Facile synthesis of biotin-labelled α-galactosylceramide as antigen for invariant natural killer T cells

Article abstract of DOI:10.1016/j.tet.2009.06.007

When iNKT cells are stimulated by the CD1d presented exogenous α-galactosylceramide, a myriad of cytokines are released that trigger a variety of immune responses. However, the invisibility and poor aqueous solubility of α-galactosylceramide during bioass

Full text of DOI:10.1016/j.tet.2009.06.007

Tetrahedron 65 (2009) 6390–6395
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Tetrahedron
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Facile synthesis of biotin-labelled a-galactosylceramide as antigen for invariant
natural killer T cells
,
Chengfeng Xia a b, Wenpeng Zhang b, Yalong Zhang b, Robert L. Woodward b, Jinhua Wang b,
,
Peng G. Wang b
*
a State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, China
b Departments of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
When iNKT cells are stimulated by the CD1d presented exogenous a-galactosylceramide, a myriad of
Received 14 January 2009
Received in revised form 28 May 2009
Accepted 3 June 2009
cytokines are released that trigger a variety of immune responses. However, the invisibility and poor
aqueous solubility of -galactosylceramide during bioassays have rendered it difficult to study the
aforementioned biological responses. Accordingly, a biotin-labelled -galactosylceramide was prepared
to facilitate detection of this antigen in the immune process. The problematic low solubility was also
remedies via incorporation of a truncated acyl chain.
a
a
Available online 10 June 2009
Keywords:
Natural killer T cells
Ó 2009 Elsevier Ltd. All rights reserved.
a
-Galactosylceramide
Biotin
CD1d
1. Introduction
Such stimulation of cytokine release is initiated in part by the
glycolipid antigen,
a-Galactosylceramide (a-GalCer) (Fig. 1).
In adaptive immunity, T helper (TH) and T cytotoxic (TC) cells
are two well-defined subpopulations of T lymphocytes which
recognize peptide antigens presented by class II major histo-
compatibility complexes (MHC) and class I MHC, respectively. In
contrast, invariant natural killer T (iNKT) cells, a newly discov-
ered subpopulation of T lymphocytes which express conserved,
a-GalCer was originally discovered in an extract from a marine
sponge and was found to strongly activate iNKT cells in very low
concentrations.2 The salient structural feature that distinguishes
exogenous
a-GalCer from endogenous mamalian glycoceramides is
the -linkage between the saccharide and ceramide moieties.
a
When this molecule presented to iNKT cells by antigen presenting
cells (APC), large amounts of cytokines are secreted, causing
a myriad downstream immune responses, including responses to
pathogens, tumors, tissue graft, allergens, and others.
The structural features of this glycolipid antigen that are es-
sential for iNKT cell activation have been rationalized through
semi-invariant ab (mouse V
a14-Ja18/Vb8 or human Va24-Ja18/
V
b
11) T cell receptors (TCR) and natural killer (NK) receptors, can
be stimulated by glycolipid antigens presented by the MHC class
I-like protein CD1d.1 It has been found that there are five classes
of such b2-microglobulin-associated CD1 proteins in humans.
These five classes are further divided into the following two
groups: group I CD1 (CD1a, CD1b and CD1c) and group II CD1
(CD1d and CD1e). In contrast, only a single class of CD1 proteins
(mCD1d) is found in mice, and it is homologous to the human
isoform CD1d. Once stimulated, the aforementioned iNKT cells
then rapidly secrete copious amounts of cytokines within only
a few hours, including the characteristic TH1 cytokine interferon-
O
HN
O
O
NH
S
OH
HN
HO
HO
HO
HO
O
C25H51
O
C7H15
NH OH
C14H29
O
g
(IFN-g) and TH2 cytokine interleukin-4 (IL-4). These cytokines
NH OH
play important roles in activating B cells, TC cells, macrophages,
and various other cells that participate in the immune response.
HO
HO
O
O
C14H29
OH
OH
1
2
* Corresponding author. Tel.: þ1 614 292 9884; fax: þ1 614 688 3106.
E-mail address: wang.892@osu.edu (P.G. Wang).
Figure 1. Structures of the optimized
belled -GalCer 2.
a-GalCer 1 (KRN7000) and designed biotin-la-
a
0040-4020/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2009.06.007

C. Xia et al. / Tetrahedron 65 (2009) 6390–6395
6391
crystal structures of
a
-GalCer/CD1d complexes. It has been found,
introduced the PMB-ether at C2, thus precluding any neighboring
group effects. After the desired product was extracted from the
reaction mixture, the crude preparation was stirred with silica gel
in methanol to remove the ether on C6 and furnish compound 5. In
order to install the azido group on the C6 position, the previously
deprotected hydroxyl group was first activated via formation of the
mesylate. Heating in the presence of sodium azide then afforded
the azido-galactose derivative 7. However, when 7 was used as
a donor for the glycosylation of ceramide 13 through activation
for example, that CD1d has a deep hydrophobic cleft that can ac-
commodate the two lipid chains.3 Specifically, the ceramide moiety
of
-GalCer in a distinct orientation for recognition by the iNKT cell
TCR. Both alkyl chains are initially inserted perpendicularly to the
a-GalCer contributes to the binding with CD1d by anchoring
a
b
-sheet platform and then extend more laterally toward the ends of
the A0 and F0 pockets. In regards to the sugar moiety, structure–
activity-relationship studies showed that the C2 hydroxyl group is
crucial for
demonstrating importance for binding with CD1d.4 Furthermore,
-glucosylceramide ( -GlcCer), in which the C4 hydroxyl group is in
an equatorial position, still showed activity in NKT cell stimulation,
although to a lesser extent than
-GalCer.5 When this position is
capped by an additional sugar, however, the resulting dihexo-
sylceramide becomes inactive.6 In contrast, the C6 position is more
flexible and can tolerate minimal modifications, such as the at-
tachment of a small molecule, without losing its activity. This fact
has been rationalized by the latest ternary crystal structure of the
a
-GalCer activity with the C3 hydroxyl group also
with N-iodosuccinimide and triflic acid, both the a- and b-isomers
were produced. In an effort to improve this anomeric selectivity, 7
was converted to the trichloroacetimide type donor 8, a molecule
which had worked well in the glycosylation of a ceramide in our
previous research.10 Specifically, the thiophenyl group was re-
moved via treatment with N-bromosuccinimide,13 after which tri-
chloroacetonitrile and 1,8-diazabicylco-[5.4.0]-undec-7-ene (DBU)
were introduced to give the donor 8.14
a
a
a
OH
HO
HO
O
OMe
SPh
O
1. NaH, PMBCl
TCR/a
-GalCer/CD1d complex, which unveiled that the C6 hydroxyl
(MeO)2CMe2
O
SPh
O
group is located on the outer of the cavity of the TCR and CD1d and
2. Silica gel
81%
pTsOH
76%
O
thus does not form any H-bonds with them.7
HO
HO
4
3
2. Discussion
OMs
OH
O
O
NaN3
MsCl, Et3N
97%
In spite of all this information, one of the lingering challenges for
studying the role of
visibility during assays. However, since the C6 position of the sugar
moiety can tolerate some small molecules, and these small mole-
cules protrude outside the complex, incorporation of a bio-visible
functional group on this position should remedy this problem
O
O
SPh
SPh
SPh
O
DMF
76%
O
a-GalCer in the immune process is its in-
PMBO
6
PMBO
5
N3
O
O
N3
O
O
1. NBS
O
without affecting the activity of
that attaching a biotin group would be a good tool to detect
-GalCer in flow cytometry studies.8 Furthermore,
-GalCer is no-
a-GalCer. Savage et al. suggested
PMBO
O
2. DBU, CCl3CN
72% for 2 steps
O
CCl3
PMBO
7
a
a
NH
8
torious for it low solubility in aqueous solutions during the assay
due to the two long hydrophobic chains. It was found, however,
that truncating the acyl chain to an 8-carbon unit could dramati-
Scheme 1. Synthesis of the 6-azido-galactosyl donor 8.
