Modification of the ceramide moiety of isoglobotrihexosylceramide on its agonist activity in stimulation of invariant natural killer T cells

Article abstract of DOI:10.1021/jm0701066

Isoglobotrihexosylceramide (iGb3) is an endogenous antigen of mammalian cells and can stimulate invariant natural killer T (iNKT) cells to evoke autoimmune activities by the release of T helper 1 (Th1) and Th2 cytokines. Th1 cytokines are correlated with the antitumor and antiviral response, while Th2 cytokines are correlated with the amelioration of autoimmune diseases. iGb3 is a very weak agonist compared to the exogenous α-galactosylceramide; however, modification of the ceramide moiety has been advocated as one of the approaches to improve its stimulatory activity and to change the bias of release of Th1 and Th2 cytokines. Two analogues of iGb3, 2H-iGb3 and HO-iGb3 with different ceramide moieties, were synthesized. Bioassay results showed that HO-iGb3 was much more effective in stimulating iNKT cells than iGb3 at low concentration. The assay also showed that the CD1d/2H-iGb3 complexes are remarkably efficient in stimulating iNKT cells.

Full text of DOI:10.1021/jm0701066

J. Med. Chem. 2007, 50, 3489-3496
3489
Modification of the Ceramide Moiety of Isoglobotrihexosylceramide on Its Agonist Activity in
Stimulation of Invariant Natural Killer T Cells
Chengfeng Xia,† Jens Schu¨mann,‡ Rossy Emmanuel,‡ Yalong Zhang,† Wenlan Chen,† Wenpeng Zhang,†
Gennaro De Libero,‡ and Peng George Wang*,†
Departments of Biochemistry and Chemistry, The Ohio State UniVersity, 876 Biological Sciences Building, 484 West 12th AVenue,
Columbus, Ohio 43210, and Experimental Immunology, Department of Research, UniVersity Hospital Basel, CH-4031 Basel, Switzerland
ReceiVed January 26, 2007
Isoglobotrihexosylceramide (iGb3) is an endogenous antigen of mammalian cells and can stimulate invariant
natural killer T (iNKT) cells to evoke autoimmune activities by the release of T helper 1 (Th1) and Th2
cytokines. Th1 cytokines are correlated with the antitumor and antiviral response, while Th2 cytokines are
correlated with the amelioration of autoimmune diseases. iGb3 is a very weak agonist compared to the
exogenous R-galactosylceramide; however, modification of the ceramide moiety has been advocated as one
of the approaches to improve its stimulatory activity and to change the bias of release of Th1 and Th2
cytokines. Two analogues of iGb3, 2H-iGb3 and HO-iGb3 with different ceramide moieties, were synthesized.
Bioassay results showed that HO-iGb3 was much more effective in stimulating iNKT cells than iGb3 at
low concentration. The assay also showed that the CD1d/2H-iGb3 complexes are remarkably efficient in
stimulating iNKT cells.
Introduction
Invariant natural killer T (iNKTa) cells are a subpopulation
of T lymphocytes and express conserved, semi-invariant Râ
(mouse VR14-JR18/Vâ8 or human VR24-JR18/Vâ11) T cell
receptors (TCR) and natural killer (NK) receptors. They can be
stimulated by the glycolipid antigens that are presented by major
histocompatibility complex class-I-like protein CD1d for specific
recognition. Upon stimulation, iNKT cells rapidly secrete
copious cytokines (Figure 1). The interferon-γ (IFN-γ) and
interleukin-2 (IL-2) belong to Th1-type cytokines, tend to
produce the proinflammatory response, and are responsible for
fighting bacterial, parasitic, and viral infections. However, the
Th1 response also causes multiple sclerosis, lupus, and type 1
diabetes. Th2-type cytokines, including IL-4 and IL-10, can
result in an immunomodulatory response. In excess, Th2
responses, however, will counteract the Th1 mediated autoim-
mune diseases.1
Figure 1. (a) Schematic illustration for the event of iNKT cells
Agelasphins are a kind of R-galactosylceramide (R-GalCer)
stimulation by glycoceramide. The glycoceramide is presented by the
that were isolated from the marine sponge Agelas mauritianus
CD1d of APC to the receptor of iNKT cells. Upon glycoceramide
stimulation, iNKT cells produce the Th1 and Th2 cytokines. (b) The
structure of isoglobotrihexosylceramide (iGb3) consists of a trisaccha-
in 1992. They are the first exogenous antigens that could be
presented by CD1d to stimulate iNKT cells.2 After decades of
searching, isoglobotrihexosylceramide (iGb3) (Figure 1) was
discovered as an endogenous mammalian glycosphingosine that
can also stimulate iNKT cells via presentation by CD1d.3 The
most distinctive difference between agelasphins and iGb3 is that
the agelasphins contain an R-linkage between the sugar and
ceramide, while iGb3 has a â-linkage.
ride and ceramide with a â-linkage and is the endogenous antigen of
iNKT cells.
