An improved synthesis of dansylated α-galactosylceramide and its use as a fluorescent probe for the monitoring of glycolipid uptake by cells

Article abstract of DOI:10.1016/j.carres.2011.02.014

A highly efficient synthesis of the biologically important fluorescent probe dansyl α-GalCer is presented. Key in our strategy is the incorporation of the fluorescent dansyl group at an early stage in the synthesis to facilitate in the monitoring and purification of intermediates via TLC and flash column chromatography, respectively, and the use of a high yielding α-selective glycosylation reaction between the phytosphingosine lipid and a galactosyl iodide donor. The ability of dansyl α-GalCer to activate iNKT cells and to serve as a fluorescent marker for the uptake of glycolipid by dendritic cells is also presented.

Full text of DOI:10.1016/j.carres.2011.02.014

Carbohydrate Research 346 (2011) 914–926
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
An improved synthesis of dansylated
a-galactosylceramide and its use as a
fluorescent probe for the monitoring of glycolipid uptake by cells
Janice M. H. Cheng a,b, Stephanie H. Chee a, Deborah A. Knight a, Hans Acha-Orbea c, Ian F. Hermans a,d
,
a,
Mattie S. M. Timmer b, , Bridget L. Stocker
⇑
⇑
a Malaghan Institute of Medical Research, PO Box 7060, Wellington, New Zealand
b School of Chemical and Physical Sciences, Victoria University of Wellington, PO Box 600, Wellington, New Zealand
c Department of Biochemistry CIIL, University of Lausanne, Chemin des Boveresses 155, 1066 Epalinges, Switzerland
d School of Biological Sciences, Victoria University of Wellington, PO Box 600, Wellington, New Zealand
a r t i c l e i n f o
a b s t r a c t
Article history:
A highly efficient synthesis of the biologically important fluorescent probe dansyl
Key in our strategy is the incorporation of the fluorescent dansyl group at an early stage in the synthesis
to facilitate in the monitoring and purification of intermediates via TLC and flash column chromatogra-
a
-GalCer is presented.
Received 16 December 2010
Received in revised form 14 February 2011
Accepted 15 February 2011
Available online 19 February 2011
phy, respectively, and the use of a high yielding
sphingosine lipid and a galactosyl iodide donor. The ability of dansyl
a
-selective glycosylation reaction between the phyto-
-GalCer to activate iNKT cells
a
and to serve as a fluorescent marker for the uptake of glycolipid by dendritic cells is also presented.
Keywords:
Ó 2011 Elsevier Ltd. All rights reserved.
a-GalCer
Cancer
Fluorescent probe
Synthesis
Glycolipid
1. Introduction
known as invariant natural killer T (iNKT) cells.4–6 This discovery
was remarkable and provided the first evidence that glycolipids,
In 1993, the pharmaceutical division of Kirin Breweries isolated
a series of novel -galactosyl ceramides from the marine sponge
Agelas mauritianus.1,2 Of the series, agelasphin-9b (AGL-9b, 1,
Fig. 1), consisting of a galactosyl moiety -linked to a ceramide
like their protein counterparts, can be presented by APCs and
recognised by T cells to invoke an immune response.
a
Given the therapeutic potential of
a-GalCer, much effort has
a
been spent in developing robust routes for its synthesis.3,7–10 In
containing an N-acylated phytosphingosine backbone, exhibited
potent anti-tumour activity against in vivo models of several mur-
ine tumour cells and later served as the parent compound for sub-
sequent analogue syntheses and structure–activity studies.3
During the course of this structure–activity work, KRN7000 (2)
was found to have similar anti-tumour activity to 1 and, due to
its easier synthesis, was deemed the more suitable candidate for
addition, a number of a-GalCer-derivatives containing a fluoro-
phore11–13 or a biotinylated probe11,14,15 have been prepared with
the objective of using these substrates to better understand the
mechanism of iNKT cell activation by
ability of -GalCer to bind to CD1d and activate iNKT cells has been
a
-GalCer.13,16,17 Though the
a
robustly studied, only little information is known about lipid traf-
ficking and how this influences presentation by CD1d and activa-
tion of iNKT cells.18 Accordingly, many groups have focussed on
clinical use. Since then, KRN7000, now widely known as
a-galacto-
syl ceramide ( -GalCer), has been reported to have potential in the
a
derivatising the lipid portion of a-GalCer with a reporter probe
treatment of several diseases including cancer, malaria, type I dia-
betes, and multiple sclerosis, and equally importantly, has been
shown to exert its therapeutic activity via its ability to bind to
CD1d [a member of the CD1 family of proteins found on the surface
of antigen presenting cells (APCs)] and activate a subset of T cells
in order to address such issues.12–14 However, as CD1d binds lipid
chains within deep hydrophobic pockets,19 it has been proposed
that the addition of a label on the lipid may interfere with
a-Gal-
Cer-CD1d association,17 and moreover, may influence intracellular
trafficking.18,20–22 Conversely, modelling of the CD1d-
a-GalCer
complex suggests that the hydroxyl groups at C400 and C600 on gal-
actose are not involved in complex formation.23 This theory is also
supported by the preparation of a a-GalCer analogue containing an
additional a
-linked galactose at the C600 position which was shown
⇑
Corresponding authors. Tel.: +64 4 499 6914x813; fax: +64 4 499 6915 (B.L.S.).
E-mail addresses: mattie.timmer@vuw.ac.nz (M.S.M. Timmer), bstocker@mala-
to stimulate iNKT cells without the need for processing.24 Taken as
a whole, it is generally accepted that the CD1d-glycolipid-NKT cell
ghan.org.nz (B.L. Stocker).
0008-6215/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2011.02.014

J. M. H. Cheng et al. / Carbohydrate Research 346 (2011) 914–926
2. Results and discussion
915
OH
NH OH
OH
OH
HO
HO
O
O
8
2.1. Synthesis of dansylated a-GalCer
HO
O
Our retrosynthetic analysis for the preparation of dansylated a-
GalCer 3 is presented in Scheme 1. We chose to prepare 3 from the
mono-lipidated derivative 4, itself formed via the coupling of
dansylated galactosyl iodide donor 5 to the phytosphingosine
backbone 6. Donor 5 and acceptor 6 are in turn both readily pre-
1, Agelasphin-9b (AGL-9b)
pared from D-galactose (7). The reasoning behind these key discon-
OH
HO
HO
nects are twofold. First, though it is possible to couple the complete
ceramide lipid backbone to a suitably protected galactose donor,
the ceramide is a particularly poor acceptor and yields for these
glycosylations are typically modest (ca. 25–55%). This is a general
phenomenon largely independent of the type of galactose donor
used.8,15,27–31 There are also advantages to be gained by incorpo-
rating the fluorescent dansyl group at an early stage in the synthe-
sis for it has been well documented that molecules containing a
chromophore are more easily monitored by TLC and purified by
flash column chromatography.32 Accordingly, in contrast to other
O
O
8
NH OH
HO
O
OH
2
, KRN7000 or α-Galactosyl ceramide (α-GalCer)
O
S
NH
syntheses of fluorescent a
-GalCer probes,11–13 we envisioned a gly-
cosylation reaction involving the use of a fluorescent galactose do-
nor, such as 5.
With our synthetic strategy in place, our first goal was the prep-
aration of the phytosphingosine backbone 6 (Scheme 2). Here, our
lipid synthesis commenced with the selective tritylation of D-gal-
NMe2
O
HO
O
O
8
HO
NH OH
OH
HO
O
actose (7) at the primary position,33 followed by installation of
an isopropylidene at the 3- and 4-positions to give the diol 8 in
85% (over two steps).34 Diol 8 was then treated with a solution
of NaIO4 in THF/H2O, which resulted in cleavage of the diol and for-
mation of the formate ester 9 in quantitative yield. As a temporary
protecting group, the formate at the 4-position was a pivotal step
3
, Dansyl α-GalCer
Figure 1.
a-Galactosyl ceramide and derivatives.
in our strategy as it prevented cyclisation to the corresponding D-
receptor interaction tolerates the appendage of small molecules at
C600 and that this is an ideal position to attach a fluorescent
reporter group. Indeed, this was the conclusion made by Zhou
et al. who developed a strategy for the preparation of dansylated
lyxofuranose derivative, which in our hands, gave only modest
yields (50–60%) when subject to the subsequent Wittig reaction.
Treatment of ester 9 with excess ylide (BuLi, 2.4 equiv, phospho-
nium bromide, 2.5 equiv), however, gave the corresponding alkene
10 in good (78%) yield and in an E:Z ratio of approximately 1:10
based on 1H NMR analysis. Subsequent conversion of alkene 10
to the required phytosphingosine backbone then followed via
quantitative hydrogenation, using 1.5 wt % Pd/C, and treatment
with triflic anhydride to give the intermediate triflate, which was
converted to azide 11 in situ. During the installation of the azide
functionality, it was found that the best yields were obtained when
the reaction was preformed at ꢀ15 °C as higher temperatures re-
sulted in the elimination of the triflate and the formation of the
corresponding olefinic by-product. Selective removal of the trityl
ether in the presence of the isopropylidene group was then at-
tempted. This transformation has proven problematic in the past,7
a
-GalCer derivative 3.11
Despite there being much interest in
a-GalCer, the need for a
more efficient synthesis of an appropriately labelled fluorescent
a-GalCer derivative remains. Indeed, our own desire to understand
more about the mechanism by which
a-GalCer is transferred to
resident dendritic cells (DCs) during cancer immunotherapy,25,26
drove us to devise an improved strategy for the synthesis of the
dansylated
a-GalCer derivative 3 (Fig. 1). Our objective was to
achieve a robust synthesis of 3 with high reaction yields through-
out, and to assess the ability of 3 to both activate iNKT cells and to
serve as a fluorescent reporter group. The results of these studies
are reported herein.
O
O
S
NAc
O
NMe2
BnO
BnO
O
S
NH
O
O
S
NH
NMe2
O
O
NMe2
O
BnO
BnO
BnO
HO OH
I
HO
O
C25H51
5
O
C12H25
O
N3
HO
OH
+
BnO
HO
NH OH
OH
O
HO
N3
O
O
7
O
C12H25
O
HO
C12H25
OH
3
4
6
Scheme 1. Retrosynthetic analysis for the preparation of dansylated a-GalCer 3.

916
J. M. H. Cheng et al. / Carbohydrate Research 346 (2011) 914–926
O
HO
HO
OTr
OH
O
O
i, ii
OTr
O
iii
O
O
O
OH
OH
O
OH
8
OH
O
7
9
iv
N3
N3
O
OH
O
vii
v, vi
O
TrO
HO
TrO
C12H25
C12H25
C12H25
O
O
O
10
6
11
Scheme 2. Reagents and conditions: (i) TrCl, pyridine, rt, 48 h; (ii) acetone, CuSO4, rt, 3 d, 85% (two steps); (iii) NaIO4, THF, H2O, 0 °C to rt, overnight, quant.; (iv) BuLi
(2.4 equiv), BrPh3PC13H27 (2.5 equiv), THF, ꢀ78 °C, 15 min, then 9, ꢀ78 °C to rt, 4.5 h, 78%; (v) H2, Pd/C, THF, rt, overnight, quant.; (vi) Tf2O, CH2Cl2, pyridine, ꢀ15 °C, 45 min,
then NaN3, DMF, ꢀ15 °C to rt, overnight, 81%; (vii) TFA, TES, Me2C(OMe)2, CH2Cl2, 0 °C, 2 min, 63%.
O
O
S
NH
O
NMe2
BnO
BnO
BnO
BnO
N3
OH
O
HO
HO
HO
HO
I
vi
iv, v
i, ii, iii
O
O
OH
BnO
OMe
BMe
OMe
OH
HO
OMe
14
13
7
12
vii
O
S
NH
O
O
O S
NAc
O
O
O
S
NAc
O
NMe2
C12H25
NMe2
BnO
BnO
BnO
BnO
O
NMe2
BnO
BnO
x
viii, ix
C12H25
N3
N3
BnO
BnO
O
O
O
O
BnO
OAc
O
O
16
4
15
xi, xii
O
S
NH
O
S
NH
NMe2
NMe2
O
O
BnO
BnO
O
C25H51
O
C25H51
NH
O
O
xiv
xiii
BnO
BnO
NH
C12H25
OH
3
BnO
BnO
O
O
O
C12H25
O
OH
17
18
Scheme 3. Regents and conditions: (i) acetone, ZnCl2, H2SO4, rt, 24 h, 92%; (ii) PPh3, I2, Imid., 90 °C, 5 h, 85%; (iii) AcCl, MeOH, 40 °C, 72 h, 92%; (iv) NaN3, DMF, 80 °C, 5 h, 82%;
(v) BnBr, NaH, DMF, 0 °C to rt, 2 h, 99%; (vi) PMe3, THF, 0 °C to rt, 1 h, then dansyl chloride, 1 M NaOH, 0 °C to rt, 22 h, 93%; (vii) AcOH, Ac2O, H2SO4, 0 °C, 3 h, quant.; (viii) TMSI,
CH2Cl2, 0 °C, 1 h; (ix) TBAI, DiPEA, benzene, 6, 70 °C, 20 h, 94% (two steps); (x) NaOMe, MeOH, CH2Cl2, rt, 3 d, quant.; (xi) PMe3, 1 M NaOH, 0 °C to rt, 21 h; (xii) HOC(O)C25H51
EDCI, DMAP, CH2Cl2, rt, 43 h, 68% (two steps); (xiii) AcOH, H2O, THF, 50 °C, 72 h, 74%; (xiv) H2, Pd(OH)2/C, CHCl3, EtOH, rt, 17 h, 62%.
,
and resulted in the authors adopting a four-step deprotection/
reprotection strategy to generate a modified 3-O, 4-O-benzylated
phytosphingosine derivative. In view of the lability of the isopro-
pylidene group, we treated azide 11 with TFA and TES, then added
2,2-dimethoxypropane to the reaction vessel to reinstall the iso-
propylidine, should it be cleaved under the acidic reaction condi-
did not lead to any improvements in yield. The use of boron tri-
chloride or formic acid to cleave the trityl group resulted in a lower
yield of product. Despite this, in sum, the core phytosphingosine
backbone 6 was prepared in seven steps and in a good overall yield
of 34%. This represents one of the most efficient syntheses of the
phytosphingosine backbone to date.35,36
tions. Indeed, using this approach, phytosphingosine
6
was
Having completed the synthesis of the required phytosphingo-
sine lipid, we then turned our attention to the preparation of the
dansylated galactose donor. Our strategy commenced with the for-
mation of the common azide (13)11 using a modified sequence
of reactions that first led to the formation of iodide 12 via the
obtained in a respectable 63% yield. Numerous attempts were then
made to further optimise the reaction including the addition of
MeOH (to scavenge the liberated TES cation, which was sometimes
found to be present on the 1-position of the lipid), however, this