With the desired donor in hand, ceramide acceptor 13 was
prepared from the commercially available phytosphingosine 9 us-
ing the procedures previously established for the 26-carbon acyl
chain10 (Scheme 2). This entailed introduction of the short acyl
chain through condensation with the octoic acid N-hydroxy-
succinimide ester to give the amide 10. Subsequent selective pro-
tection of the primary hydroxyl group with trityl chloride in
pyridine followed by protection of the two secondary hydroxyl
groups with benzoyl groups afforded 12. Such protection of the
secondary hydroxyl groups was carried out so as to avoid formation
cally improve the solubility of
a-GalCer without changing iNKT cell
stimulation profile.9 Herein, we accordingly report the facile syn-
thesis and bioassay of biotin-labelled
acyl chain (Fig. 1).
a-GalCer 2 with a truncated
The formation of the
a-isomer is the key in the synthesis of the
biotin-labelled -GalCer 2. Both of the protecting groups on car-
a
bohydrate part and ceramide part play important effects on the
outcomes of glycosidation. We already noticed that electron with-
drawing protecting groups on the ceramide part favor the forma-
tion of
a
-linkage during the glycosidation, while the electron
of the b-isomer from an SN1 reaction. Lastly, removal of trityl group
donating groups prefer the formation of
b
-isomer.10 The protecting
group on the C2 position of carbohydrate part that doesn’t exert
a neighboring group effect is also crucial i our synthetic design. The
use of ether groups is the widely accepted method for this purpose
as they are easily introduced and possess versatile deprotection
protocols. For example, the benzyl ether group can be conveniently
removed by palladium catalyzed hydrogenation or sodium medi-
ated reduction in ammonia. However, due to the sulfur atom in
biotin, these hydrogenation-dependent protecting groups are not
compatible. Therefore, the p-methoxybenzyl (PMB) group was se-
lected since it can be removed not only by hydrogenation, but also
oxidation with DDQ or CAN, as well as by mild acidic conditions.
Accordingly, preparation of the C6-azido galactosyl donor
commenced with treatment of the thiophenyl compound 311 with
a trace amount of p-toluenesulfonic acid in 2,2-dimethoxylpropane
(Scheme 1). This afforded protection of the C3 and C4 hydroxyl
groups as a five-membered isopropylidene ketal ring, while the C6
hydroxyl group was protected as the 1-methoxyl-1-methylethyl
ether.12 Subsequent treatment with NaH followed by PMBCl
O
C7H15
NH2 OH
C14H29
OH
9
NH OH
TrtCl, DMAP
C7H15CO2Su
HO
HO
C14H29
THF
89%
92%
OH
10
O
C7H15
O
O
C7H15
NH OH
C14H29
pTsOH
BzCl, DMAP
NH OBz
TrtO
TrtO
HO
pyr.
86%
MeOH
92%
C14H29
OBz
OH
11
12
C7H15
NH OBz
C14H29
OBz
13
Scheme 2. Synthesis of the short acyl chain acceptor 13.

6392
C. Xia et al. / Tetrahedron 65 (2009) 6390–6395
was achieved via treatment with p-toluenesulfonic acid in
methanol.
In the final sequence, glycosylation of acceptor 13 with donor 8
was performed by using TMSOTf as a promoter in a mixed solvent
composed of Et2O and THF (Scheme 3). These conditions afforded
by A20/CD1 cells which express CD1d on the surface. When the
DN3A4-1.2 hybridoma cells are co-cultured with the A20/CD1 cells,
cytokine IL-2 will be secreted. The results in Figure 2A indicate that
although biotin-labelled a-GalCer 2 is less efficient than 1, it still can
stimulate iNKT hybridoma cells to release cytokine IL-2. They also
further prove that the iNKT cells can tolerate some small molecules
linked on the C6 position of the sugar moiety.
only the a-isomer of the glycolipid in a moderate yield. Attempts to
subsequently reduce the azido group to the amine via the Staudinger
method failed due to the formation of the phosphinimine, a species
which proved too stable to be decomposed under mild conditions.15
It was intriguing, however, that when the two benzoyl groups in the
phytosphingosine were first hydrolyzed, reduction of the azido
group with triphenylphospine proceeded smoothly. The resultant
amine 16 was treated with the biotin ester 17 to afford compound 18.
Since the PMB group can be removed under acidic condition, we then
tried to deprotect both of the PMB and isopropylidene functionalities
with p-toluenesulfonic acid or trifluoroacetic acid. However, TLC
showed that only a trace amount of final product formed while many
by-products were generated. The complexity of this reaction might
arise from an issue with the PMB group because some of the by-
products were still UV visible and existed even after prolonging the
reaction time or increasing the amount of acid. To overcome this
problem, these two protecting groups were accordingly removed
separately. Specifically, the PMB was first deprotected by oxidation
with DDQ,16 after which the isopropylidene group was removed by
treatment with p-toluenesulfonic acid to furnish the final product 2
in 51% yield over two steps.