(SAR).4 It was found that modification of the ceramide chains
could polarize the secretion of Th1 and Th2 cytokines. The
glycoceramide OCH, which is an R-GalCer analogue, is
structurally distinct from other R-GalCer analogues in having
a substantially shorter sphingosine chain.5 OCH can stimulate
iNKT cells to preferentially produce Th2 cytokines. Another
glycoceramide C20:2, which contains two double bonds in the
acyl chain, can induce strong Th1 production and induce cellular
proliferation.6
Crystallographic studies of the CD1d/R-GalCer complexes
illustrated that the ceramide moiety of R-GalCer contributes to
the binding with CD1d by anchoring R-GalCer in a distinct
orientation.7 Both alkyl chains are initially inserted perpendicu-
larly to the â-sheet platform and then extend more laterally
toward the ends of the A′ and F′ pockets (Figure 2). A specific
Since the discovery of R-GalCer, numerous researchers have
struggled to demonstrate its structure-activity-relationship
* To whom correspondence should be addressed. Phone: (+1) 614-292-
9884. Fax: (+1) 614-688-3106. E-mail: wang.892@osu.edu.
† The Ohio State University.
‡ University Hospital Basel.
a Abbreviations: iGb3, isoglobotrihexosylceramide; iNKT, invariant
natural killer T; Th1, T helper 1; IFN-γ, interferon-γ; IL-2, interleukin-2;
DDQ, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone; DMAP, dimethyl-
aminopyridine; EDCI, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide
hydrochloride; TMSOTf, trimethylsilyltrifluoromethane sulfonate; ELISA,
enzyme linked immunosorbent serologic assay.
10.1021/jm0701066 CCC: $37.00 © 2007 American Chemical Society
Published on Web 07/04/2007

3490 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15
Xia et al.
Figure 2. Crystal structure of R-GalCer with CD1d. There are two
pockets in the CD1d molecule. The acyl chain is located in the A′
pocket, and the sphingosine chain is located in the F′ pocket. The sugar
head stands outside for recognition by the TCR.
Figure 3. Two iGb3 analogues with different ceramides.
trisaccharide donor 10, which was readily prepared from
1-benzyllactose 3 (Scheme 1). According to our published
protocol, the 3′′-OH of lactose 3 was activated by the formation
of a dibutylstannylene acetal.11 Regioselective monoalkylation,
in a one-pot manner, by subjection to 4-methoxybenzyl chloride
(PMBCl) afforded compound 4. The remaining hydroxy groups
were protected with a bulky pivaloyl group by treatment with
pivaloyl chloride and DMAP in pyridine at 70 °C. The use of
a bulky pivaloyl group is important for avoiding the formation
of the orthoester in the glycosylation with the lipid. Selective
deprotection of the PMB group was achieved by treatment
with DDQ to release the 3′′-OH to afford the disaccharide
acceptor 6.
The glycosylation between the perbenzylated phenylthiogly-
coside donor 7 and the lactosyl acceptor 6 was carried out at
-30 °C under activation with N-iodosuccinimide (NIS)/triflic
acid to produce the protected trisaccharide 8. The structure of
trisaccharide 8 was confirmed by the JH1-H2 ) 2.4 Hz at δ 5.51
ppm, indicating an R linkage. The trisaccharide 8 was subjected
to Pd(OH)2 under hydrogen atmosphere to remove all the benzyl
groups. The newly formed free hydroxy groups were then
protected with acetyl groups by using acetic anhydride and
pyridine to provide compound 9. The anomeric acetyl group
was selectively removed by treatment with benzylamine in THF.