J. M. H. Cheng et al. / Carbohydrate Research 346 (2011) 914–926
917
selective installation of isopropylidenes at the 1,2 and 3,4 positions
of -galactose (7), subsequent installation of the iodide at the pri-
amine, using Staudinger methodology, then coupled the product
to hexacosanoic acid via an EDCI/DMAP condensation protocol to
D
mary position, and then simultaneous cleavage of the isopropylid-
enes and Fisher glycosylation using a 5% solution of AcCl in MeOH
(Scheme 3). This route provided a convenient means by which to
prepare gram quantities of iodide 13 in excellent overall yield
(72%, three steps) and without the need for column chromatogra-
phy. The iodide in 12 was then displaced to give the intermediate
azide, which was subsequently benzylated to give the fully pro-
tected azide 13, again in excellent overall yield.
give fully protected dansylated a-GalCer 17 in good (68%) overall
yield. Removal of the isopropylidene group occurred smoothly to
give 18 in 74% yield. Finally, global deprotection, again using
Pd(OH)2/C as the catalyst, gave dansyl
a-GalCer 3 in 62% yield,
which glowed bright white under UV light (k = 366 nm). Though
glycolipids such as 3 have been reported to be notoriously difficult
to solubilise and hence assign,51,52 using a variety of 2D NMR spec-
troscopy techniques (COSY, HSQC, HMBC) with spectra recorded in
pyridine-d5, all 13C and 1H resonances were fully assigned. Further-
more, the dansyl group of 3 allowed for the ready detection of the
compound by way of HPLC analysis (see Supplementary data).
With the objective of installing the fluorescent group at an early
stage in the synthesis, the dansyl group was then incorporated
onto the galactose residue 13 in a two-step one-pot procedure
involving the reduction of the azide to an amine using Staudinger
conditions10 and subsequent treatment with dansyl chloride in the
presence of 1 M NaOH. Using this strategy, dansylated galactose 14
was prepared in excellent (93%) yield. Dansylated galactose 14 is a
bright-green oil, and as such, is readily identified during flash col-
umn chromatography whereby the yellow-green band can be fol-
lowed by eye during the purification process. Furthermore,
incorporation of the dansyl flurophore also aids in the monitoring
of the reaction by TLC analysis with 14 glowing bright white under
UV light (k = 254 nm). Treatment of 14 with a solution of AcOH,
Ac2O and H2SO4 at 0 °C for 3 h then resulted in the quantitative for-
mation of acetate 15. Here, it should be noted that in addition to
the conversion of the anomeric methoxy to the acetate, the amine
of the dansyl group was also acetylated. Though not optimal, the
over-acetylation of dansyl groups has been previously reported37
and we were confident that the superfluous acetyl could be readily
removed during a subsequent step of the synthesis. A dramatic
change in colour was also observed upon monitoring the reaction
by TLC analysis (k = 254 nm), with the methyl glycoside 14 being
bright white, whilst the corresponding acetate 15 was mauve
(see Supplementary data). As with all dansylated compounds pre-
pared, acetate 15 was a fluorescent green colour, which allowed for
its ready purification by flash column chromatography and illus-
trates the added value of the early incorporation of the dansyl-
group into the synthetic plan.
Thus, in summary, dansyl
steps from the commercially available and inexpensive
a
-GalCer 3 was prepared in 14 linear
-galactose
D
and in 16% overall yield. This represents a highly efficient synthesis
of this important biological probe and is comparable to the work of
Zhou et al. whereby the probe was prepared in <10% yield from 6-
azido-6-deoxy-1,2:3,4-di-O-isopropylidene-a-D-
galactopyranose.11
2.2. The effects of
a-GalCer 3 on the stimulation of iNKT cells
in vitro and in vivo
Having developed an efficient synthesis of dansyl
we then set about to investigate whether 3 could activate iNKT
cells in a manner similar to that of its parent compound, -GalCer
(2).6,7,9,11,28,30,45 This was achieved by analysing levels of the cyto-
kine IL-2 released by the V
14+ iNKT cell hybridoma4 DN32.D3 fol-
a-GalCer 3,
a
a
lowing in vitro stimulation with the glycolipid compounds and by
examining the expression of the co-stimulatory molecule, CD86, on
splenic DC and B cells after injection of the compounds into mice.9
The latter assay is based on the observation that CD86 is upregu-
lated as a consequence of iNKT cells interacting with, and subse-
quently driving, DC and B cell maturation.53,54
To assess the ability of 3 to stimulate DN32.D3 iNKT cell hybrid-
oma cells, titrated amounts of dansyl
a-GalCer 3, or a-GalCer 2,
were incubated for 24 h with the hybridoma cells in the presence
of a constant number of murine DCs from the immortalised line,
DC2114,55 and supernatants were then collected and analysed for
IL-2. Proliferation of the IL-2-dependent cell line HT-2 was used
Forging ahead with our synthesis, we thus converted glycosyl
donor 15 to the corresponding glycosyl iodide via methodology
based on seminal work by Gervay-Hague.38–41 Though a number
of galactosyl donors have been prepared and coupled to the phyto-
as a read-out of IL-2 levels. As illustrated (Fig. 2A), both
2 and dansyl -GalCer 3 stimulated release of IL-2 in a dose-depen-
dent manner, with -GalCer 2 driving a higher level of this cyto-
kine. This observation is consistent with literature precedent for
weaker cytokine profiles generated by similar C600-substituted
GalCer derivatives, but is nonetheless indicative that dansyl -Gal-
Cer 3 is indeed capable of iNKT cell activation.11,15 The ability of
dansyl -GalCer 3 to stimulate iNKT cells in vivo was assessed by
injecting mice with 200 ng of dansyl -GalCer 3 or -GalCer 2,
a-GalCer
sphingosine backbone en route to the preparation of
Cer,7,9,10,27–29,42–45 few of these glycosylation reactions have been
reported to be both high-yielding and highly
-selective.38,46–48
a
-Gal-
a
a
a
To this end, we chose the glycosyl iodide route of Gervay-Hague
a-
and were delighted to report that glycolipid 4 could be prepared
a
in 94% yield and as only the a
-anomer, as determined by a 1H J1,2
coupling constant of 3.2 Hz and a 13C–1H (1JCH) coupling of
172.2 Hz.49
a
a
a
With glycolipid 4 in hand, we then set about installing the C26
acyl chain. First, the acetyl on the sulfonimide was removed using
Zemplén conditions50 to give the desired sulfonamide 16 in quan-
titative yield. Both glycolipid 4 and sulfonamide 16 had identical Rf
values (0.40; PE/EA, 2/1, v/v), though were different colours when
observed via TLC analysis. With glycolipid 4 being mauve in colour
(UV light, k = 254 nm) and sulfonamide 16 bright white, this
greatly aided in the monitoring of the reaction. The isopropylidene
group was then removed and the diol product subjected to hydro-
genation conditions using Pd(OH)2/C in the presence of HCl in an
attempt to simultaneously remove the benzyl groups and reduce
the azide to an amine. Numerous attempts were made to achieve
this transformation, yet despite the addition of HCl to protonate
the resultant amine and prevent poisoning of the palladium cata-
lyst, only partial debenzylation was observed. In view of this, we
changed our strategy and first reduced the azide in 16 to the
and then determining the expression of the maturation marker
CD86 on the surface of splenic B cells and DCs by flow cytometry
20 h later. As shown, administration of fluorescent analogue 3
stimulated the maturation of B cells and DCs (Fig. 2B) with activi-
ties resembling that of the parent compound 2. To confirm that this
in vivo activity was mediated by iNKT cells, the experiment was re-
peated in CD1d deficient mice. Here, no up-regulation of CD86 was
observed on DCs (Fig. 2C), confirming that dansyl
-GalCer 2, activates APCs by stimulating iNKT cells in a CD1d
dependent manner.
a-GalCer 3, like
a
2.3. Uptake of
a-GalCer by dendritic cells
Having established that dansyl
a-GalCer 3 binds to CD1d and
activates iNKT cells in a manner similar to that of
a-GalCer 2, we
then set out to investigate the ability of dansyl
a-GalCer 3 to

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J. M. H. Cheng et al. / Carbohydrate Research 346 (2011) 914–926
Figure 2. Dansyl
a
-GalCer 3 stimulates iNKT cells in vitro and in vivo: (A) production of IL-2 by iNKT cells (DN32.D3) in the presence of titrated doses of
a
-GalCer 2 or dansyl
-GalCer 2 (n = 3) or
a
-GalCer 3 (n = 3), and spleens were removed 20 h after injection for antibody labelling and flow cytometry. The expression of CD86 on CD11c+ dendritic cells and
a-GalCer 3; (B) dansyl a-GalCer 3 drives the maturation of splenic DCs and B cells in vivo. C57BL/6 mice were injected intravenously with 200 ng of a
dansyl
B220 B cells were assessed; (C) 200 ng of dansyl a
-GalCer 3 was injected intravenously into wild type (C57BL/6) and CD1dꢀ/ꢀ mice, and spleens were removed 20 h after
injection for antibody labelling and flow cytometry. The spleen of an untreated wild type (C57BL/6) mouse was used as control. The expression of CD86 on CD11c+ dendritic
cells was assessed. Mean fluorescence intensities (MFI) SD are presented.
function as a fluorescent reporter group, which has previously not
been reported. The wavelengths of maximum absorbance and
emission of 3 were determined by spectrofluorimetry to be
343 nm and 510 nm, respectively, and these maxima were used
in further analyses. The DC cell line, DC2114 was incubated with
4 lg of dansyl a-GalCer 3 for 24 h and uptake of the fluorescent
glycolipid was analysed by flow cytometry (Fig. 3). A distinct shift
in mean fluorescence intensity was observed for cells treated with
dansyl
a-GalCer 3, but not for those treated with a-GalCer 2 or
controls (untreated and vehicle), illustrating the ability of dansyl