N3
O
O
C7H15
O
NaOMe
TMSOTf
O
NH OBz
8
+
13
PMBO
Et2O/THF
-25 °C
55%
MeOH, reflux
71%
O
C14H29
OBz
14
N3
NH2
O
O
O
C7H15
NH OH
O
C7H15
NH OH
C14H29
O
O
PPh3
PhH/H2O
O
O
PMBO
PMBO
O
O
C14H29 86%
OH
16 OH
15
O
O
NH
O
O
HN
O
NH
N
NH
O
S
NH
O
O
17
S
Figure 2. (A) Stimulating the mouse iNKT hybridoma cells with the a-galactosylcer-
O
C7H15
NH OH
C14H29
O
amides 1 and 2. The release of IL-2 cytokine was measured by ELISA assay. (B) De-
tecting the glycosylceramide 1 and 2 loaded A20/CD1 cells by flow cytometry.
Et3N, THF
85%
O
PMBO
O
To prove the visibility of biotin-labelled a-GalCer 2, the A20/CD1
cells treated with 2 were stained by fluorescein isothiocyanate
OH
18
O
HN
S
(FITC) conjugated stravidin and analyzed by flow cytometry. As
shown in Figure 2B, A20/CD1 cells with either vehicle or a-GalCer 1
could not be stained by stravidin–FITC. However, a significant FITC
positive A20/CD1 cells population could be detected when they
O
NH
1. DDQ
DCM/H2O
HN
HO
HO
O
C7H15
O
2. pTsOH, MeOH
51% for 2 steps
were co-cultured with biotin-labelled
a-GalCer 2. This assay in-
NH OH
dicates that compound 2 can be detected after being presented on
the cell surface. Compound 2 can be further used to characterize
which proteins, organelles, cells and organs are involved in glyco-
lipids trafficking, presentation and recognition.
HO
O
C14H29
2
OH
Scheme 3. Preparation of the biotin-labelled
a-GalCer 2.
The V
a14i NKT hybridoma DN3A4-1.2 cells, which produce high
3. Conclusion
amounts of IL-2 cytokines, were used to measure the stimulatory
activities of biotin-labelled glycolipid antigen with a previously
reported assay.6 It’s discovered that the acyl chain truncated com-
pound 2 can be easily dissolved in aqueous without detergent, while
the 26-carbon compound 1 (KRN7000) is very poorly soluble and
normally different detergents are required to facilitate its dissolu-
tion. Specifically, the glycolipid antigens are up taken and presented
In summary, an efficient method of preparation of C6 biotin-
labelled
a-galactosylceramide was developed. The convergent
synthetic pathway included the preparation of a 6-azido-galactosyl
donor and the di-benzoyl protected ceramide acceptor. The yield of
donor preparation is 33%, and acceptor is 65%, respectively. The
following glycosidation and further installation of biotin group give

C. Xia et al. / Tetrahedron 65 (2009) 6390–6395
6393
the final product in 15% yield. To improve the solubility of the gly-
cosylceramide in bioassay, a short acyl chain was incorporated. The
assay results indicated that the appending of a small probe on C6 did
added methanesulfonyl chloride (0.30 mL, 3.9 mmol) dropwise at
0 ꢀC. After stirred for 30 min, the reaction was allowed to warm to
room temperature and continued stirring for another 2 h. The re-
action mixture was diluted with CH2Cl2 and washed with water.
Dried over anhydrous Na2SO4 and purified by silica gel chroma-
tography (hexane/ethyl acetate 3:1) to give 1.29 g of 6 as white solid
not affect the presentation of
and recognition by TCR of iNKTcells. This biotin-labelled
a
-galactosylceramide by CD1d of APC
-GalCer 2
a
can be easily detected during assays such as flow cytometry.
in 97% yield. 1H NMR (500 MHz, CDCl3)
d
7.49 (d, J¼7.2 Hz, 2H),
4. Experiments
4.1. General
7.35–7.25 (m, 5H), 6.89 (d, J¼7.2 Hz, 2H), 4.75 (d, J¼11.1 Hz, 1H),
4.73 (d, J¼8.9 Hz, 1H), 4.62 (d, J¼11.1 Hz, 1H), 4.49 (dd, J¼11.4,
8.2 Hz, 1H), 4.39 (dd, J¼11.4, 3.7 Hz, 1H), 4.33 (t, J¼6.0 Hz, 1H), 4.19
(dd, J¼5.9, 2.1 Hz, 1H), 4.05 (dt, J¼6.9, 3.5 Hz, 1H), 3.82 (s, 3H), 3.56
(dd, J¼8.9, 6.1 Hz,1H), 2.92 (s, 3H),1.45 (s, 3H),1.36 (s, 3H); 13C NMR
All solvents were dried with solvent-purification system from
Innovative Technology, Inc. All reagents were obtained from com-
mercial sources and used without further purification. Analytical
TLC was carried out on silica gel 60 F254 aluminum-backed plates
(E. Merck). The 230–400 mesh size of the same absorbent was
utilized for all chromatographic purifications. 1H and 13C NMR
spectra were recorded at the indicated field strengths. The high-
resolution mass spectra were collected at The Ohio State University
Campus Chemical Instrumentation Center.
(125 MHz, CDCl3)
d 159.4, 133.6, 131.5, 129.9, 129.7, 129.0, 127.5,
113.8, 110.6, 85.7, 79.1, 73.9, 73.0, 72.96, 69.2, 55.3, 37.5, 27.6, 26.2;
HRMS calcd for C24H30O8S2Na ([MþNa]þ) 533.1280, found
533.1293.