Finally, the donor trichloroacetimidate 10 was obtained by
treatment with trichloroacetonitrile and 1,8-diazabicyclo[5.4.0]-
under-7-ene (DBU).
hydrogen-bond network was observed between CD1d and
glycoceramide. Binding was assisted by these interactions,
specifically between three conserved residues (mouse Asp153,
Asp80, and Thr156; human Asp151, Asp80, and Thr154) in
CD1d and the 2′-OH on the galactose ring, the 3-OH on the
sphingosine chain, and the 1′-O glycosidic linkage. Aromatic
residues Tyr73 (A′ pocket), Phe77, and Trp133 (F′ pocket) make
extensive van der Waals contacts with the glycoceramide,
stabilizing both alkyl chains after insertion into their individual
binding pockets. The phytosphingosine moiety fully occupies
the F′ pocket, which can accommodate linear alkyl chains of
up to C18. In pocket A′, a linear hydrophobic compound has
been found, which acts as a “spacer lipid” similar to the
detergent molecules initially noted in the CD1b phosphatidyli-
nositol structure.8 Such a factor could stabilize the hydrophobic
binding pocket in the absence of an antigenic groove-filling
ligand, such as a full-length R-GalCer for CD1d or a glucose
monomycolate for CD1b.9
To the best of our knowledge, only a few analogues of iGb3
have been synthesized to explore its SAR.10 Herein, we report
the synthesis of two novel iGb3 analogues with different
ceramides and their associated stimulatory activity of iNKT
cells.
Results and Discussion
On the basis of the structure-activity relationship studies of
R-GalCer, we assumed that varying the lipid portion of iGb3
would change its ability to bind with CD1d. This would further
affect its activities in stimulating iNKT cells and could polarize
iNKT cell release toward either the Th1 or the Th2 cytokines.
The difference between the lipid moieties of endogenous iGb3
and exogenous R-GalCer is that the former is D-erythrosphin-
gosine, while the latter is phytosphingosine. The D-erythrosph-
ingosine has a double bond between C4 and C5, whereas the
phytosphingosine has an extra hydroxy group on C4. The
additional hydroxy group of phytosphingosine may form extra
hydrogen bonds with CD1d and thereby increase the stability
of the CD1d/glycoceramide complex. This additional hydroxyl
group may also effect the orientation of the sugar head on the
surface of CD1d for recognition by the T cell receptor (TCR)
of iNKT cells. The trans double bond of D-erythrosphingosine
has some function in fixing the conformation of lipid. Reduction
of the double bond will increase the rotatable flexibility of the
lipid, thus altering the rate of complex formation.
Since the H-bonding of the amide group will reduce the
reactivity of the ceramide acceptor, the azidosphingosine 11 was
used as the acceptor.12 After extensive experimentation with a
variety of Lewis acids, we found that glycosylation catalyzed
by TMSOTf at -20 °C in the presence of 4 Å molecular sieves
worked extremely well with â-selective glycosylation to give
compound 12 in excellent yield (Scheme 2). To incorporate the
fatty acid on the lipid, the azide was reduced to an amine
quantitatively with triphenylphosphine-H2O in benzene at
60 °C. The crude amine was subjected to amide formation with
cerotic acid in the presence of EDCI in THF to provide the
protected glycoceramide 13. The compound was then hydro-
genated to remove the PMB protecting group and reduce the
double bond on the lipid. Finally, the acetyl and pivaloyl
protecting groups were removed under reflux conditions with
a catalytic amount of NaOMe in anhydrous methanol to achieve
1 in high yield.
To prepare the iGb3 analogue that has the phytosphingosine
type ceramide instead of the D-erythrosphingosine type ceramide,
azidophytosphingosine 16 was used as an acceptor (Scheme 3).
It was easily prepared from commercially available phytosph-
ingosine 14 by conducting a diazotransfer reaction with CuSO4
as the catalyst13 to give the azido lipid 15.14 The primary
hydroxyl group was selectively protected with trityl chloride,
Initially, the structure-activity relationship study of the
ceramide of iGb3 focused on the functional groups on the lipid
part. Two iGb3 analogues, 2H-iGb3 (1) and HO-iGb3 (2), were
designed on the basis of the above rationale (Figure 3). Both
of the two iGb3 analogues could be synthesized from the same

Modification of the Ceramide Moiety
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15 3491
Scheme 1. Synthesis of Trisaccharide Donora
a Reagents and conditions: (a) (i) Bu2SnO, MeOH, reflux; (ii) PMBCl, Bu4NI, benzene, reflux; (b) PivCl, DMAP (cat.), pyridine, 52% for three steps;
(c) DDQ, H2O, DCM, 76%; (d) NIS, TfOH, DCM, -30 °C, 84%; (e) (i) H2, Pd/C, MeOH; (ii) Ac2O, pyridine, 97%; (f) (i) BnNH2, THF; (ii) CCl3CN, DBU,
DCM, 78%.
