); HRMS-ESI
[M+Na]+ calcd for C50H99NO9Na: 880.7218. Found
880.7186. The 1H and 13C NMR spectra of this material
were in good agreement to that reported.15,23
4.29. Materials and methods for DC maturation assays
4.29.1. Mice. C57BL/6 were from breeding pairs orig-
inally obtained from Jackson Laboratories, Bar Harbor,
Maine. Also used were CD1dꢀ/ꢀ mice,39 which are
devoid of CD1d-restricted iNKT cells.
4.29.2. Administration of glycolipids. Each glycolipid
(5 mg) was solubilized in 500 lL of chloroform/
MeOH/water at a ratio of 10:10:3, and then diluted to
200 lg/mL in 0.5% Tween/phosphate-buffered saline
(PBS). For injections, each glycolipid was diluted in
PBS and injected into the lateral tail vein.
4.27. (2S,3S,4R)-1-(2,3-Di-O-benzyl-a-D-galactopyranos-
yloxy)-3,4-di(benzyloxy)-2-hexacosanoylamino-octadeca-
6-ene (E:Z, 4:1) (33)
A solution of HFÆpy (approx 38 M, 0.85 mL, 32 mmol)
was added to a stirred solution of 32 (350 mg,
0.258 mmol) in THF (10 mL) cooled in an ice/water
bath. After 1 h the cold mixture was diluted with EtOAc
(100 mL) and washed with a satd aq soln of NaHCO3
(50 mL). After drying and filtration, the solvent was
removed and the residue purified by column chromato-
graphy on silica gel. Elution with EtOAc/light petroleum
(1:1) afforded the title compound 33 (248 mg,
0.204 mmol, 79%) as an oil; 1H NMR (300 MHz,
CDCl3) d 7.38–7.18 (m, 20H), 5.74 (d, J = 8.9 Hz,
1H), 5.59–5.35 (m, 2H), 4.84 (d, J = 3.1 Hz, 1H), 4.80–
4.37 (m, 9H), 4.02 (br s, 1H), 3.94–3.50 (m, 9H), 2.75–
2.33 (m, 4H), 2.10–1.95 (m, 2H), 1.93–1.82 (m, 2H),
1.55–1.40 (m, 2H), 1.39–0.97 (m, 62H), 0.96–0.75 (m,
6H); 13C NMR (75 MHz, CDCl3), major isomer, d
173.1, 138.5, 138.4, 138.1, 132.5, 128.6, 128.5, 128.0,
127.9, 127.8, 127.7, 125.1, 99.6, 80.4, 79.0, 77.6, 76.0,
73.5, 73.2, 72.7, 71.7, 69.9, 69.5, 68.6, 62.9, 50.5, 36.9,
32.0, 29.8, 29.5, 29.4, 28.1, 27.6, 25.8, 22.7, 14.2;
HRMS-ESI [M+Na]+ calcd for C78H121NO9Na:
1238.8939. Found 1238.8947.
4.29.3. Phenotyping DC from spleen. Antibody staining
and flow cytometry were used to examine the expression
of maturation markers on dendritic cells in the spleen
following injection of glycolipids. Splenocyte prepara-
tions were prepared by gentle teasing of splenic tissue
through gauze in Iscove’s Modified Dulbecco’s Medium
with 2 mM glutamine, 1% penicillin–streptomycin,
5 · 10ꢀ5 M 2-mercapto-ethanol and 5% foetal bovine
serum (all Invitrogen, Auckland, New Zealand), fol-
lowed by lysis of red blood cells with RBC lysis buffer
(Puregene, Gentra Systems, Minneapolis, MN, USA).
Antibody staining was performed in PBS, 2% foetal
bovine serum and 0.01% sodium azide. The anti-FccRII
monoclonal antibody 2.4G2 was used at 10 mg/mL to
inhibit non-specific staining. Monoclonal antibodies
(all BD Biosciences Pharmingen, San Jose, CA, USA)
were used to examine expression of the maturation
markers CD40, CD80 and CD86 on CD11c+ dendritic
cells.
4.29.4. Analysis of cytokine release into serum. Blood
was collected from the lateral vein at different time inter-
vals after glycolipid administration. Serum was collected
after blood had clotted, and the levels of cytokines
IL-12p70, IL-4 and IFN-c were assessed by cytokine
bead array technology (BD Biosciences Pharmingen),
according to the manufacturer’s instructions.
4.28. (2S,3S,4R)-1-O-(a-D-galactopyranosyl)-2-hexa-
cosanoylamino-3,4-octadecandiol (1)
To a solution of 33 in MeOH/CH2Cl2 (4:1) was added
Pd(OH)2 on carbon (20%, 120 mg). The suspension
was stirred under a hydrogen atmosphere for 3.5 h after

2798
A. Lee et al. / Carbohydrate Research 341 (2006) 2785–2798
13. Natori, T.; Koezuka, Y.; Higa, T. Tetrahedron Lett. 1993,
Acknowledgements
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The authors wish to thank the New Zealand Foundation
for Research, Science and Technology for financial
support (grant C08X0209), the New Zealand Health
Research Council for the award of the Sir Charles
Hercus Fellowship (to I.F.H.) and the Degussa-Stiftung
and the Hanseatische Stiftung for financial support (for
S.J.). Professor Robin Ferrier assisted in the preparation
of the manuscript.
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Supplementary data
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