4.1.4. Phenyl 1-thio-2-O-p-methoxybenzyl-3,4-O-isopropylidene-6-
deoxy-6-azido-b-D-galactopyranoside (7)
To a solution of the above compound 6 (1.28 g, 2.5 mmol) in
DMF (10 mL) was added NaN3 (0.85 g, 13.0 mmol). The resulting
suspension was stirred at 80 ꢀC for 36 h. The solvent was removed
in vacuo, and the residue was dissolved in ethyl acetate, washed
with water twice. The organic layer was separated and dried over
anhydrous Na2SO4, purified by silica gel chromatography (hexane/
ethyl acetate 5:1) to give 0.87 of 7 as white solid in 76% yield. 1H
4.1.1. Phenyl 1-thio-3,4-O-isopropylidene-6-O-(1-methoxyl-1-
methylethyl)-b-D-galactopyranoside (4)
To a solution of phenyl 1-thiogalactoside 3 (2.7 g, 10.0 mmol) in
2,2-dimethoxylpropane (25 mL) was added p-toluenesulfonic acid
monohydrate (150 mg, 0.8 mmol). The resulting mixture was stir-
red for 4 h at room temperature. The reaction was quenched by
addition of Et3N (0.5 mL). After removal of the solvent, the residue
was dissolved in ethyl acetate, and washed with water. The organic
layer was dried over anhydrous Na2SO4 and concentrated. The
residue was purified by silica gel chromatography (hexane/ethyl
acetate 3:1) to give 2.8 g of 4 as colorless oil in 76% yield. 1H NMR
NMR (500 MHz, CDCl3)
d
7.56 (m, 2H), 7.36 (d, J¼8.6 Hz, 2H), 7.32
(m, 3H), 6.91 (d, J¼8.6 Hz, 2H), 4.77 (d, J¼11.0 Hz, 1H), 4.69 (d,
J¼9.1 Hz, 1H), 4.66 (d, J¼11.0 Hz, 1H), 4.31 (t, J¼6.0 Hz, 1H), 4.16 (dd,
J¼5.9, 2.1 Hz, 1H), 3.85 (m, 1H), 3.83 (s, 3H), 3.65 (dd, J¼12.8, 7.9 Hz,
1H), 3.56 (dd, J¼9.0, 6.1 Hz, 1H), 3.42 (dd, J¼12.9, 5.1 Hz, 1H), 1.46 (s,
3H), 1.38 (s, 3H); 13C NMR (125 MHz, CDCl3)
d 159.4, 133.5, 132.3,
(500 MHz, CDCl3)
d
7.60 (m, 2H), 7.35 (m, 3H), 4.48 (d, J¼10.2 Hz,
129.9, 129.86, 128.9, 127.6, 113.8, 110.4, 86.5, 79.2, 77.6, 75.1, 73.6,
73.1, 55.3, 51.3, 27.6, 26.3; HRMS calcd for C23H27N3O5S Na
([MþNa]þ) 480.1569, found 480.1582.
1H), 4.22 (dd, J¼5.5, 2.1 Hz, 1H), 4.10 (t, J¼6.7 Hz, 1H), 3.91 (ddd,
J¼7.1, 5.4, 2.1 Hz, 1H), 3.77 (dd, J¼9.9, 7.0 Hz, 1H), 3.69 (dd, J¼9.9,
5.2 Hz, 1H), 3.59 (ddd, J¼9.8, 6.9, 2.4 Hz, 1H), 3.25 (s, 3H), 2.49 (br s,
1H), 1.44 (s, 3H), 1.40 (s, 3H), 1.39 (s, 3H), 1.35 (s, 3H); 13C NMR
4.1.5. 2-O-p-Methoxybenzyl-3,4-O-isopropylidene-6-deoxy-6-
(125 MHz, CDCl3)
d
132.4, 128.9, 127.9, 110.2, 100.2, 87.8, 79.1, 76.1,
azido-a-D-galactopyranosyl-(1,1)-trichloroacetimide (8)
73.8, 71.6, 60.5, 48.6, 28.1, 26.3, 24.4.
A solution of the thiogalactose 7 (0.36 g, 0.79 mmol) in a mix-
ture of acetone and water (9:1, 10 mL) at 0 ꢀC was treated with NBS
(0.32 g, 1.80 mmol) for 1 h. The reaction was quenched by addition
solid NaHCO3. The organic solvent was removed under reduced
pressure. The residue was dissolved in ethyl acetate and washed
with saturated aqueous NaHCO3 and brine. The organic layer was
dried over anhydrous Na2SO4 and purified by silica gel chroma-
tography (hexane/ethyl acetate 2:1) to give 0.26 g of product as
white solid in 91% yield.
To a solution of the above compound (0.26 g, 0.71 mmol) in dry
CH2Cl2 (8 mL) were added CCl3CN (0.71 mL, 7.1 mmol) and DBU
(54 mL, 0.36 mmol) successively. After stirring for 2 h at room
temperature, the solvent was evaporated and the residue was pu-
rified by silica gel chromatography (hexane/ethyl acetate 6:1) to
give 0.28 g of 8 was white solid in 80% yield. 1H NMR (500 MHz,
4.1.2. Phenyl 1-thio-2-O-p-methoxybenzyl-3,4-O-isopropylidene-
b
-D-galactopyranoside (5)
A solution of galactose derivative 4 (1.9 g, 5.2 mmol) in dry DMF
(30 mL) was treated with 60% NaH (0.25 g, 6.2 mmol) for 30 min.
PMBCl (0.84 mL, 6.2 mmol) was added by syringe and the reaction
mixture was continued stirring for 5 h. The solvent was removed in
vacuo. The residue was dissolved in ethyl acetate, washed with
water. The organic layer was separated and dried over anhydrous
Na2SO4. After concentrated, it was dissolved in methanol, stirred
with silica gel (5 g) for 1 h. When TLC showed the reaction is com-
pleted, it was concentrated and purified by silica gel chromatogra-
phy (hexane/ethyl acetate 4:1) to give 1.8 g of 5 as colorless oil in 81%
yield. 1H NMR (500 MHz, CDCl3)
d
7.53 (m, 2H), 7.36 (d, J¼8.5 Hz,
2H), 7.33–7.27 (m, 3H), 6.90 (d, J¼8.5 Hz, 2H), 4.77 (d, J¼11.0 Hz,1H),
4.66 (d, J¼9.5 Hz, 1H), 4.63 (d, J¼11.0 Hz, 1H), 4.30 (t, J¼6.0 Hz, 1H),
4.20 (dd, J¼5.7, 1.7 Hz, 1H), 3.96 (dd, J¼11.6, 7.3 Hz, 1H), 3.83 (s, 3H),
3.81 (m,1H), 3.77 (dd, J¼11.6, 3.9 Hz,1H), 3.54 (dd, J¼9.4, 6.3 Hz,1H),
CDCl3)
d
8.67 (s, 1H), 7.29 (d, J¼8.4 Hz, 2H), 6.89 (d, J¼8.4 Hz, 2H),
6.39 (d, J¼3.5 Hz,1H), 4.75 (d, J¼11.8 Hz,1H), 4.65 (d, J¼11.8 Hz,1H),
4.47 (t, J¼6.4 Hz, 1H), 4.39 (m, 1H), 4.27 (dd, J¼6.0, 2.4 Hz, 1H), 3.82
(s, 3H), 3.80 (m, 1H), 3.57 (dd, J¼12.7, 7.6 Hz, 1H), 3.42 (dd, J¼12.8,
5.6 Hz, 1H), 1.44 (s, 3H), 1.37 (s, 3H); 13C NMR (125 MHz, CDCl3)
1.44 (s, 3H), 1.37 (s, 3H); 13C NMR (125 MHz, CDCl3)
d 159.4, 133.5,
132.0,131.5,130.0,129.9,129.89,129.1,128.9,127.5,114.1,113.8,110.4,
86.0, 79.8, 78.0, 76.7, 73.9, 73.1, 62.6, 55.3, 27.8, 26.3; HRMS calcd for
C23H28O6SNa ([MþNa]þ) 455.1504, found 455.1498.
d 161.0, 159.4, 129.9, 129.5, 129.4, 114.0, 113.8, 110.0, 94.3, 91.2, 74.7,
74.0, 73.0, 72.5, 69.8, 55.3, 50.8, 27.5, 26.0; HRMS calcd for
C19H23Cl3N4O6Na ([MþNa]þ) 531.0581, found 531.0591.