Scheme 2. Synthesis of 1a
a Reagents and conditions: (a) TMSOTf, DCM, -25 °C, 87%; (b) (i) PPh3, H2O, benzene, 50 °C; (ii) C25H51CO2H, EDCl, DCM, 95%; (c) (i) 10% Pd/C,
H2, EtOH; (ii) NaOMe, MeOH, reflux, 82%.
Scheme 3. Synthesis of 2a
a Reagents and conditions: (a) TfN3, CuSO4, DCM/MeOH/H2O, 89%; (b) (i) TrtCl, DMAP, pyr, 50 °C; (ii) NaH, BnBr, DMF; (iii) pTsOH, MeOH, 68%;
(c) TMSOTf, DCM, -25 °C, 81%; (d) (i) PPh3, H2O, benzene, 50 °C; (ii) C25H51CO2H, EDCI, DCM, 84%; (e) (i) H2, 10% Pd/C, MeOH; (ii) NaOMe,
MeOH, reflux, 76%.
after which the two secondary hydroxy groups were protected
with benzyl bromide. Finally, removal of the trityl group under
acidic conditions afforded the lipid acceptor 16.15 Under the
activation of TMSOTf, the azido acceptor 16 was glycosylated
with donor 10 to produce glycolipid 17. The azide was reduced
and the cerotic acid was introduced, as in the preparation of
13, to generate the protected glycoceramide 18. Finally, the
two benzyl groups were removed by hydrogenation with 10%
Pd/C in methanol under hydrogen atmosphere and the remain-
ing esters were hydrolyzed with NaOMe in refluxing MeOH to
give 2.
IFN-γ (Th1-type cytokine) and IL-4 (Th2-type cytokine)
productions were measured to evaluate the abilities of the iGb3
analogues to stimulate iNKT cells. The relative potencies of
iGb3, 1, and 2 were measured with the use of human VR24i
NKT cell clone (JS7) and hCD1d-transfected human B lym-

3492 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15
Xia et al.
Figure 4. iNKT cells release IFN-γ and IL-4 cytokines when activated by iGb3 analogues.
phoblastoid (C1R). The release of cytokines was measured by
enzyme linked immunosorbent serologic assay (ELISA) from
cocultured supernatants of the iNKT cells and antigen presenting
cells (APC) with different concentrations of glycoceramides
(Figure 4). The results showed that 1 was slightly more active
in stimulation of iNKT cells in low concentration relative to
iGb3. At higher concentrations (e.g., 20 µg/mL), very efficient
stimulation of iNKT cells was observed. These results indicated
that reduction of the double bond on the lipid to the saturated
bond increased the flexibility of the glycolipids, which made it
easier for 1 to adopt the appropriate confirmation to enter the
F′ pocket of CD1d. An impressive stimulation was observed
when using 2 to activate iNKT cells. Both Th1 and Th2
cytokines were released when human iNKT cells were stimu-
lated with 2 at 10 ng/mL (100-fold lower concentration than
iGb3). This may result from the greater stability of the 2 and
CD1d complex because the 4-OH of the phytosphingosine can
form an additional hydrogen bond with the F′ pocket. The results
are in agreement with the observation by Kronenberg et al.16
They examined the interactions between the solubilized form
of the TCR and CD1d-glycolipd complex by surface plasmon
resonance. The equilibrium dissociation constants (KD), based
on TCR binding at equilibrium, are 0.35 µM for KRN7000
(same ceramide as 2) and 1.12 µM for 4-deoxy-GalCer (same
ceramide as 1).
replacement of the D-erythrosphingosine moiety with phytosph-
ingosine led to the generation of 2. Two iGb3 analogues with
different ceramides were synthesized by first constructing the
trisaccharide scaffold and then by introducing the lipid part.
The release of cytokines by iNKT cells involves two stages.
The glycoceramide is first loaded on CD1d, and then the CD1d/
glycoceramide complex will bind to iNKT cells.17 Any develop-
ment of the glycoceramides that can improve one of the two
stages will change the status of cytokine production by iNKT
cells. The saturated lipid part of 1 increased its rotational
flexibility, which was beneficial to its uploading on CD1d.
Bioassay results showed that 2 is much more efficient in
stimulating iNKT cells compared to iGb3 at very low concen-
trations. Introduction of the additional hydroxyl group on the
ceramide part of iGb3 may improve the stability of the CD1d/
glycoceramide complex.