J. M. H. Cheng et al. / Carbohydrate Research 346 (2011) 914–926
919
Figure 3. Cells that have taken up dansyl
a
-GalCer 3 can be detected by flow cytometry. 1 ꢂ 106 cells (DC2114 cell line) were incubated for 24 h with 4
lg of dansyl
a
-GalCer
3,
a-GalCer 2 or vehicle only. Cells were excited with the UV laser, and detected by the Hoechst Blue-A detector.
a
-GalCer 3 to be used as a fluorescent probe to detect sub-popula-
drich, 20 wt %), EtOAc (Pancreac), hexanes (Fisher Scientific),
petroleum ether (Pure Science), MeOH (Pure Science), CHCl3 (Panc-
reac), EtOH (absolute, Pure Science), NaOH (Pure Science), NaHCO3
(Pure Science), NaCl (Pancreac) were used as received. All solvents
were removed by evaporation under reduced pressure. Reactions
were monitored by TLC-analysis on Macherey-Nagel silica gel
tions of DCs that present the glycolipid.
3. Conclusion
In summary we have presented an efficient synthesis of the bio-
coated plastic sheets (0.20 mm, with fluorescent indicator UV254
)
logically important probe, dansyl a-GalCer 3. En route to the syn-
with detection by UV-absorption (short wave UV—254 nm; long
wave UV—366 nm), by dipping in 10% H2SO4 in EtOH followed by
charring at ꢁ150 °C, by dipping in I2 in silica, or by dipping into
a solution of ninhydrin in EtOH followed by charring at ꢁ150 °C.
Column chromatography was performed on Pure Science silica
thesis of this glycolipid, we developed a highly efficient synthesis
of the phytosphingosine lipid acceptor. In addition, we found that
the early incorporation of the fluorescent group greatly aids in the
monitoring and purification of reaction products. The ability of
dansyl
cent probe for the analysis of glycolipid uptake by DCs has also
been illustrated. Given the importance of -GalCer 2, it is antici-
pated that dansyl -GalCer 3 will find wide application as a molec-
a-GalCer 3 to activate iNKT cells and to be used as a fluores-
gel (40–63 lm). AccuBOND II ODS-C18 (Agilent) was used for re-
verse phase chromatography. High-resolution mass spectra were
recorded on a Waters Q-TOF Premier™ Tandem Mass Spectrometer
using postive electro-spray ionisation. Optical rotations were re-
corded using a Perkin–Elmer 241 polarimeter or Autopol II (Ru-
dolph Research Analytical) at 589 nm (sodium D-line). Infrared
spectra were recorded as thin films using a Bruker Tensor 27 FTIR
spectrometer equipped with an Attenuated Total Reflectance (ATR)
sampling accessory and are reported in wave numbers (cmꢀ1). Nu-
clear magnetic resonance spectra were recorded at 20 °C in D2O or
CDCl3 using either a Varian INOVA operating at 500 MHz or Varian
VNMRS operating at 600 MHz. Chemical shifts are given in ppm (d)
relative to TMS. NMR peak assignments were made using COSY,
HSQC and HMBC 2D experiments. Melting points were obtained
using the Gallenkamp Melting Point Apparatus. Spectrofluorimetry
measurements were carried out on the Horiba Jobin-Yvon Fluoro-
log-3 spectrofluorometer using the three dimensional (3D) scan-
ning mode. High-pressure liquid chromatography was performed
on Waters 600 liquid chromatograph with a photodiode array
a
a
ular tool for the better understanding of
a-GalCer trafficking and
presentation by CD1d.
4. Experimental
4.1. General
Unless otherwise stated all reactions were performed under N2.
Prior to use, THF (Pancreac) was distilled from LiAlH4, pyridine was
dried over 4 Å molecular sieves (4 Å MS), acetone (Pure Science)
was dried over 3 Å MS, CH2Cl2 (Pancreac) was distilled from
P2O5, TMSI (Aldrich) was distilled from antimony powder (M&B)
and stored over Cu(s) powder (Hopkin & Williams), and H2O and
benzene (Fisher Scientific) were distilled. Trityl chloride (Acros),
CuSO4 (Fluka), NaIO4 (M&B), anhydrous Et2O (Pancreac), n-BuLi
(Aldrich), 1-bromotridecane (Aldrich), PPh3 (Merck), Pd/C (Aldrich,
10 wt %), Tf2O (Aldrich), NaN3 (BDH), anhydrous DMF (Acros), TFA
(Aldrich), triethylsilane (Fluka), Me2C(OMe)2 (Aldrich), ZnCl2 (Al-
drich), H2SO4 (Lab-Scan), I2 (BDH), imidazole (Aldrich), AcCl
(B&M), BnBr (Fluka), NaH (Avocado Research Chemicals, 60% dis-
persion in mineral oil), PMe3 (Aldrich, 1 M in THF), dansyl chloride
(Acros), AcOH (Ajax Finechem), Ac2O (Peking Reagent), TBAI (Rie-
del-de Haen), DiPEA (Aldrich), NaOMe (Janssen Chimica),
detector (Waters 2996) and a C-18 column (Waters Xbridge 5 lm).
4.2. 3,4-O-Isopropylidene-6-O-triphenylmethyl-D-
galactopyranoside (8)
Trityl chloride (16.4 g, 58.8 mmol) was added to a solution of D-
galactose (7) (10 g, 55.5 mmol) in pyridine (150 mL). The reaction
mixture was stirred at 50 °C for 48 h and concentrated in vacuo.
C25H51COOH (Acros), EDCI (Aldrich), DMAP (Merck), Pd(OH)2/C (Al-

920
J. M. H. Cheng et al. / Carbohydrate Research 346 (2011) 914–926
The resulting residue was purified by chromatography through a
short silica column and the tritylated galactose was eluted with
acetone and crystallised from EtOH to afford the tritylated galact-
ose (23.4 g, 55.4 mmol, quant.) [Rf: 0.17 (EtOAc)] as a white pow-
der. To a solution of tritylated galactose (15 g, 35.5 mmol) in
anhydrous acetone (750 mL) was added copper sulfate (60 g,
380 mmol). After stirring the reaction mixture for 3 d at room tem-
perature, carbon was then added and the solution filtered through
Celite. The filtrate was concentrated in vacuo and purified by flash
column chromatography. Elution with 1:1 (v/v) hexanes/EtOAc
afforded the title compound 8 (13.9 g, 30.1 mmol, 85%, two steps)
solved in distilled THF (130 mL) at ꢀ78 °C and stirred for 15 min.
To the bright orange mixture, a solution of formate ester 9 (co-
evaporated with dry toluene ꢂ 3) in THF (55 mL) was added. The
reaction mixture was stirred at ꢀ78 °C for 1 h, then warmed to
room temperature and stirred for a further 3.5 h before being
quenched with NH4Cl solution (150 mL), concentrated, and redis-
solved in ethyl acetate (100 mL). The organic layer was washed
with saturated NaHCO3 solution (100 mL), water (100 mL) and
brine (100 mL), dried (MgSO4), filtered and concentrated in vacuo.
The resulting residue was purified by silica gel column chromatog-
raphy and the desired product eluted with 8:1 (v/v) petroleum
ether/EtOAc to afford alkene 10 as a clear oil (2.57 g, 4.29 mmol,
78%) in an approximate E:Z, 1:10. Rf: 0.33 (PE/EA, 5/1, v/v);
as a clear oil. Rf: 0.65 (EtOAc); ½a D20
¼ þ18:0 (c 1.0, CHCl3); IR (film)
ꢃ
3406, 3059, 3034, 2986, 2936, 1597, 1491, 1449, 1380, 1219, 1162,
1132, 1079, 1032, 909, 872, 814, 776, 732, 705 cmꢀ1
(500 MHz, CDCl3) 7.48–7.46 (m, 6H, CPh3, CH-o), 7.30 (t,
Jo,m = Jm,p = 7.4 Hz, 6H, CPh3, CH-m), 7.26–7.23 (m, 3H, CPh3, CH-
p), 5.23 (t, J1,2 = J1,OH = 3.7 Hz, 1H, H-1 ), 4.34–4.29 (m, 3H, H-3,
;
1H NMR
½
a 2D0
ꢃ
¼ ꢀ22:2 (c 1.0, CHCl3); IR (film) 3559, 3058, 3023, 2924,
2854, 1598, 1491, 1449, 1380, 1214, 1162, 1073, 898, 880, 763,
746, 705 cmꢀ1 1H NMR (500 MHz, CDCl3) d 7.48–7.44 (m, 6H,
d
;
a
CPh3, CH-o), 7.33–7.29 (m, 6H, CPh3, CH-m), 7.26–7.23 (m, 3H,
CPh3, CH-p), 5.59 (dd, J5,6 = 11.3 Hz, J6,7 = 6.0 Hz, 1H, H-6), 5.55
(dd, J5,6 = 11.3 Hz, J4,5 = 7.7 Hz, 1H, H-5), 4.92 (dd, J4,5 = 7.7 Hz,
J3,4 = 6.9 Hz, 1H, H-4), 4.23 (dd, J3,4 = 6.9 Hz, J2,3 = 4.3 Hz, 1H, H-3),
3.77 (pent, J1,2 = J2,3 = J2,OH = 5.4 Hz, 1H, H-2), 3.18 (dd,
J1a,1b = 9.4 Hz, J1a,2 = 5.0 Hz, 1H, H-1a), 3.13 (dd, J1a,1b = 9.4 Hz,
J1b,2 = 6.4 Hz, 1H, H-1b), 2.03–1.98 (m, 1H, H-7a), 1.82–1.77 (m,
1H, H-7a), 1.51 (s, 3H, CH3 iPr), 1.41 (s, 3H, CH3 iPr), 1.33–1.23
(m, 20H, H-8–H-17), 0.90 (t, J17,18 = 7.0 Hz, 3H, H-18); 13C NMR
(125 MHz, CDCl3) d 143.9 (C-i, CPh3), 135.4 (C-6), 128.7 (CH-o,
CPh3), 127.8 (CH-m, CPh3), 127.0 (CH-p, CPh3), 125.0 (C-5), 108.4
(Cq iPr), 86.7 (Cq CPh3), 77.5 (C-3), 73.0 (C-4), 69.2 (C-2), 65.0 (C-
1), 31.9, 29.70, 29.67, 29.62, 29.52, 29.50, 29.4, 29.3, 27.6, 22.7
(C-7–C-17), 27.4, 25.1 (2 ꢂ CH3 iPr), 14.2 (C-18); HRMS(ESI) m/z
calcd for [C40H54O4+Na]+: 621.3920, obsd.: 621.3926.
H-4, H-5), 3.87–3.85 (m, 1H, H-2), 3.02 (dd, J6a,6b = 9.1 Hz,
J6a,5 = 6.4 Hz, 1H, H-6a), 3.33 (dd, J6a,6b = 9.1 Hz, J6b,5 = 6.4 Hz, 1H,
H-6a), 3.02 (d, JOH,1 = 3.6 Hz, 1H, 1-OH), 2.45–2.44 (m, 1H, 2-OH),
1.44 (s, 3H, CH3 iPr), 1.36 (s, 3H, CH3 iPr); 13C NMR (125 MHz,
CDCl3) d 143.9 (C-i, CPh3), 128.8 (C-o, CPh3), 127.8 (C-m, CPh3),
127.0 (C-p, CPh3), 109.5 (Cq iPr), 91.2 (C-1), 86.8 (Cq CPh3), 75.4
(C-3), 72.9 (C-4), 69.1 (C-2), 67.9 (C-5), 62.8 (C-6), 27.4, 25.7
(2 ꢂ CH3 iPr); HRMS(ESI) m/z calcd for [C28H30O6+Na]+: 485.1940,
obsd.: 485.1949.
4.3. 4-O-Formyl-2,3-O-isopropylidene-5-O-triphenylmethyl-D-
lyxose (9)
A solution of NaIO4 (0.46 g, 2.14 mmol) in H2O (4 mL) was
added to 8 (0.66 g, 1.42 mmol) dissolved in 4 mL THF at 0 °C. After
stirring at room temperature overnight, the reaction mixture was
diluted with EtOAc (15 mL) and washed with water (20 mL), satu-
rated NaHCO3 solution (20 mL) and brine (20 mL), dried (MgSO4),
filtered and concentrated in vacuo to afford the title compound 9
(0.65 g, 1.42 mmol, quant.) as a clear oil, which was carried
through without further purification. Rf: 0.48 (PE/EA, 1/1, v/v); IR
(film) 3450, 3060, 3025, 2989, 2938, 2360, 2341, 2252, 1732,
1597, 1491, 1449, 1383, 1218, 1160, 1076, 987, 907, 764, 729,
4.5. 2-Azido-3,4-O-isopropylidene-1-O-triphenylmethyl-
arabino-octadecane-1,3,4-triol (11)
D-
Pd/C (1.5 wt %) was added to
a solution of 10 (2.52 g,
4.21 mmol) in distilled THF (42 mL) and stirred under H2 (g) over-
night at room temperature. The reaction mixture was then filtered
through Celite and concentrated to afford 3,4-O-isopropylidene-1-
O-triphenylmethyl-D-arabino-octadecane-1,2,3,4-tetraol as a clear
oil (2.53 g, 4.21 mmol, quant.), which was used without further
701, 648 cmꢀ1 1H NMR (500 MHz, CDCl3) d 9.60 (d, J1,2 = 2.4 Hz,
;
1H, H-1), 8.00 (s, 1H, HCO2), 7.44–7.41 (m, 6H, CPh3, CH-o), 7.34–
7.30 (m, 6H, CPh3, CH-m), 7.27–7.24 (m, 3H, CPh3, CH-p), 5.18 (q,
J3,4 = J4,5a = J4,5b = 4.9 Hz, 1H, H-4), 4.74 (dd, J2,3 = 7.7 Hz,
J3,4 = 4.2 Hz, 1H, H-3), 4.31 (dd, J2,3 = 7.7 Hz, J1,2 = 2.4 Hz, 1H, H-2),
3.40 (dd, J5a,5b = 9.8 Hz, J4,5a = 5.6 Hz, 1H, H-5a), 3.37 (dd,
J5a,5b = 9.8 Hz, J4,5b = 5.6 Hz, 1H, H-5b), 1.56 (s, 3H, iPr CH3), 1.42
(s, 3H, iPr CH3); 13C NMR (125 MHz, CDCl3) d 201.1 (C-1), 159.7
(C-6), 128.6 (CH-o, CPh3), 127.9 (CH-m, CPh3), 127.2 (CH-p, CPh3),
111.1 (Cq iPr), 87.2 (Cq CPh3), 80.2 (C-2), 77.0 (C-3), 69.5 (C-4),
61.8 (C-5), 26.8, 25.1 (2 ꢂ CH3 iPr); HRMS(ESI) m/z calcd for
[C28H28O6+Na]+: 483.1784, obsd.: 483.1793.
purification. Rf: 0.44 (PE/EA, 5/1, v/v); ½a D20
¼ ꢀ4:0 (c 1.0, CHCl3);
ꢃ
IR (film) 3560, 3060, 3023, 2923, 2853, 1598, 1491, 1449, 1379,
;
1216, 1073, 899, 872, 762, 746, 705 cmꢀ1 1H NMR (500 MHz,
CDCl3) 7.47 (d, Jo,m = 7.7 Hz, 6H, CPh3, CH-o), 7.31 (t,
d
Jo,m = Jm,p = 7.7 Hz, 6H, CPh3, CH-m), 7.27–7.23 (m, 3H, CPh3, CH-
p), 4.15 (dd, J2,3 = 6.0 Hz, J3,4 = 3.8 Hz, 1H, H-3), 4.11–4.07 (m, 1H,
H-4), 3.74 (dt, J1,2 = 9.5 Hz, J2,3 = 6.0 Hz, 1H, H-2), 3.24–3.19 (m,
2H, H-1a, H-1b), 2.34 (d, JOH,2 = 5.9 Hz, 1H, OH), 1.71–1.64 (m,
2H, H-5), 1.51–1.28 (m, 24H, H-6–H-17), 1.46 (s, 3H, iPr CH3),
1.37 (s, 3H, iPr CH3), 0.89 (t, J17,18 = 6.9 Hz, 3H, H-18); 13C NMR
(125 MHz, CDCl3) d 143.9 (C-i, CPh3), 128.7 (CH-o, CPh3), 127.8
(CH-m, CPh3), 127.1 (CH-p, CPh3), 107.7 (Cq iPr), 86.8 (Cq CPh3),
77.5 (C-4), 77.0 (C-3), 69.0 (C-2), 65.2 (C-1), 31.9, 29.72, 29.68,
29.63, 29.62, 29.57, 29.39, 26.8, 22.7 (C-5–C-17), 27.4, 25.2
(2 ꢂ CH3 iPr), 14.2 (C-18); HRMS(ESI) m/z calcd for [C40H56O4+Na]+:
623.4076, obsd.: 623.4080. 3,4-O-Isopropylidene-1-O-triphenyl-
4.4. 3,4-O-Isopropylidene-1-O-triphenylmethyl-D-arabino-
octadec-5-en-1,2,3,4-tetraol (10)
A mixture of 1-bromotridecane (7.0 mL, 27.4 mmol) and tri-
phenylphosphine (7.18 g, 27.4 mmol) was heated at 140 °C under
N2 atmosphere for 5 h. The reaction mixture was allowed to cool
to room temperature and formed a solid gel. The gel was dissolved
in boiling dry acetone (36 mL) and dry diethyl ether (87 mL) was
added and the compound left to crystallise, which greatly im-
proved in subsequent batches via the use of seed crystals. The crys-
tals were filtered under N2 atmosphere to afford BrPh3PC13H27
(11.1 g, 21.1 mol, 77%) as white needles. n-BuLi (6.6 mL,
13.2 mmol) was added to BrPh3PC13H27 (7.0 g, 13.7 mmol) dis-
methyl-D-arabino-octadecane-1,2,3,4-tetraol (1.59 g, 2.65 mmol)
was then co-evaporated with dry toluene (ꢂ3), dissolved in a mix-
ture of dry CH2Cl2 (8 mL) and distilled pyridine (0.54 mL) and
cooled to ꢀ15 °C. Triflic anhydride (0.67 mL, 3.97 mmol) was
added dropwise over 5 min and stirred at ꢀ15 °C for 45 min to af-
ford the triflate intermediate [Rf: 0.56 (PE/EA, 5/1, v/v)]. Dry DMF
(27 mL) was cooled to ꢀ15 °C and added to the reaction mixture,
followed by the addition of sodium azide (0.69 g, 10.61 mmol).
The reaction mixture was then warmed to room temperature,