4.1.3. Phenyl 1-thio-2-O-p-methoxybenzyl-3,4-O-isopropylidene-6-
O-methanesulfonyl-b-D-galactopyranoside (6)
4.1.6. (2S,3S,4R)-1-O-Triphenylmethyl-2-octanoylamino-
octadecan-1,3,4-triol (11)
To a solution of the 6-hydroxyl-galactopyranoside 5 (1.13 g,
2.6 mmol) and Et3N (0.72 mL, 5.2 mmol) in dry CH2Cl2 (15 mL) was
To a solution of amide 10 (1.84 g, 4.15 mmol) in pyridine (40 mL)
were added TrtCl (5.78 g, 20.7 mmol) and DMAP (100 mg,

6394
C. Xia et al. / Tetrahedron 65 (2009) 6390–6395
0.82 mmol) and stirred at 50 ꢀC for overnight. The solution was
concentrated under reduced pressure. The residue was dissolved by
EtOAc and washed by water. The organic layer was dried over an-
hydrous Na2SO4. The concentrated residue was purified by silica gel
chromatography (hexane/ethyl acetate 4:1) to give 2.62 g of 11 in
0.11 mmol) was added by syringe and continued stirring for 2 h.
The reaction was quenched by addition Et3N (0.2 mL) and filtered
through Celite pad. The filtrate was concentrated and the residue
was purified by silica gel chromatography (hexane/ethyl acetate
7:1) to give 218 mg of 14 as white solid in around 55% yield. 1H
92% yield as colorless oil. 1H NMR (400 MHz, CDCl3)
d
7.42–7.44 (m,
NMR (500 MHz, CDCl3) d 8.06 (m, 2H), 7.97 (m, 2H), 7.64 (m, 1H),
6H), 7.31–7.35 (m, 6H), 7.25–7.28 (m, 3H), 6.06 (d, J¼8.4 Hz, 1H),
4.27 (m, 1H), 3.59 (m, 1H), 3.52 (m, 2H), 3.42–3.35 (m, 2H), 3.15 (d,
J¼8.4 Hz, 1H), 2.28 (d, J¼7.6 Hz, 1H), 2.17 (t, J¼7.6 Hz, 2H), 1.71–1.60
(m, 4H), 1.45 (m, 2H), 1.32–1.25 (m, 28H), 0.90 (t, J¼6.4 Hz, 6H); 13C
7.57 (m, 1H), 7.51 (m, 2H), 7.40 (m, 2H), 7.22 (d, J¼8.6 Hz, 2H),
6.77 (d, J¼8.6 Hz, 2H), 6.73 (d, J¼9.5 Hz, 1H), 5.72 (dd, J¼9.3,
2.8 Hz, 1H), 5.37 (m, 1H), 4.72 (d, J¼3.5 Hz, 1H), 4.66 (d, J¼11.8 Hz,
1H), 4.63 (m, 1H), 4.59 (d, J¼11.8 Hz, 1H), 4.28 (t, J¼6.1 Hz, 1H),
4.17 (m, 1H), 4.11 (dd, J¼5.8, 2.4 Hz, 1H), 3.83 (dd, J¼10.8, 3.1 Hz,
1H), 3.77 (s, 3H), 3.70 (dd, J¼10.9, 3.6 Hz, 1H), 3.53 (dd, J¼13.3,
8.3 Hz, 1H), 3.52 (dd, J¼7.0, 3.4 Hz, 1H), 3.38 (dd, J¼12.84.8 Hz,
1H), 2.24 (t, J¼7.8 Hz, 2H), 1.93 (m, 2H), 1.65 (m, 2H), 1.43–1.26
(m, 32H), 1.36 (s, 3H), 1.31 (s, 3H), 0.90 (t, J¼7.2 Hz, 3H), 0.89 (t,
NMR (100 MHz, CDCl3)
d 173.2, 143.2, 128.5, 128.1, 127.4, 87.7, 75.6,
73.2, 65.9, 63.0, 50.4, 36.9, 33.3, 31.9, 31.7, 29.7, 29.6, 29.4, 29.3, 29.0,
25.8, 25.7, 22.7, 22.6, 14.1, 14.06; HRMS calcd for C45H67NO4Na
([MþNa]þ) 708.4968, found 708.4962.
4.1.7. (2S,3S,4R)-1-O-Triphenylmethyl-3,4-O-dibenzoyl-2-
octanoylamino-octadecan-1,3,4-triol (12)
J¼7.1 Hz, 3H); 13C NMR (125 MHz, CDCl3)
d 173.1, 166.3, 165.4,
159.3, 133.4, 133.0, 130.1, 130.0, 129.8, 129.75, 129.7, 129.5, 128.6,
128.4, 113.7, 109.6, 98.4, 75.2, 75.1, 74.0, 73.4, 72.4, 72.3, 68.2, 67.8,
55.2, 51.2, 48.4, 36.8, 31.9, 31.8, 29.7, 29.69, 29.67, 29.64, 29.6,
29.57, 29.4, 29.3, 29.1, 28.2, 27.7, 26.1, 25.8, 25.7, 22.7, 22.68, 14.14,
14.1; HRMS calcd for C57H82N4O11Na ([MþNa]þ) 1021.5878, found
1021.5881.