J. M. H. Cheng et al. / Carbohydrate Research 346 (2011) 914–926
921
stirred overnight, and quenched by adding ice water (120 mL). The
desired product was extracted with EtOAc (2 ꢂ 150 mL) and the
combined organic layers washed with saturated NaHCO3 solution
(200 mL), water (200 mL) and brine (200 mL), dried (MgSO4), fil-
tered and concentrated in vacuo. The residue was purified by silica
gel flash column chromatography and eluted with 1.5% EtOAc/hex-
anes to afford the title compound 11 (1.35 g, 2.16 mmol, 81%) as an
addition of 10% aq Na2S2O4. The product was extracted with EtOAc,
and the combined organic layers were washed with brine, dried
(MgSO4), filtered and concentrated. Distillation of the residue gave
6-deoxy-6-iodo-1,2:3,4-di-O-isopropylidene-a-D-galactopyranose
as a yellow oil (3.14 g, 8.48 mmol, 85%). AcCl (1.8 mL) was added
dropwise to a mixture of 6-deoxy-6-iodo-1,2:3,4-di-O-isopropyli-
dene-a-D-galactopyranose (3.14 g, 8.48 mmol) in MeOH (60 mL).
amorphous white solid. Rf: 0.61 (PE/EA, 5/1, v/v); ½a D25
ꢃ
¼ þ7:0 (c
After stirring for 2 d, the mixture was concentrated in vacuo and
the residue crystallised from MeOH, to give the title compound
12 (2.37 g, 7.80 mmol, 92%) as white crystals. Mp 175.0–175.6 °C,
1.0, CHCl3), Lit:56
½
a 2D6
ꢃ
¼ þ9:0 (c 1.0, CHCl3); IR (film) 3060, 2923,
2853, 2097, 1598, 1491, 1379, 1370, 1246, 1219, 1156, 1073,
1034, 900, 869, 762, 744, 703 cmꢀ1 1H NMR (500 MHz, CDCl3) d
;
Lit:59 162 °C; Rf = 0.43 (MeOH/EtOAc, 1/9, v/v); ½a 2D1
ꢃ
¼ þ129:0 (c
7.51 (d, Jo,m = 7.3 Hz, 6H, CPh3, CH-o), 7.32 (t, Jo,m = Jm,p = 7.6 Hz,
6H, CPh3, CH-m), 7.26 (d, Jo,m = 7.1 Hz, 3H, CPh3, CH-p), 4.13–4.09
(m, 1H, H-4), 3.89 (dd, J2,3 = 9.5 Hz, J3,4 = 5.5 Hz, 1H, H-3), 3.55
(dd, J1a,1b = 9.9 Hz, J1a,2 = 2.5 Hz, 1H, H-1a), 3.50 (dt, J2,3 = 9.5 Hz,
J1a,2 = 2.5 Hz, 1H, H-2), 3.37 (dd, J1a,1b = 9.9 Hz, J1b,2 = 7.8 Hz, 1H,
H-1b), 1.59–1.28 (m, 26H, H-5–H-17), 1.27 (s, 3H, iPr CH3), 1.24
(s, 3H, iPr CH3), 0.90 (t, J17,18 = 6.9 Hz, 3H, H-18); 13C NMR
(125 MHz, CDCl3) d 143.8 (C-i, CPh3), 128.7 (CH-o, CPh3), 127.9
(CH-m, CPh3), 127.1 (CH-p, CPh3), 108.1 (Cq CPh3), 87.3 (Cq iPr),
77.9 (C-4), 75.9 (C-3), 64.6 (C-1), 60.8 (C-2), 32.0, 29.73, 29.69,
29.66, 29.64, 29.57, 29.4, 29.3, 26.5, 22.7 (C-5–C-17), 28.0, 25.7
(2 ꢂ CH3 iPr), 14.2 (C-18); HRMS(ESI) m/z calcd for [C40H55N3O3+
Na]+: 648.4141, obsd.: 648.4145.
1.0, H2O), ½a 2D1
ꢃ
¼ þ122:1 (c 1.0, MeOH), Lit:59
½ ꢃ
a 2D2
¼ þ142:0 (c
1.0, H2O); IR (film) 3354, 3227, 3007, 2953, 2935, 2906, 2836,
1454, 1417, 1360, 1347, 1297, 1257, 1200, 1145, 1132, 1102,
1078, 1026, 999, 935, 886, 854, 790, 721, 691, 657 cmꢀ1 1H NMR
;
(500 MHz, D2O)
d 4.69 (d, J1,2 = 3.6 Hz, 1H, H-1), 3.97 (dd,
J3,4 = 3.3, J4,5 = 1.2 Hz, 1H, H-4), 3.90 (ddd, J4,5 = 1.2 Hz, J5,6a = 5.3,
J5,6b = 8.0 Hz 1H, H-5), 3.75 (dd, J1,2 = 3.6, J2,3 = 10.2 Hz, 1H, H-2),
3.69 (dd, J2,3 = 10.2, J3,4 = 3.3 Hz, 1H, H-3), 3.46 (s, 3H, OMe), 3.37
(dd, J5,6a = 5.3, J6a,6b = 10.2 Hz, 1H, H-6a), 3.33 (dd, J5,6b = 8.0,
J6a,6b = 10.2 Hz, 1H, H-6b); 13C NMR (125 MHz, D2O) d 100.2 (C-
1), 71.4 (C-5), 70.4 (C-4), 69.9 (C-2), 68.4 (C-3), 54.6 (OMe), 2.3
(C-6). HRMS(ESI) m/z calcd for [C7H13IO5+Na]+: 326.9700, obsd.:
326.9709.
4.6. 2-Azido-3,4-O-isopropylidene-
D-ribo-octadecane-1,3,4-triol
4.8. Methyl 6-azido-2,3,4-tri-O-benzyl-6-deoxy-a-D-
(6)
galactopyranoside (13)
Triethylsilane (91.8
tion of azide 11 (181 mg, 0.289 mmol) (co-evaporated with dry tol-
l
L, 0.58 mmol) was added to a stirred solu-
Sodium azide (4.28 g, 0.066 mmol) was added to iodide 12
(5.01 g, 0.016 mmol) dissolved in dry DMF (300 mL). After stirring
overnight at room temperature, MeOH (150 mL) was added and
the reaction mixture was concentrated in vacuo. The residue was
purified by silica gel flash column chromatography and the product
eluted with 8% MeOH/CH2Cl2. The residue was crystallised from
uene ꢂ 3) in dry CH2Cl2 (5 mL) at 0 °C. Trifluoroacetic acid (TFA)
(90 lL, 1.17 mmol) was added very slowly, allowing the reaction
mixture to return to a colourless solution before adding the next
drop. After stirring for 2 min, 2,2-dimethoxypropane (7 L,
l
0.057 mmol) was added and the reaction mixture stirred for a fur-
ther 1 min before being quenched with saturated NaHCO3 solution
(50 mL). The product was extracted with CH2Cl2 (2 ꢂ 50 mL) and
the combined organic layers washed with water (80 mL) and brine
(80 mL), dried (MgSO4), filtered and concentrated in vacuo. The
resulting residue was purified by silica gel flash column chroma-
tography and eluted with 5:1 (v/v) petroleum ether/EtOAc to af-
ford the title compound 6 (69.3 mg, 0.18 mmol, 63%) as an
MeOH to afford methyl 6-azido-6-deoxy-
a-D-galactopyranoside
(2.96 g, 14 mmol, 82%) as white crystals. Mp 173.8–174.1 °C,
Lit:60
172–173 °C;
Rf = 0.43
(MeOH/EtOAc,
1/9,
v/v);
½
a 2D3
ꢃ
¼ þ144:0 (c 1.0, H2O), Lit:61
½ ꢃ
a 2D2
¼ þ154:0 (c 0.8, H2O); IR
(film) 3371, 3238, 2934, 2091, 1642, 1459, 1349, 1297, 1245,
1137, 1107, 1080, 1029, 961, 791, 730, 698 cmꢀ1
;
1H NMR
(500 MHz, CDCl3) d 4.73 (d, J1,2 = 3.7 Hz, 1H, H-1), 3.90 (ddd,
J5,6a = 8.8 Hz, J5,6b = 4.0 Hz, J4,5 = 1.1 Hz, 1H, H-5), 3.79 (dd,
J3,4 = 3.3 Hz, J4,5 = 1.1 Hz, 1H, H-4), 3.77 (dd, J2,3 = 10.1 Hz,
J1,2 = 3.7 Hz, 1H, H-2), 3.72 (dd, J2,3 = 10.1 Hz, J3,4 = 3.3 Hz, 1H, H-
3), 3.58 (dd, J6a,6b = 12.8 Hz, J5,6a = 8.8 Hz, 1H, H-6a), 3.44 (s, 3H,
OCH3), 3.27 (dd, J6a,6b = 12.8 Hz, J5,6b = 4.0 Hz, 1H, H-6b); 13C NMR
(125 MHz, CDCl3) d 100.2 (C-1), 70.0, 69.83, 69.77 (C-3, C-4, C-5),
68.6 (C-2), 54.3 (OCH3), 51.3 (C-6); HRMS(ESI) m/z calcd for
[C7H13N3O5+Na]+: 242.0753, obsd.: 242.0749. Methyl 6-azido-6-
amorphous white solid. Rf: 0.21 (PE/EA, 5/1, v/v); ½a D20
¼ þ24:0 (c
ꢃ
1.0, CHCl3); IR (film) 3426, 2923, 2853, 2098, 1465, 1370, 1247,
1220, 1170, 1063, 909, 869, 734 cmꢀ1
4.21–4.17 (m, J3,4 = 5.7 Hz, H-4), 4.0 (ddd, J1a,1b = 11.8 Hz,
J1a,OH = 5.1 Hz, J1a,2 = 4.4 Hz, 1H, H-1a), 3.97 (dd, J2,3 = 9.6 Hz,
J3,4 = 5.7 Hz, 1H, H-3), 3.87 (ddd, J1a,1b = 11.8 Hz, J1b,OH
;
1H NMR (500 MHz, CDCl3)
d
=
6.6 Hz, J1b,2 = 5.6 Hz, 1H, H-1b), 3.48 (ddd, J2,3 = 9.6 Hz,
J1b,2 = 5.6 Hz, J1a,2 = 4.4 Hz, 1H, H-2), 2.20 (dd, J1b,OH = 6.6 Hz,
J1a,OH = 5.1 Hz, 1H, OH), 1.63–1.55 (m, 3H, H-5a, H-5b, H-6a), 1.43
(s, 3H, iPr CH3), 1.34 (s, 3H, iPr CH3), 1.41–1.26 (m, 23H, H-6b,
H-7–H-17), 0.88 (t, J17,18 = 7.0 Hz, 3H, H-18); 13C NMR (125 MHz,
CDCl3) d 108.4 (Cq iPr), 77.7 (C-4), 76.7 (C-3), 63.9 (C-1), 61.2
(C-2), 31.9, 29.70, 29.69, 29.68, 29.66, 29.65, 29.60, 29.58, 29.53,
29.39, 29.36, 26.52, 22.7 (C-5–C-17), 28.0, 25.5 (2 ꢂ CH3 iPr), 14.1
(C-18); HRMS(ESI) m/z calcd for [C21H41N3O3+Na]+: 406.3046,
obsd.: 406.3049.
deoxy-a-D-galactopyranose (1.53 g, 6.98 mmol) was then co-evap-
orated from dry DMF (ꢂ1), dissolved in dry DMF (150 mL) and
cooled to 0 °C. Benzyl bromide (3.3 mL, 27.9 mmol) was added fol-
lowed by sodium hydride (60% in oil suspension) (1.40 g,
34.9 mmol) and the reaction mixture allowed to warm to rt. After
stirring for 2 h, the reaction was quenched with MeOH (20 mL) and
concentrated in vacuo. The residue was taken up in EtOAc (100 mL)
and the mixture was washed with water (100 mL), saturated NaH-
CO3 solution (100 mL), water (100 mL) and brine (100 mL), dried
(MgSO4), filtered and concentrated in vacuo. The residue was puri-
fied by silia gel flash column chromatography and eluted with 20:1
(v/v) petroleum ether/EtOAc to afford the title compound 13
(3.37 g, 6.88 mmol, 99%) as a clear oil. Rf: 0.72 (PE/EA, 1/1, v/v);
4.7. Methyl 6-deoxy-6-iodo-
a
-
D
-galactopyranoside (12)
-galactopyra-
To a mixture of 1,2:3,4-di-O-isopropylidene-
a-D
nose57,58 (2.6 g, 10 mmol), PPh3 (3.93 g, 15 mmol) and imidazole
(1.36 g, 20 mmol) in dry THF (100 mL), was added iodine (3.81 g,
15 mmol) in small portions. After refluxing for 1 h, the reaction
mixture was cooled to room temperature, and quenched by the
½
a 2D0
ꢃ
¼ þ4:6 (c 1.0, CHCl3); IR (film) 2937, 2098, 1453, 1351,
1124, 1094, 1045, 789, 735 cmꢀ1 1H NMR (500 MHz, CDCl3) d
;
7.44–7.31 (m, 15H, Harom), 5.03 (d, Ja,b = 11.4 Hz, 1H, CH-a, 4-O-
Bn), 4.93 (d, Ja,b = 11.7 Hz, 1H, CH-a, 3-O-Bn), 4.87 (d, Ja,b = 12.1 Hz,

922
J. M. H. Cheng et al. / Carbohydrate Research 346 (2011) 914–926
1H, CH-a, 2-O-Bn), 4.78 (d, Ja,b = 11.7 Hz, 1H, CH-b, 3-O-Bn), 4.72 (d,
Ja,b = 12.1 Hz, 1H, CH-b, 2-O-Bn), 4.72 (d, J1,2 = 3.7 Hz, 1H, H-1), 5.03
(d, Ja,b = 11.4 Hz, 1H, CH-b, 4-O-Bn), 4.07 (dd, J2,3 = 10.1 Hz,
J1,2 = 3.7 Hz, 1H, H-2), 3.96 (dd, J2,3 = 10.1 Hz, J3,4 = 2.6 Hz, 1H, H-3),
3.83 (dd, J5,6a = 8.3 Hz, J5,6b = 4.7 Hz, 1H, H-5), 3.80 (d, J3,4 = 2.6 Hz,
1H, H-4), 3.53 (dd, J6a,6b = 12.7 Hz, J5,6a = 8.3 Hz, 1H, H-6a), 3.43 (s,
3H, OCH3), 2.94 (dd, J6a,6b = 12.7 Hz, J5,6b = 4.7 Hz, 1H, H-6b); 13C
NMR (125 MHz, CDCl3) d 138.7 (C-i, 3-O-Bn), 138.4 (C-i, 2-O-Bn),
138.1 (C-i, 4-O-Bn), 128.52, 128.50, 128.4, 128.1, 128.0, 127.8,
127.7, 127.6 (8 ꢂ CHarom), 98.8 (C-1), 79.0 (C-3), 76.3 (C-2), 75.3
(C-4), 74.6 (CH2, 4-O-Bn), 73.70, 73.65 (2 ꢂ CH2, 2-O-Bn, 3-O-Bn),
69.8 (C-5), 55.5 (OCH3), 51.5 (C-6); HRMS(ESI) m/z calcd for
[C28H31N3O5+Na]+: 512.2161, obsd.: 512.2159.
(MgSO4), filtered and concentrated in vacuo. The residue was puri-
fied by silica gel flash column chromatography and eluted with 3:1
(v/v) petroleum ether/EtOAc to afford the title compound 13
(605 mg, 0.79 mmol) as a lime green foam. Rf: 0.66 (PE/EA, 1/1,
v/v); Glows mauve on TLC (k = 254 nm); ½a D23
¼ þ65:8 (c 1.0,
ꢃ
CHCl3); IR (film) 3064, 3030, 2940, 2872, 1750, 1705, 1454, 1342,
1229, 1135, 1102, 1051, 1009, 788, 739, 699 cmꢀ1
;
1H NMR
(500 MHz, CDCl3) d 8.64 (br s, 1H, CH-2 Dan), 8.30 (d, JCH-3,CH-4
=
7.4 Hz, 1H, CH-4 Dan), 7.85 (br s, 1H, CH-8 Dan), 7.58–7.53 (m,
2H, CH-3, CH-7 Dan), 7.44–7.29 (m, 15H, Harom), 7.23 (br s, 1H,
CH-6 Dan), 6.35 (d, J1,2 = 3.7 Hz, 1H, H-1), 5.11 (d, Ja,b = 11.6 Hz,
1H, CH-a, 4-O-Bn), 4.91 (d, Ja,b = 11.6 Hz, 1H, CH-a, 3-O-Bn), 4.82
(d, Ja,b = 11.6 Hz, 1H, CH-b, 3-O-Bn), 4.72 (s, 2H, CH-a, CH-b, 2-O-
Bn), 4.69 (d, Ja,b = 11.6 Hz, 1H, CH-b, 4-O-Bn), 4.29 (dd,
J5,6a = 8.6 Hz, J5,6b = 2.5 Hz, 1H, H-5), 4.18 (dd, J2,3 = 10.0 Hz,
J1,2 = 3.7 Hz, 1H, H-2), 4.10 (dd, J6a,6b = 15.2 Hz, J5,6 = 8.6 Hz, 1H,
H-6a), 4.07 (br d, J3,4 = 2.6 Hz, 1H, H-4), 3.96 (dd, J2,3 = 10.0 Hz,
J3,4 = 2.6 Hz, 1H, H-3), 3.85 (dd, J6a,6b = 15.2 Hz, J5,6b = 2.5 Hz, H1,
H-6b), 2.93 (s, 6H, NMe2), 2.18 (s, 3H, NAc), 2.15 (s, 3H, OAc); 13C
NMR (125 MHz, CDCl3) d 170.9 (C@O NAc), 169.7 (C@O OAc),
138.4 (C-i, 3-O-Bn), 138.3 (C-i, 4-O-Bn), 137.9 (C-i, 2-O-Bn), 134.7
(C-4a Dan), 131.2 (C-2 Dan), 129.2 (C-1/C-8a Dan), 128.53,
128.50, 128.45, 128.42, 128.40, 128.38, 128.01, 127.94, 127.89,
127.86, 127.82, 127.74, 127.72, 127.61, 127.5 (15 ꢂ CHarom),
123.5 (C-3 Dan), 115.5 (C-6 Dan), 90.2 (C-1), 78.9 (C-3), 75.2 (C-
2), 74.9 (C-4), 74.6 (CH2, 4-O-Bn), 73.5 (CH2, 3-O-Bn), 73.4 (CH2,
2-O-Bn), 72.1 (C-5), 47.7 (C-6), 45.5 (NMe2), 24.7 (NAc), 21.2
(OAc), HRMS(ESI) m/z calcd for [C43H46N2O9S+Na]+: 789.2822,
obsd.: 789.2819.
4.9. Methyl 2,3,4-tri-O-benzyl-6-deoxy-6-
[N-(5-[dimethylamino]napth-1-ylsulfonyl)
‘amido]-a-D-galactopyranoside (14)
Trimethylphosphine (1 M in THF) (10.2 mL, 10.2 mmol) was
added to a solution of 13 (1.0 g, 2.04 mmol) (co-evaporated with
toluene ꢂ 3) in distilled THF (10 mL) at 0 °C. After stirring for
15 min at 0 °C and 45 min at rt, the reaction mixture was cooled
to 0 °C and dansyl chloride (1.38 g, 5.11 mmol) was added followed
by the addition of NaOH (1 M, 8.17 mL, 8.17 mmol) over 10 min.
The reaction was then allowed to warm to rt and after 6 h, further
dansyl chloride (0.83 g, 3.06 mmol) and NaOH (1 M, 2.04 mL,
2.04 mmol) were added. After stirring for a further 16 h, the solu-
tion was diluted with EtOAc (50 mL), and the organic layer washed
with water (50 mL) and brine (50 mL), dried (MgSO4), filtered and
concentrated in vacuo. The residue was purified by silica gel flash
column chromatography and eluted with 1:1 (v/v) petroleum
ether/EtOAc to afford the title compound 14 (1.32 g, 1.89 mmol,
93%) as a lime green foam. Rf: 0.61 (PE/EA, 1/1, v/v); Glows white
4.11. 2-Azido-3,4-O-isopropylidene-1-O-(2,3,4-tri-O-benzyl-6-
deoxy-6-[N-(5-[dimethylamino]napth-1-ylsulfonyl)acetimido]-
a-D
-galactopyranosyl)-D-ribo-octadecane-1,3,4-triol (4)
on TLC (k = 254 nm); ½a D19
¼ þ9:2 (c 1.0, CHCl3); IR (film) 3293,
ꢃ
3063, 3030, 2988, 2349, 1588, 1574, 1497, 1326, 1200, 1143,
Iodotrimethylsilane (65.3
lL, 0.46 mmol) was added to a solu-
791, 735, 698 cmꢀ1; 1H NMR (500 MHz, CDCl3) d 8.55 (d, JCH-2,CH-3
8.8 Hz, 1H, CH-2 Dan), 8.13 (d, JCH-3,CH-4 = 7.4 Hz, 1H, CH-4 Dan),
=
tion of 15 (237 mg, 0.31 mmol) (co-evaporated with toluene ꢂ 3)
in dry CH2Cl2 (2 mL) at 0 °C. After stirring for 1 h at 0 °C, the reac-
tion mixture was concentrated in vacuo, azeotroped with toluene
(ꢂ3) and redissolved in dry benzene (3 mL). In a separate flask,
molecular sieves (4 Å, 50 mg), TBAI (342 mg, 0.93 mmol) and Di-
8.08 (d, JCH-7,CH-8 = 8.7 Hz, 1H, CH-8 Dan), 7.54 (dd, JCH-7,CH-8
8.7 Hz, JCH-6,CH-7 = 7.7 Hz, 1H, CH-7 Dan), 7.50 (d, JCH-2,CH-3
=
=
8.8 Hz, 1H, CH-8 Dan), 7.38–7.26 (m, 15H, Harom), 7.20–7.17 (m,
3H, Harom, CH-6 Dan), 4.84 (d, Ja,b = 11.7 Hz, 1H, CH-a, 4-O-Bn),
4.77 (d, Ja,b = 12.0 Hz, 1H, CH-a, 2-O-Bn), 4.76 (d, Ja,b = 11.5 Hz,
1H, CH-a, 3-O-Bn), 4.61 (d, Ja,b = 12.0 Hz, 2H, CH-b, 2-O-Bn, CH-b,
3-O-Bn), 4.47 (d, J1,2 = 3.8 Hz, 1H, H-1), 4.29 (dd, JNH,6a = 8.4 Hz,
JNH,6b = 4.6 Hz, 1H, NH Dan), 3.86 (dd, J1,2 = 3.8 Hz, J2,3 = 9.4 Hz,1H,
H-2), 3.65 (dd, J2,3 = 9.4 Hz, J3,4 = 2.7 Hz, 1H, H-3), 3.64 (br s, 1H,
H-4), 3.48 (t, J5,6 = 6.6 Hz, 1H, H-5), 3.23 (s, 3H, OCH3), 2.99–2.77
(m, 2H, H-6a, H-6b), 2.88 (s, 6H, NMe2); 13C NMR (125 MHz, CDCl3)
d 138.5, 138.3 (2 ꢂ C-i, 2-O-Bn, 3-O-Bn), 137.9 (C-i, 4-O-Bn), 134.9
(C-4a Dan), 130.4 (C-2 Dan), 129.5, 128.9, 128.6, 128.5, 128.4,
128.3, 128.1, 127.8, 127.7, 127.6 (10 ꢂ CHarom), 123.4 (C-3 Dan),
118.9 (C-8 Dan), 115.4 (C-6 Dan), 98.7 (C-1), 79.0 (C-3), 76.1 (C-
2), 74.1 (CH2, 4-O-Bn), 73.7 (C-4), 73.63, 73.59 (2 ꢂ CH2, 2-O-Bn,
3-O-Bn), 68.5 (C-5), 55.4 (OCH3), 45.5 (NMe2), 43.3 (C-6); HRMS(E-
SI) m/z calcd for [C40H44N2O7S+Na]+: 719.2756, obsd.: 719.2760.
PEA (53.8 lL, 0.31 mmol) were added to a solution of the lipid
acceptor 6 (39.1 mg, 0.10 mmol) (co-evaporated from toluene ꢂ 3)
in dry benzene (1 mL) at room temperature. After stirring the reac-
tion mixture at 70 °C for 15 min, the glycosyl iodide was cannulat-
ed into the flask containing the lipid acceptor and stirred at 70 °C
for 20 h. The reaction mixture was then diluted with EtOAc
(10 mL), cooled to 0 °C and filtered. The filtrate was concentrated
and the resulting residue purified by silica gel flash column chro-
matography. Elution with 7:1 (v/v) hexanes/EtOAc afforded the de-
sired glycolipid 4 (103.9 mg, 0.095 mmol, 94%) as a lime green oil.
Rf: 0.40 (PE/EA, 2/1, v/v); Glows mauve on TLC (k = 254 nm);
½
a 2D4
ꢃ
¼ þ40:9 (c 1.0, CHCl3); IR (film) 3064, 3031, 2923, 2853,
2789, 2099, 1704, 1572, 1455, 1347, 1231, 1147, 1096, 1045,
787, 735, 697 cmꢀ1; 1H NMR (500 MHz, CDCl3) d 8.58 (d, JCH-2,CH-3
8.6 Hz, 1H, CH-2 Dan), 8.29 (d, JCH-3,CH-4 = 7.3 Hz, 1H, CH-4 Dan),
7.75 (d, JCH-7,CH-8 = 8.6 Hz, 1H, CH-8 Dan), 7.53 (t, JCH-2,CH-3
=
=
4.10. 1-O-Acetyl-2,3,4-tri-O-benzyl-6-deoxy-6-[N-(5-
JCH-7,CH-8 = 8.6 Hz, 2H, CH-3, CH-7 Dan), 7.43–7.2.8 (m, 15H, Harom),
7.19 (d, JCH-6,CH-7 = 7.5 Hz, 1H, CH-6 Dan), 5.11 (d, Ja,b = 11.7 Hz, 1H,
CH-a, 40-O-Bn), 5.00 (d, J1,2 = 3.2 Hz, 1H, H-1), 4.91 (d, Ja,b = 11.7 Hz,
1H, CH-a, 20-O-Bn), 4.80 (d, Ja,b = 12.0 Hz, 1H, CH-a, 30-O-Bn), 4.79
(d, Ja,b = 11.7 Hz, 1H, CH-b, 20-O-Bn), 4.74 (d, Ja,b = 12.0 Hz, 1H,
CH-b, 30-O-Bn), 4.68 (d, Ja,b = 11.7 Hz, 1H, CH-b, 40-O-Bn), 4.25–
4.18 (m, 3H, H-1a, H-50, H-3), 4.15–4.05 (m, 4H, H-60a, H-4, H-30,
[dimethylamino]napth-1-ylsulfonyl)acetimido]-
a-D-
galactopyranoside (15)
Concentrated sulfuric acid (63.1
lL, 1.18 mmol) was added
dropwise to solution of 14 (550 mg, 0.79 mmol) in Ac2O
a
(2.4 mL, 25.3 mmol) and acetic acid (0.32 mL, 5.53 mmol) at 0 °C.
After stirring at 0 °C for 1 h and at rt for 2 h, the solution was
diluted with CH2Cl2 (50 mL) and washed with water (50 mL),
saturated NaHCO3 solution (50 mL) and brine (100 mL), dried
H-20), 4.00 (br s, 1H, H-40), 3.85 (dd, J6a ,6b = 14.8 Hz, J5,6b = 2.9 Hz,
1H, H-60b), 3.74 (dd, J1a,1b = 10.8 Hz, J1b,2 = 5.2 Hz, 1H, H-1b), 3.41
(ddd, J2,3 = 9.5 Hz, J1a,2 = 5.2 Hz, J1b,2 = 2.5 Hz, 1H, H-2), 2.89 (s,
0
0
0