To a solution of 11 (2.8 g, 4.08 mmol) in pyridine (40 mL) were
added BzCl (2.8 mL, 24.5 mmol) and DMAP (49 mg, 0.4 mmol) and
the reaction mixture was stirred for 8 h. The solution was con-
centrated under reduced pressure. The residue was partitioned
between ethyl acetate and water. The organic layer was separated
and washed with cooled 1 N HCl, saturated aqueous NaHCO3 and
brine. After dried over anhydrous Na2SO4, it was purified by silica
gel chromatography (hexane/ethyl acetate 15:1) to give 3.14 g of 12
4.1.10. 2-O-p-Methoxybenzyl-3,4-O-isopropylidene-6-deoxy-6-
azido-a-D-galactopyranosyl-(1,1)-(2S,3SR,4R)-2-octanoylamino-
in 86% yield as colorless oil. 1H NMR (400 MHz, CDCl3)
d 7.98 (d,
octadecan-1,3,4-triol (15)
J¼8.0 Hz, 2H), 7.90 (d, J¼7.6 Hz, 2H), 7.62–7.55 (m, 2H), 7.42 (dd,
J¼14.8, 7.2 Hz, 4H), 7.33–7.31 (m, 6H), 7.19–7.17 (m, 9H), 6.02 (d,
J¼9.6 Hz, 1H), 5.81 (dd, J¼8.8, 2.4 Hz, 1H), 5.37 (m, 1H), 4.60 (m,
1H), 3.35 (dd, J¼12.2, 4.5 Hz, 1H), 3.31 (dd, J¼12.1, 3.6 Hz, 1H),
2.23–2.16 (m, 2H), 1.91–1.86 (m, 2H), 1.68–1.62 (m, 2H), 1.40–1.23
A
solution of the protected glycoceramide 14 (200 mg,
0.2 mmol) and freshly prepared NaOMe (11 mg, 0.2 mmol) in dry
MeOH (5 mL) was heated to refluxing for 24 h. After cooled to
room temperature, it was neutralized by Dowex ion-exchange
resin and filtered. The filtrate was concentrated and the residue
was purified by silica gel chromatography (hexane/ethyl acetate
3:1) to give 112 mg of 15 as white solid in 71% yield. 1H NMR
(m, 32H), 0.91–0.88 (m, 6H); 13C NMR (100 MHz, CDCl3)
d 172.8,
166.4, 165.1, 143.3, 133.1, 132.9, 130.2, 129.9, 129.8, 129.76, 128.6,
128.4, 129.36, 127.8, 127.0, 86.8, 74.2, 72.8, 61.7, 48.6, 36.9, 31.9,
31.7, 29.7, 29.66, 29.6, 29.57, 29.5, 29.4, 29.1, 28.5, 25.7, 25.69, 22.7,
22.66, 14.1, 14.09; HRMS calcd for C59H75NO6Na ([MþNa]þ)
916.5492, found 916.5497.
(500 MHz, CDCl3)
d
7.28 (d, J¼8.6 Hz, 2H), 6.89 (d, J¼8.6 Hz, 2H),
6.33 (d, J¼8.5 Hz, 1H), 4.86 (d, J¼3.8 Hz, 1H), 4.79 (d, J¼11.6 Hz,
1H), 4.61 (d, J¼11.6 Hz, 1H), 4.32 (dd, J¼7.2, 5.8 Hz, 1H), 4.27 (m,
1H), 4.16 (dd, J¼5.7, 2.5 Hz, 1H), 4.07 (m, 1H), 4.00 (dd, J¼10.5,
3.9 Hz, 1H), 3.83 (m, 1H), 3.81 (s, 3H), 3.57–3.53 (m, 3H), 3.49 (dd,
J¼6.2, 3.7 Hz, 1H), 3.40 (dd, J¼12.9, 5.5 Hz, 1H), 2.20 (t, J¼7.0 Hz,
2H), 1.63 (m, 2H), 1.49–1.26 (m, 34H), 1.43 (s, 3H), 1.35 (s, 3H), 0.89
4.1.8. (2S,3S,4R)-3,4-O-Dibenzoyl-2-octanoylamino-octadecan-
1,3,4-triol (13)
To a solution of 12 (1.8 g, 2.0 mmol) in CH2Cl2 and MeOH (2:1,
20 mL) were added p-toluenesulfonic acid monohydrate (400 mg,
2.1 mmol) and stirred for 3 h. The solution was quenched by ad-
dition Et3N (0.2 mL). The solution was concentrated in vacuo. The
concentrated residue was purified by silica gel chromatography
(hexane/ethyl acetate 5:1) to give 1.2 g of 13 in 92% yield as color-
(t, J¼6.7 Hz, 6H); 13C NMR (125 MHz, CDCl3)
d 173.1, 159.6, 129.8,
129.3, 113.9, 109.7, 98.3, 76.1, 76.06, 75.4, 73.3, 73.2, 72.4, 69.9, 67.3,
36.8, 33.4, 31.9, 31.7, 29.7, 29.66, 29.3, 29.1, 27.9, 26.2, 25.9, 25.8,
22.7, 22.6, 14.1, 14.05; HRMS calcd for C43H74N4O9Na ([MþNa]þ)
813.5354, found 813.5352.
less oil. 1H NMR (500 MHz, CDCl3)
d
8.04 (d, J¼1.0 Hz, 2H), 7.95 (d,
4.1.11. 2-O-p-Methoxybenzyl-3,4-O-isopropylidene-6-deoxy-6-
amino-a-D-galactopyranosyl-(1,1)-(2S,3S,4R)-2-octanoylamino-
octadecan-1,3,4-triol (16)
J¼1.0 Hz, 2H), 7.63 (t, J¼7.5 Hz, 1H), 7.49–7.54 (m, 3H), 7.38 (d,
J¼7.5 Hz, 2H), 6.50 (d, J¼9.5 Hz, 1H), 5.46 (dd, J¼9.5, 2.5 Hz, 1H),
5.39 (dt, J¼9.3, 3.1 Hz, 1H), 4.41 (tt, J¼9.4, 2.6 Hz, 1H), 3.66 (dd,
J¼12.0, 2.0 Hz, 2H), 2.95 (br, 1H), 2.29 (t, J¼7.5 Hz, 2H), 2.03 (m, 2H),
1.70 (m, 2H), 1.24–1.46 (m, 32H), 0.88 (J¼7.0 Hz, 6H); 13C NMR
A solution of the azido-glycoceramide 15 (160 mg, 0.20 mmol)
and PPh3 (79 mg, 0.30 mmol) in benzene and trace amount of water
was heated to 50 ꢀC for 6 h. The solvent was evaporated and the
residue was purified by silica gel chromatography (ethyl acetate to
dichloromethane/methanol 5:1) to give 132 mg of amine 16 in 86%
(125 MHz, CDCl3)
d 173.3, 167.0, 166.4, 133.8, 133.1, 130.0, 129.9,
129.7, 129.2, 128.7, 128.4, 74.0, 73.7, 50.0, 36.9, 31.9, 31.7, 29.7, 29.68,
29.65, 29.6, 29.57, 29.4, 29.36, 29.3, 29.1, 28.5, 25.8, 22.7, 22.6, 14.1,
14.07; HRMS calcd for C40H61NO6Na ([MþNa]þ) 674.4397, found
674.4401.