J. M. H. Cheng et al. / Carbohydrate Research 346 (2011) 914–926
923
6H, NMe2), 2.18 (s, 3H, NAc), 1.67–1.54 (m, 4H, H-5, H-6), 1.43 (s,
4.13. 1-O-(2,3,4-Tri-O-benzyl-6-deoxy-6-[N-(5-[dimethyl-
amino]napth-1-ylsulfonyl)amido]- -galactopyranosyl)-2-
hexacosanoylamido-3,4-O-isopropylidene-D-ribo-octade-
3H, CH3 iPr), 1.30 (s, 3H, CH3 iPr), 1.41–1.27 (m, 22H, H-7–H-17),
0.89 (t, J17,18 = 7.0 Hz, 3H, H-18); 13C NMR (125 MHz, CDCl3) d
170.8 (C@O NAc), 152.2 (C-5 Dan), 138.9 (C-i, 30-O-Bn), 138.8 (C-
i, 20-O-Bn), 138.6 (C-i, 40-O-Bn), 134.5 (C-4a Dan), 131.5 (C-2
Dan), 131.4 (C-4 Dan), 129.9, (C-1 Dan), 129.6 (C-8a Dan), 128.7
(C-7 Dan), 128.43, 128.40, 128.37, 128.2, 127.7, 127.6, 127.53,
127.52, 127.43 (9 ꢂ CHarom), 123.1 (C-3 Dan), 117.8 (C-8 Dan),
115.2 (C-6 Dan), 108.1 (Cq iPr), 98.5 (C-10), 78.8 (C-20), 77.8 (C-4),
76.5 (C-30), 75.5 (C-40), 75.0 (C-3), 74.5 (CH2, 40-O-Bn), 73.7 (CH2,
20-O-Bn), 72.7 (CH2, 30-O-Bn), 70.0 (C-50), 69.2 (C-1), 59.3 (C-2),
47.8 (C-60), 45.4 (NMe2), 31.9, 29.71, 29.68, 29.67, 29.66, 29.64,
29.61, 29.4, 26.6, 22.7 (C-5–C-17), 28.3, 25.8 (2 ꢂ CH3 iPr), 24.8
(NAc), 14.1 (C-18); HRMS(ESI) m/z calcd for [C62H83N5O10S+Na]+:
1112.5758, obsd.: 1112.5767.
a-D
cane-1,3,4-triol (17)
Trimethylphosphine (1 M in THF) (0.17 mL, 0.17 mmol) was
added to a solution of 16 (34.7 mg, 0.033 mmol) (co-evaporated
with toluene ꢂ 3) in distilled THF (0.2 mL) at 0 °C. After stirring
the reaction mixture for 15 min at 0 °C and 45 min at rt, the
solution was cooled to 0 °C and 1 M NaOH (aq) solution
(0.33 mL, 0.33 mmol) added drop wise. The reaction mixture
was then stirred at rt for 21 h, then diluted with EtOAc
(20 mL), washed with water (2 ꢂ 20 mL) and brine (20 mL),
dried (MgSO4) and filtered. Concentration in vacuo afforded
the amine [Rf: 0.11 (toluene/EtOAc, 3/2, v/v)] as a green oil,
which was used without further purification. A solution of hex-
acosanoic acid (33.5 mg, 0.084 mmol), EDCI (16.2 mg,
0.084 mmol), and DMAP (0.4 mg, 0.0034 mmol) in dry CH2Cl2
(1 mL) was added to the amine dissolved in dry CH2Cl2
(1 mL). After stirring at room temperature for 43 h, the reaction
mixture was diluted with EtOAc (20 mL) and washed with sat-
urated NaHCO3 solution (20 mL), water (20 mL) and brine
(20 mL), dried (MgSO4) and filtered. The filtrate was concen-
trated in vacuo and the residue was purified by silica gel col-
umn chromatography. Elution with 5:1 (v/v) hexanes/EtOAc
afforded the desired product 17 (31.9 mg, 0.023 mmol, 68%) as
a lime green oil. Rf: 0.79 (toluene/EtOAc, 3/2, v/v); Glows white
4.12. 2-Azido-3,4-O-isopropylidene-1-O-(2,3,4-tri-O-benzyl-6-
deoxy-6-[N-(5-[dimethylamino]napth-1-ylsulfonyl)amido]-
a-D-
galactopyranosyl)- -ribo-octadecane-1,3,4-triol (16)
D
Glycolipid 4 (42.5 mg, 0.039 mmol) was dissolved in a mixture
of MeOH/CH2Cl2 (3:1, v/v) and to this was added NaOMe until the
solution reached pH 9.0. After stirring at room temperature for 3 d
in the dark, while maintaining pH 9.0 via the addition of NaOMe.
The solution was then diluted with EtOAc (15 mL), and washed
with saturated NH4Cl solution (20 mL), water (20 mL) and brine
(20 mL). The organic layer was dried (MgSO4), filtered and con-
centrated in vacuo to afford the title compound 16 (41 mg, quant.)
as a lime green oil, which was used without further purification.
Rf: 0.40 (PE/EA, 2/1, v/v); Glows white on TLC (k = 254 nm);
on TLC (k = 254 nm); ½a D22
¼ þ4:3 (c 1.0, CHCl3); IR (film) 2923,
ꢃ
2853, 2361, 1650, 1541, 1455, 1330, 1218, 1145, 1092, 1052,
791, 697 cmꢀ1
7.4 Hz, 1H, CH-2 Dan), 8.19 (br s, 1H, CH-4 Dan), 8.16 (d,
JCH-7,CH-8 = 7.3 Hz, 1H, CH-8 Dan), 7.56 (t, JCH-6,CH-7 = JCH-7,CH-8
8.1 Hz, 1H, CH-7 Dan), 7.52 (t, JCH-2,CH-3 = JCH-3,CH-4 = 8.0 Hz, 1H,
CH-3 Dan), 7.41–7.23 (m, 15H, arom), 7.20 (d, JCH-6,CH-7
; =
1H NMR (500 MHz, CDCl3) d 8.55 (d, JCH-2,CH-3
½
a 2D1
ꢃ
¼ þ10:5 (c 1.0, CHCl3); IR (film) 3309, 3031, 2923, 2853,
2360, 2099, 1574, 1454, 1328, 1220, 1145, 1094, 1047, 791, 734,
697 cmꢀ1 1H NMR (500 MHz, CDCl3)
8.55 (d, JCH-2,CH-3
=
;
d
Dan = 8.4 Hz, 1H, CH-2 Dan), 8.17 (d, JCH-3,CH-4 Dan = 7.3 Hz, 1H,
CH-4 Dan), 8.09 (d, JCH-7,CH-8 Dan = 8.7 Hz, 1H, CH-8 Dan), 7.56
(dd, JCH-7,CH-8 Dan = 8.7 Hz, JCH-6,CH-7 Dan = 7.6 Hz, 1H, CH-7 Dan),
7.53 (dd, JCH-2,CH-3 Dan = 8.4 Hz, JCH-3,CH-4 Dan = 7.3 Hz, 1H, CH-3
Dan), 7.38–7.26 (m, 13H, Harom), 7.20–7.18 (m, 3H, Harom, CH-6
Dan), 4.86 (d, Ja,b = 11.8 Hz, 1H, CH-a, 40-O-Bn), 4.79 (d,
Ja,b = 12.0 Hz, 1H, CH-a, 30-O-Bn), 4.77 (d, J1,2 = 3.6 Hz, 1H, H-10),
4.75 (d, Ja,b = 11.9 Hz, 1H, CH-a, 2-O-Bn), 4.66 (d, Ja,b = 11.9 Hz,
1H, CH-b, 2-O-Bn), 4.63 (d, Ja,b = 12.0 Hz, 1H, CH-b, 3-O-Bn), 4.53
H
=
7.1 Hz, 1H, CH-6 Dan), 5.89 (d, JNH,2 = 9.5 Hz, 1H, NH C26),
4.92 (br s, 1H, NH Dan), 4.88 (d, Ja,b = 11.5 Hz, 1H, CH-a, 400-O-
Bn), 4.89 (d, J1,2 = 3.6 Hz, 1H, H-100), 4.79 (d, Ja,b = 11.4 Hz, 1H,
CH-a, 200-O-Bn), 4.76 (d, Ja,b = 11.7 Hz, 1H, CH-a, 300-O-Bn), 4.71
(d, Ja,b = 11.7 Hz, 1H, CH-b, 300-O-Bn), 4.64 (d, Ja,b = 11.4 Hz, 1H,
CH-b, 200-O-Bn), 4.53 (d, Ja,b = 11.5 Hz, 1H, CH-b, 400-O-Bn),
4.18–4.14 (m, 1H, H-2), 4.07 (t, J2,3 = J3,4 = 6.9 Hz, 1H, H-3),
00 00
00 00
4.04–4.01 (m, 1H, H-4), 3.96 (dd, J2 ,3 = 10.0 Hz, J1 ,2 = 3.6 Hz,
(d, Ja,b = 11.8 Hz, 1H, CH-b, 4-O-Bn), 4.34 (dd, J6 b, NH = 8.3 Hz, J6 a,
NH = 4.5 Hz, 1H, NH), 4.13 (p, J4,5 = 4.2 Hz, 1H, H-4), 4.04 (dd,
1H, H-200), 3.85 (dd, J1a,1b = 11.6 Hz, J1a,2 = 4.6 Hz, 1H, H-1a),
0
0
3.82 (br s, 1H, H-400), 3.77 (dd, J2 ,3 = 10.0 Hz, J3 ,4 = 2.7 Hz,
00 00
00 00
1H, H-300), 3.71 (t, J5 ,6 a = 6.6 Hz, 1H, H-500), 3.59 (dd,
00 00
J2,3 = 9.8 Hz, J3,4 = 5.4 Hz, 1H, H-3), 3.95 (dd, J1a,1b = 10.6 Hz, J1a, 2
=
2.5 Hz, 1H, H-1a), 3.91 (dd, J2 ,3 = 10.1 Hz, J1,2 = 3.6 Hz, 1H, H-20),
J1a,1b = 11.6 Hz, J1b,2 = 2.6 Hz, 1H, H-1b), 2.90 (s, 6H, NMe2),
0
0
3.79 (dd, J2 ,3 = 10.1 Hz, J3 ,4 = 2.7 Hz, 1H, H-30), 3.70 (br s, 1H, H-
40), 3.69–3.68 (m, 1H, H-50), 3.61 (dd, J1a,1b = 10.6 Hz,
J1b,2 = 6.4 Hz, 1H, H-1b), 3.38 (ddd, J2,3 = 9.8 Hz, J1b,2 = 6.4 Hz,
2.87–2.85 (m, 2H, H-600a, H-600b), 2.14 (dt, J2´ a,2b = 14.7 Hz,
0
0
0
0
´
J´ = 7.5 Hz, 1H, H-20a), 2.06 (dt, J´
= 14.7 Hz, J2´ a,3 = 7.5 Hz,
´
2a,2b
´
´
2a,3
1H, H-20b), 1.71 (br s, 2H, CH2, H-5), 1.59 (m, 2H, H-30), 1.47–
J1a,2 = 2.5 Hz, 1H, H-2), 2.92–2.81 (dd, J5,6 a = 7.1 Hz, J6 a,NH = 4.5 Hz,
1.43 (m, 68H, CH2, H-6–H-17, H-40–H-250), 1.43 (s, 3H, CH3
0
0
H-60a),
2.83
(ddd,
J6 a,6 b = 13.6 Hz,
J6 b,NH = 8.3 Hz,
iPr), 1.32 (s, 3H, CH3 iPr), 0.89 (t, J17,18 = J25,26 = 7.0 Hz, 6H, H-
´
0
0
0
1H,
´
J5,6 b = 6.1 Hz, 1H, H-60b), 2.89 (s, 6H, NMe2), 1.64–1.27 (m, 26H,
H-5–H-17), 1.45 (s, 3H, CH3 iPr), 1.31 (s, 3H, CH3 iPr), 0.89 (t,
J17,18 = 7.0 Hz, 3H, H-18); 13C NMR (125 MHz, CDCl3) d 152.1 (C-
5 Dan), 138.7, 138.6 (2 ꢂ C-i, 20-O-Bn, 30-O-Bn), 138.0 (C-i, 40-O-
Bn), 134.7 (C-4a Dan), 130.5 (C-2 Dan), 129.9 (C-1/C-8a Dan),
129.6 (C-4 Dan), 129.5 (C-1/C-8a Dan), 128.9, 128.6, 128.4
(3 ꢂ CHarom) 128.30 (C-7 Dan), 128.25, 128.22, 127.64, 127.63,
127.54, 127.48 (6 ꢂ CHarom), 123.1 (C-3 Dan), 118.8 (C-8 Dan),
115.2 (C-6 Dan), 108.3 (Cq iPr), 98.5 (C-10), 78.6 (C-30), 77.8 (C-
4), 76.3 (C-20), 75.2 (C-3), 74.1 (CH2, 40-O-Bn), 73.8 (C-40), 73.7
(CH2, 30-O-Bn), 72.9 (CH2, 20-O-Bn), 69.5 (C-1), 69.2 (C-50), 59.5
(C-2), 45.4 (NMe2), 43.3 (C-60), 31.9, 29.72, 29.69, 29.68, 29.64,
29.61, 29.4, 29.3, 26.6, 22.7 (C-5–C-17), 28.3, 25.7 (2 ꢂ CH3 iPr),
14.1 (C-18); HRMS(ESI) m/z calcd for [C60H81N5O9S+Na]+:
1070.5653, obsd.: 1070.5657.
18, H-260); 13C NMR (125 MHz, CDCl3) d 172.6 (C@O), 151.8
(C-5 Dan), 138.4 (C-i, 300-O-Bn), 138.2 (C-i, 200-O-Bn), 138.1 (C-
i, 400-O-Bn), 134.7 (C-4a/C-8a Dan), 130.3 (C-2 Dan), 129.8 (C-
4a/C-8a Dan), 129.6 (C-8 Dan), 128.8, 128.72, 128.66, 128.60,
128.56, 128.5, 128.4, 128.15, 128.1, 128.0, 127.94, 127.88,
127.7, 127.6, 127.5 (15 ꢂ CHarom), 128.21 (C-7 Dan), 123.3 (C-3
Dan), 119.1 (C-1 Dan), 115.2 (C-6 Dan), 107.8 (Cq iPr), 99.3
(C-100), 79.1 (C-300), 77.7 (C-4), 76.6 (C-3), 76.5 (C-200), 74.2
(CH2, 400-O-Bn), 73.7 (CH2, 200-O-Bn), 73.4 (C-400), 73.0 (CH2, 300-
O-Bn), 69.4 (C-1), 69.1 (C-500), 49.1 (C-2), 45.5 (NMe2), 43.3
(C-600), 36.9 (C-20), 31.95, 31.94, 31.6, 29.8, 29.73, 29.71, 29.70,
29.67, 29.65, 29.50, 29.47, 29.42, 29.39, 29.37, 28.83, 26.8,
22.7 (C-5–C-17, C-30–C-250), 27.8, 25.73 (2 ꢂ CH3 iPr), 25.72
(CH2, C-300), 14.1 (2 ꢂ CH3, C-18, C-260); HRMS(ESI) m/z calcd
for [C86H133N3O10S+Na]+: 1422.9609, obsd.: 1422.9615.
0

924
J. M. H. Cheng et al. / Carbohydrate Research 346 (2011) 914–926
4.14. 1-O-(2,3,4-Tri-O-benzyl-6-deoxy-6-[N-(5-[dimethyl-
amino]napth-1-ylsulfonyl)amido]- -galactopyranosyl)-2-
hexacosanoylamido- -ribo-octadecane-1,3,4-triol (18)
MeOH/CH2Cl2. This afforded the target compound 3 (4.9 mg,
4.49 mmol, 62%) as a yellow oil. Rf: 0.22 (CH2Cl2/MeOH, 92/8, v/
a
-D
D
v); Glows white on TLC (k = 366 nm); ½a D22
¼ þ40:0 (c 0.1, pyri-
ꢃ
dine); IR (film) 3320, 2918, 2851, 2359, 1653, 1634, 1560, 1457,
Glycolipid 17 (31.9 mg, 0.023 mmol) was dissolved in a mixture
of 8:4:1 (v/v/v) AcOH/H2O/toluene and stirred at 50 °C for 1 d after
which analysis by TLC revealed the presence of the starting mate-
rial. The reaction mixture was concentrated in vacuo (to remove
the acetone by product), then redissolved in the same mixture of
solvents and stirred for a further day. This procedure was repeated
until all starting material was converted to product via TLC analy-
sis. The reaction mixture was then concentrated in vacuo and the
residue was purified by silica gel flash column chromatography.
Elution with 1:2 (v/v) hexanes/EtOAc afforded the diol 18
(23.0 mg, 0.017 mmol, 74%) as a lime green oil. Rf: 0.22 (PE/EA, 3/
1310, 1144, 1030, 791, 684 cmꢀ1; UV kmaxAbs = 343 nm, kmax-
Em = 510 nm; 1H NMR (600 MHz, pyridine-d5) d 9.04 (d, JCH-2,CH-3
=
8.7 Hz, 1H, CH-2 Dan), 8.61–8.57 (m, 2H, NH C26, CH-4 Dan), 8.54
(d, JCH-7,CH-8 = 8.6 Hz, 1H, CH-8 Dan), 7.54 (dd, JCH-7,CH-8 = 8.6 Hz,
JCH-6,CH-7 = 7.4 Hz, 1H, CH-7 Dan), 7.51 (dd, JCH-2,CH-3 = 8.7 Hz,
JCH-3,CH-4 = 7.6 Hz, 1H, CH-3 Dan), 7.12 (d, JCH-6,CH-7 = 7.4 Hz, 1H,
CH-6 Dan), 5.45 (d, J1 ,2 = 3.9 Hz, 1H, H-100), 5.23 (m, 1H, H-2),
00 00
4.54 (dd, J1a,1b = 10.9 Hz, J1a,2 = 5.6 Hz, 1H, H-1a), 4.52 (dd,
J2 ,3 = 9.9 Hz, J1 ,2 = 3.9 Hz, 1H, H-200), 4.51 (t, J5,6a = J5,6b = 6.6 Hz,
00 00
00 00
1H, H-500), 4.35–4.30 (m, 3H, H-3, H-4, H-400), 4.26 (dd,
J2 ,3 = 9.9 Hz, J3 ,4 = 3.3 Hz, 1H, H-300), 4.19 (dd, J1a,1b = 10.9 Hz,
J1b,2 = 4.8 Hz, 1H, H-1b), 3.85 (br s, 2H, H-600a, H-600b), 2.50 (dt,
J2´ a,2b = 14.8 Hz, J´ = 7.8 Hz, 1H, H-20a), 2.46 (dt, J2´a,2b = 14.8 Hz,
00 00
00 00
2, v/v); Glows white on TLC (k = 254 nm); ½a D21
¼ þ13:7 (c 1.0,
ꢃ
CHCl3); IR (film) 3357, 3032, 2922, 2852, 2360, 1643, 1536, 1498,
´
´
´
2a,3
1312, 1143, 1092, 1050, 909, 789, 733, 697 cmꢀ1
;
1H NMR
J2b,3
´
´
= 7.8 Hz, 1H, H-20b), 2.34–1.24 (m, 72H, CH2, H-5–H-17, H-
(500 MHz, CDCl3) d 8.54 (d, JCH-2,CH-3 = 8.5 Hz, 1H, CH-2 Dan),
30–H-250), 0.86 (t, J17,18 = J25,26 = 7.0 Hz, 6H, H-18, H-260); 13C
´
´
8.10 (d, JCH-3,CH-4 = 7.4 Hz, 1H, CH-4 Dan), 8.07 (d, JCH-7,CH-8
8.6 Hz, 1H, CH-8 Dan), 7.56 (dd, JCH-7,CH-8 = 8.6 Hz, JCH-6,CH-7
7.8 Hz, 1H, CH-7 Dan), 7.51 (dd, JCH-2,CH-3 = 8.5 Hz, JCH-3,CH-4
=
=
=
NMR (150 MHz, pyridine-d5) d 173.7 (C@O), 152.4 (C-5 Dan),
137.8 (C-4a Dan), 130.8 (C-1 Dan), 130.6 (C-8a Dan), 130.3 (C-8
Dan), 129.3 (C-4 Dan), 128.5 (C-3 Dan), 123.5 (C-7 Dan), 120.7
(C-2 Dan), 115.9 (C-6 Dan), 101.4 (C-100), 76.8 (C-3), 72.8 (C-4),
71.42 (C-500), 71.37 (C-300), 71.2 (C-400), 70.2 (C-200), 68.5 (C-1),
51.5 (C-2), 45.52 (NMe2), 45.16 (C-600), 37.1 (C-20), 34.6 (C-5),
32.42, 32.41, 30.8, 30.7, 30.5, 30.4, 30.34, 30.33, 30.32, 30.31,
30.29, 303.27, 30.26, 30.23, 30.21, 30.19, 30.14, 30.10, 29.92,
29.90, 26.8, 26.7, 23.24, 23.23 (C-6–C-17, C-30–C-250), 14.6
(2 ꢂ CH3, C-18, C-260); HRMS(ESI) m/z calcd for [C62H111N3O10S+
Na]+: 1112.7888, obsd.: 1112.7878.
7.4 Hz, 1H, CH-3 Dan), 7.41–7.27 (m, 13H, Harom), 7.22–7.18 (m,
3H, Harom, CH-6 Dan), 6.31 (d, JNH,2 = 8.3 Hz, 1H, NH C26), 4.88 (d,
Ja,b = 11.7 Hz, 1H, CH-a, 400-O-Bn), 4.83 (d, Ja,b = 11.7 Hz, 1H, CH-a,
200-O-Bn), 4.81 (d, Ja,b = 11.2 Hz, 1H, CH-a, 300-O-Bn), 4.75 (d,
Ja,b = 11.2 Hz, 1H, CH-b, 30-O-Bn), 4.74 (d, J1 ,2 = 3.7 Hz, 1H, H-100),
00 00
4.66 (d, Ja,b = 11.7 Hz, 1H, CH-b, 200-O-Bn), 4.53 (d, Ja,b = 11.7 Hz,
1H, CH-b, 400-O-Bn), 4.51–4.49 (m, 1H, NH Dan), 4.30 (dd,
00 00
J2,3 = 8.1 Hz, J1a/b,2 = 3.7 Hz, 1H, H-2), 3.95 (dd, J2 ,3 = 10.0 Hz,
J1 ,2 = 3.7 Hz, 1H, H-200), 3.87–3.84 (m, 3H, H-1a, H-400, H-500), 3.80
00 00
(dd, J2 ,3 = 10.0 Hz, J3 ,4 = 2.4 Hz, 1H, H-300), 3.76 (dd,
J1a,1b = 10.5 Hz, J1b,2 = 4.1 Hz, 1H, H-1b), 3.65–3.64 (m, 1H, 3-OH),
3.54–3.51 (m, 2H, H-3, H-4), 2.89 (s, 6H, NMe2), 2.83 (ddd,
00 00
00 00
4.16. Materials and methods for cell proliferation (IL-2) assay,
in vivo DC maturation assay and in vitro treatment of dendritic
cells
J6 a,6 b = 14.1 Hz, J5,6 a = 7.1 Hz, J6 a,NH = 4.4 Hz, 1H, H-600a), 2.77
00
00
00
00
(ddd, J6 a,6 b = 14.1 Hz, J6 b,NH = 8.1 Hz, J5,6 b = 6.1 Hz, 1H, H-600b),
2.56–2.55 (m, 1H, 4-OH), 2.25 (dt, J2´a,2b = 14.9 Hz, J2´ a,3 = 7.5 Hz,
00
00
00
00
4.16.1. Mice
´
´
Breeding pairs of the inbred strains C57BL/6 were obtained
from The Jackson Laboratories and from the Animal Resource Cen-
tre. All mice were maintained in the Biomedical Research Unit of
the Malaghan Institute of Medical Research. CD1dꢀ/ꢀ mice, which
are devoid of CD1d-restricted iNKT cells, were also used.62 The
experiments were approved by the NZ national animal ethics com-
mittee and performed according to established national guidelines.
1H, H-20a), 2.19 (dt, J2´ a,2b = 14.9 Hz, J2´ b,3 = 7.5 Hz, 1H, H-20b),
´
´
1.67–1.15 (m, 74H, CH2, H-5–H-17, H-30–H-250), 0.89 (t,
J17,18 = J25,26 = 7.0 Hz, 6H, H-18, H-260); 13C NMR (125 MHz, CDCl3)
´
´
d 173.5 (C@O), 152.0 (C-5 Dan), 138.2 (C-i, 300-O-Bn), 137.9 (C-i, 400-
O-Bn), 137.8 (C-i, 200-O-Bn), 134.1 (C-4a Dan), 130.6 (C-2 Dan),
129.9 (C-1 Dan), 129.6 (C-4 Dan), 129.5 (C-8a Dan), 128.9, 128.6,
128.54, 128.52, 128.2, 128.05, 128.00, 127.8, 127.5 (9 ꢂ CHarom),
128.4 (C-7 Dan), 123.1 (C-3 Dan), 118.8 (C-8 Dan), 115.2 (C-6
Dan), 98.8 (C-100), 79.4 (C-300), 76.3 (C-3), 75.9 (C-200), 74.2 (CH2,
400-O-Bn), 74.0 (CH2, 200-O-Bn), 73.3 (CH2, 300-O-Bn), 73.2 (C-4),
73.1 (C-400), 69.6 (C-500), 69.2 (C-1), 49.9 (C-2), 45.4 (NMe2), 43.2
(C-600), 36.8 (C-20), 33.5 (C-5), 31.9, 29.73, 29.71, 29.68, 29.67,
29.6, 29.44, 29.38, 25.9, 25.7, 22.7 (C-6–C-17, C-30–C-250), 14.1
(2 ꢂ CH3, C-18, C-260); HRMS(ESI) m/z calcd for [C83H129N3O10S+
Na]+: 1382.9296, obsd.: 1382.9296.
4.16.2. Solubilisation of glycolipid
a-GalCer (Industrial Research Ltd, New Zealand) and synthes-
ised glycolipids were tested to be endotoxin-free at the sensitivity
of 0.125 EU/mL with an endotoxin kit (Pyrotell, Limulus Amebo-
cyte Lysate). Each glycolipid (1 mg) was dissolved in 200 lL of
CHCl3/MeOH/H2O (10:10:3), heated at 37 °C for 15 min followed
by sonication for 10 min. The solution was then diluted to
200 lg/mL in 0.5% Tween/phosphate-buffered saline (PBS) and left
to sit at ꢀ4 °C overnight. The glycolipid was then heated at 80 °C
for 5 min followed by sonication for 5 min then cooled at 0 °C for
5 min (ꢂ2) and left to sit at ꢀ4 °C overnight.
4.15. 1-O-[6-Deoxy-6-[N-(5-[dimethylamino]napth-1-ylsulfo-
nyl)amido]-a-D-galactopyranosyl]-2-hexacosanoylamido-D-ribo-
octadecane-1,3,4-triol (3)
4.16.3. Cell proliferation (IL-2) assay
Pd/(OH)2 (2.5 mol %) was added to a solution of diol 18 (9.9 mg,
0.0073 mmol) in a mixture of CHCl3/EtOH (1 mL, 3/2, v/v) and the
reaction stirred under H2 (g) for 17 h at rt. The reaction mixture
was then filtered through Celite, the Celite was washed thoroughly
with CHCl3/EtOH (3/2, v/v), and the filtrate was concentrated in
vacuo. The residue was purified by silica gel flash column
chromatography (10% MeOH/CH2Cl2), followed by reverse phase
chromatography (ODS-C18 resin, product eluted with MeOH),
and finally silica gel column chromatography by elution with 5%
Dendritic cells (DC2114;55 2.5 ꢂ 104 cells) and murine NKT
hybridoma cells (DN32.D3;4 1 ꢂ 105 cells) were incubated with
various concentration of glycolipid (a-GalCer 2: 583–0.569 nM;
Dansyl
a-GalCer 3: 458–0.447 nM in twofold dilution) in 200 lL
of complete medium consisting of Iscove’s Modified Dulbecco’s
Medium (IMDM) supplemented with 2 mM glutamine (Invitro-
gen), 1% penicillin–streptomycin (Invitrogen), 5 ꢂ 10ꢀ5
M
2-mercaptoethanol (Invitrogen) and 5% foetal bovine serum