yield. 1H NMR (500 MHz, CDCl3)
d
7.26 (d, J¼8.6 Hz, 2H), 6.87 (d,
J¼8.6 Hz, 2H), 6.66 (d, J¼8.3 Hz, 1H), 4.83 (d, J¼3.6 Hz, 1H), 4.74 (d,
J¼11.8 Hz, 1H), 4.62 (d, J¼11.8 Hz, 1H), 4.28 (t, J¼6.5 Hz, 1H), 4.13
(dd, J¼5.5, 2.1 Hz, 1H), 3.98 (m, 1H), 3.79 (s, 3H), 3.75 (dd, J¼10.5,
4.1 Hz, 1H), 3.51–3.48 (m, 3H), 3.35 (br, 2H), 3.03 (dd, J¼13.1, 8.6 Hz,
1H), 2.90 (dd, J¼13.1, 3.6 Hz, 1H), 2.18 (t, J¼7.4 Hz, 2H), 1.64 (m, 3H),
1.49 (m, 1H), 1.40 (s, 3H), 1.32 (s, 3H), 1.31–1.21 (m, 32H), 0.88 (t,
4.1.9. 2-O-p-Methoxybenzyl-3,4-O-isopropylidene-6-deoxy-6-
azido-
3,4-O-dibenzoyl-octadecan-1,3,4-triol (14)
suspension of the azido-galactosyl donor
a-D-galactopyranosyl-(1,1)-(2S,3S,4R)-2-octanoylamino-
A
8
(0.28 g,
J¼6.8 Hz, 6H); 13C NMR (125 MHz, CDCl3)
d 173.4, 159.5, 129.7,
0.55 mmol), ceramide acceptor 13 (0.26 g, 0.40 mmol) and 4 Å
molecular sieve (0.5 g) in a mixture of ethyl ether and tetrahy-
drofuran (5:1, 4 mL) was stirred at room temperature for 30 min.
129.6, 113.8, 109.5, 98.1, 76.2, 75.5, 74.1, 72.7, 72.2, 68.5, 68.2, 55.2,
50.0, 42.3, 36.7, 33.7, 31.9, 31.7, 29.8, 29.78, 29.7, 29.66, 29.4, 29.3,
29.1, 28.0, 26.3, 26.0, 25.8, 22.7, 22.6, 14.1, 14.1; C43H76N2O9Na
([MþNa]þ) 787.5449, found 787.5444.
After the mixture was cooled to ꢁ25 ꢀC, TMSOTf (23
mL,

C. Xia et al. / Tetrahedron 65 (2009) 6390–6395
6395
4.1.12. 2-O-p-Methoxybenzyl-3,4-O-isopropylidene-6-deoxy-6-
biotinamino- -galactopyranosyl-(1,1)-(2S,3S,4R)-2-
A20 cells for overnight. After washing with culture medium, the
A20/CD1 cells were mixed with 50,000 V 14i NKT hybridoma
a
-D
a
octanoylamino-octadecan-1,3,4-triol (18)
The above amine 16 (40 mg, 0.052 mmol) was dissolved in THF
(1.5 mL), and Et3N (16 mL, 0.12 mmol) and biotin-OSu 17 (26 mg,
DN3A4-1.2 cells and co-cultured for 24 h. The released IL2 in the
supernatant was measured by the ELISA assay. Data is representa-
tive of 3 independent experiments.
0.072 mmol) was added. The resulting mixture was stirred at
room temperature overnight. The solvent was removed in vacuo,
and the residue was purified by silica gel chromatography
(dichloromethane/methanol 25:1) to afford 44 mg of 18 as white
4.3. Flow cytometry assay
The A20/CD1 cells were treated overnight with compounds 1, 2
and a DMSO control at the concentration of 1 mg/mL. After washing
foam in 85% yield. 1H NMR (500 MHz, CDCl3)
d
7.27 (d, J¼7.6 Hz,
2H), 6.88 (d, J¼7.6 Hz, 2H), 6.78 (br, 1H), 6.69 (br, 1H), 4.82 (d,
J¼2.6 Hz, 1H), 4.74 (d, J¼11.8 Hz, 1H), 4.61 (d, J¼11.8 Hz, 1H), 4.49
(br, 1H), 4.34–4.26 (m, 3H), 4.16 (d, J¼4.3 Hz, 1H), 4.12 (d, J¼7.8 Hz,
1H), 3.88 (m, 1H), 3.81 (s, 3H), 3.76 (m, 1H), 3.70 (m, 1H), 3.53–
3.49 (m, 3H), 3.31 (t, J¼10.8 Hz, 1H), 3.14 (m, 1H), 2.89 (d,
J¼10.9 Hz, 1H), 2.75 (d, J¼12.4 Hz, 1H), 2.24 (m, 2H), 2.20 (t,
J¼7.2 Hz, 2H), 1.76–1.58 (m, 7H), 1.53–1.44 (m, 3H), 1.41 (s, 3H),
1,34 (s, 3H), 1.32–1.23 (m, 34H), 0.89 (t, J¼6.7 Hz, 6H); 13C NMR
away the extra glycolipids in the medium, the treated cells were
stained by Stravidin–FITC conjugate following the blocking pro-
cedure. The analysis was carried out on BD FACS Calibur and the
data was analyzed by FLOWJO V.7.
Acknowledgements
This project was supported by The Ohio State University to Peng
George Wang.
(125 MHz, CDCl3)
d 173.8, 173.5, 164.0, 159.5, 129.7, 129.6, 113.9,
109.6, 98.1, 76.3, 76.1, 75.4, 73.9, 72.8, 72.2, 68.3, 66.4, 62.1, 60.3,
55.7, 55.3, 49.7, 40.5, 36.7, 35.7, 33.7, 31.9, 31.8, 29.8, 29.79, 29.76,
29.7, 29.68, 29.4, 29.3, 29.1, 28.1, 28.0, 26.3, 26.0, 25.8, 25.6, 22.7,
22.66, 14.1, 14.12; HRMS calcd for C53H90N4O11SNa ([MþNa]þ)
1013.6225, found 1013.6251.
Supplementary data
Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.tet.2009.06.007.
4.1.13. 6-Deoxy-6-biotinamino-a-D-galactopyranosyl-(1,1)-
(2S,3S,4R)-2-octanoylamino-octadecan-1,3,4-triol (2)
References and notes
A solution of protected biotin-galactosylceramide 18 (26 mg,
0.026 mmol) in a mixture of CH2Cl2 and H2O (30:1, 1.5 mL) was
treated with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (8.9 mg,
0.039 mmol) for 4 h. After neutralized with Et3N, it was purified by
column. Then it was dissolved in MeOH, and p-toluenesulfonic acid
(2.5 mg, 0.013 mmol) was added. The resulting mixture was stirred
at room temperature for 6 h. After neutralized by addition of Et3N
and concentrated, the residue was purified by silica gel chroma-
tography (chloroform/methanol/water 6:1:0.1) to give 11 mg of 2 as
1. (a) Wu, L.; Van Kaer, L. Curr. Mol. Med. 2009, 9, 4; (b) Bendelac, A. Fundam.