J. M. H. Cheng et al. / Carbohydrate Research 346 (2011) 914–926
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to 5 ꢂ 103 HT-2 cells in a flat-bottom 96-well plate, incubated at
37 °C for 24 h and incorporation of [3H]thymidine over the final
6 h was measured with a beta-counter (Wallac). A standard curve
was constructed by incubating HT-2 cells with known concentra-
tions of IL-2 (200–0 U/mL in fivefold dilution). Each sample was
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4.16.4. Antigen presenting cell maturation assay
Glycolipid diluted to 2 lg/mL and 200 ng (in 100 lL) was
administered iv to groups of C57BL/6 mice (n = 3) and spleens were
removed 20 h later. Spleens were teased through a cell strainer, red
blood cells (RBC) were lysed with RBC lysis buffer followed by anti-
body labelling for CD11c (clone HL3; BD Pharmingen), B220 (clone
RA3-6B2 conjugated to A488; BD Pharmingen), CD86 (clone GL1
conjugated to PE; eBioscience) and propidium iodide (BD Pharmin-
gen) and analysis by flow cytometry (BD FACSort). The same proto-
col was used for assessing expression of CD86 on CD11c+ dendritic
cells by CD1dꢀ/ꢀ mice.
4.16.5. Uptake of glycolipid by dendritic cells in vitro
1 ꢂ 106 DC2114 cells were incubated with 4
lg of glycolipid in
1 mL cIMDM in a flat-bottom 6-well plate at 37 °C for 24 h. The
cells were resuspended in FACS buffer, stained with viability dye
(PI; BD Pharmingen) and analysed by flow cytometry (BD LSRII
SORP; UV laser, filter 525/50, Hoechst Blue detector).
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Acknowledgement
The authors would like to thank the Cancer Society of New Zea-
land and the Health Research Council of New Zealand for financial
assistance.
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Supplementary data
Supplementary data (1H NMR and 13C NMR spectra for all new
compounds) associated with this article can be found, in the online
version, at doi:10.1016/j.carres.2011.02.014.
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