Immunol. 2008, 518 (6th Ed.); (c) Kronenberg, M. Annu. Rev. Immunol. 2005, 26,
877; (d) 46De Libero, G.; Mori, L. Nat. Rev. Immunol. 2005, 5, 485.
2. (a) Natori, T.; Koezuka, Y.; Higa, T. Tetrahedron Lett. 1993, 34, 5591; (b) Notari, T.;
Morita, M.; Akimoto, K.; Koezuka, Y. Tetrahedron 1994, 50, 2771; (c) Morita, M.;
Motoki, K.; Akimoto, K.; Natori, T.; Sakai, T.; Sawa, E.; Yamaji, K.; Koezuka, Y.;
Kobayashi, E.; Fukushima, H. J. Med. Chem. 1995, 38, 2176.
3. (a) Koch, M.; Stronge, V. S.; Shepherd, D.; Gadola, S. D.; Mathew, B.; Ritter, G.;
Fersht, A. R.; Besra, G. S.; Schmidt, R. R.; Jones, E. Y.; Cerundolo, V. Nat. Immunol.
2005, 6, 819; (b) Wu, D.; Zajonc, D. M.; Fujio, M.; Sullivan, B. A.; Kinjo, Y.;
Kronenberg, M.; Wilson, I. A.; Wong, C.-H. Proc. Natl. Acad. Sci. U.S.A. 2006, 103,
3972.
white solid in 51% yield. 1H NMR (500 MHz, pyridine-d6)
d 8.81 (t,
4. (a) Wu, D.; Xing, G.-W.; Poles, M. A.; Horowitz, A.; Kinjo, Y.; Sullivan, B.; Bod-
mer-Narkevitch, V.; Plettenburg, O.; Kronenberg, M.; Tsuji, M.; Ho, D. D.; Wong,
C.-H. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 1351; (b) Lijima, H.; Kimura, K.; Sakai,
T.; Uchimura, A.; Shimizu, T.; Ueno, H.; Natori, H.; Koezuka, Y. Bioorg. Med.
Chem. 1998, 6, 1905.
5. Kawano, T.; Cui, J.; Koezuka, Y.; Toura, I.; Kaneko, Y.; Motoki, K.; Ueno, H.;
Nakagawa, R.; Sato, H.; Kondo, E.; Koseki, H.; Taniguchi, M. Science 1997,
278, 1626.
6. Prigozy, T.; Naidenko, O.; Qasba, P.; Elewaut, D.; Brossay, L.; Khurana, A.; Natori,
T.; Koezuka, Y.; Kulkarni, A.; Kronenberg, M. Science 2001, 291, 664.
7. Borg, N. A.; Wun, K. S.; Kjer-Nielsen, L.; Wilce, M. C. J.; Pellicci, D. G.; Koh, R.;
Besra, G. S.; Bharadwaj, M.; Godfrey, D. I.; McCluskey, J.; Rossjohn, J. Nature
2007, 448, 44.
8. Zhou, X.; Forestier, C.; Goff, R. D.; Li, C.; Teyton, L.; Bendelac, A.; Savage, P. B. Org.
Lett. 2002, 4, 1267.
9. Zajonc Dirk, M.; Cantu, C., 3rd; Mattner, J.; Zhou, D.; Savage Paul, B.; Bendelac,
A.; Wilson Ian, A.; Teyton, L. Nat. Immunol. 2005, 6, 810.
10. Xia, C.; Yao, Q.; Schuemann, J.; Rossy, E.; Chen, W.; Zhu, L.; Zhang, W.; De Libero,
G.; Wang, P. G. Bioorg. Med. Chem. Lett. 2006, 16, 2196.
J¼5.2 Hz, 1H), 8.56 (d, J¼8.6 Hz, 1H), 7.53 (br, 1H), 7.44 (br, 1H), 7.01
(br, 1H), 6.38 (br, 2H), 6.13 (br, 1H), 5.49 (d, J¼3.4 Hz, 1H), 5.22 (br,
1H), 4.59 (dd, J¼10.8, 5.0 Hz, 1H), 4.57 (dd, J¼12.9, 3.5 Hz, 1H), 4.51
(t, J¼6.6 Hz, 1H), 4.47 (t, J¼6.2 Hz, 1H), 4.36 (t, J¼4.5 Hz, 1H), 4.32–
4.24 (m, 5H), 4.18 (m, 1H), 3.88 (m, 1H), 3.20 (dd, J¼11.2, 7.6 Hz, 1H),
2.93 (dd, J¼12.5, 4.9 Hz, 1H), 2.83 (d, J¼12.4 Hz, 1H), 2.48–2.43 (m,
4H), 2.26 (m, 1H), 1.90 (m, 2H), 1.83–1.74 (m, 5H), 1.67–1.57 (m, 3H),
1.42–1.09 (m, 32H), 0.84 (t, J¼6.4 Hz, 3H), 0.78 (t, J¼6.5 Hz, 3H); 13C
NMR (125 MHz, pyridine-d6)
d 173.6, 173.1, 164.2, 101.1, 76.5, 72.3,
71.0, 70.9, 70.4, 69.8, 68.3, 62.2, 60.4, 56.0, 51.0, 40.9, 40.8, 36.6,
36.0, 34.2, 31.9, 31.7, 30.2, 30.0, 29.8, 29.77, 29.7, 29.4, 29.39, 29.2,
28.8, 28.78, 26.3, 26.2, 26.0, 22.7, 22.6, 14.1, 14.0; HRMS calcd for
C42H78N4O10SNa ([MþNa]þ) 853.5336, found 853.5318.
4.2. Cytokine release assay
11. Ohlsson, J.; Magnusson, G. Carbohydr. Res. 2000, 329, 49.
12. Li, Z.; Gildersleeve, J. C. J. Am. Chem. Soc. 2006, 128, 11612.
13. Motawia, M. S.; Marcussen, J.; Moeller, B. J. Carbohydr. Chem. 1995, 14, 1279.
14. Jung, K.-H.; Hoch, M.; Schmidt, R. R. Liebigs Ann. Chem. 1989, 1099.
15. Bachi, M. D.; Vaya, J. J. Org. Chem. 1979, 44, 4393.
1 and 2 were dissolved in DMSO at a concentration of 1.0 mg/mL
and then diluted with medium to the indicated concentration. They
were pulsed to 100,000 cytoplasmic tail-deleted CD1d-transfected
16. Wu, J.; Guo, Z. J. Org. Chem. 2006, 71, 7067.