Experimental Section
All solvents were dried with a solvent-purification system from
Innovative Technology, Inc. All reagents were obtained from
commercial 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
1
utilized for all chromatographic purifications. H 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.
However, the modification of the lipid part of iGb3 by
reducing the double bond or introducing an extra hydroxy group
did not polarize the release of cytokines. Both of the Th1 and
Th2 cytokines were produced when the iNKT cells were
incubated with APC and iGb3 analogues. The reported modi-
fications of R-GalCer to change the bias of Th1 and Th2
secretion were mostly successful from the lipids instead of the
polar portion of ceramide. To obtain the selectivity toward either
Th1 or Th2 cytokines response, more investigation is needed
for the lipid part of the ceramide.
Benzyl 2,4,6-Tri-O-pivaloyl-3-(4-methoxybenzyl)-â-D-galac-
topyranosyl-(1f4)-2,3,6-tri-O-pivaloyl-â-D-glucopyranoside (5).
A suspension of benzyllactose 3 (13.0 g, 30.1 mmol) and Bu2SnO
(9.0 g, 36.1 mmol) in anhydrous MeOH (150 mL) was heated to
reflux and stirred for 6 h. The solvent was removed in vacuo. Then
the residue was dissolved in dry benzene (150 mL). p-Methoxy-
benzyl chloride (4.9 mL, 36.1 mmol), tetrabutylammonium iodide
(4.43 g, 12.0 mmol), and 4 Å molecular sieves (5 g) were added.
The resulting mixture was heated to reflux for another 6 h and
then cooled to room temperature. The suspension was filtered
through a Celite pad and the filtrate was concentrated and
chromatographied (6:1 chloroform-methanol) to afford 10.6 g of
crude product 4 (64% yield).
Conclusion
In summary, we described a SAR study guided by iGb3, an
endogenous antigen for iNKT cells. Reduction of the double
bond of the D-erythrosphingosine moiety gave 1. Furthermore,
Pivaloyl chloride (5.4 mL, 43.47 mmol) was added to a solution
of the above crude (3.00 g, 5.43 mmol) and 4-dimethylaminopy-

Modification of the Ceramide Moiety
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15 3493
ridine (50 mg) in dry pyridine (20 mL). The reaction mixture was
heated for 36 h at 70 °C and then allowed to cool to room
temperature. The solvent was removed in vacuo. The residue was
dissolved in EtOAc (100 mL), washed with 1 N HCl (60 mL),
saturated aqueous NaHCO3 (60 mL), and brine (50 mL) succes-
sively, dried (Na2SO4), and evaporated. The crude product was
purified by silica gel flash chromatography (1:2 EtOAc-hexane)
Acetyl 2,3,4,6-Tetra-O-acetyl-r-D-galactopyranosyl-(1f3)-
2,4,6-tri-O-pivaloyl-â-D-galactopyranosyl-(1f4)-2,3,6-tri-O-piv-
aloyl-â-D-glucopyranoside (9). Palladium hydroxide (200 mg) was
added to a solution of trisaccharide 8 (1.35 g, 0.93 mmol) in MeOH
(15 mL), and the reaction mixture was stirred under hydrogen
balloon pressure for 12 h. The palladium hydroxide was then filtered
through a Celite pad and concentrated in vacuo. The crude residue
was dissolved in anhydrous pyridine (15 mL). Acetic anhydride
(0.7 mL) and DMAP (40 mg) were added, and the solution was
stirred for 10 h. The solvent was removed in vacuo, and the residue
was dissolved in EtOAc (40 mL). The organic layer was washed
with 1 N HCl (30 mL), saturated aqueous NaHCO3 (30 mL), and
brine (30 mL) successively, dried over Na2SO4, concentrated in
vacuo, and purified by silica gel flash chromatography (1:2 EtOAc-
1
to give compound 5 (4.70 g 82%) as a clear oil. H NMR (400
MHz, CDCl3): δ 7.32 (m, 5H), 7.13 (d, J ) 8.6 Hz, 2H), 6.85
(d, J ) 8.6 Hz, 2H), 5.51 (d, J ) 2.5 Hz, 1H), 5.24 (t, J ) 9.4 Hz,
1H), 5.06 (dd, J ) 8.4, 9.4 Hz, 1H), 4.96 (dd, J ) 8.4, 9.4 Hz,
1H), 4.84 (d, J ) 11.4 Hz, 1H), 4.59 (m, 4H), 4.42 (d, J ) 9.4 Hz,
1H), 4.27 (m, 2H), 4.13 (m, 2H), 3.88 (m, 2H), 3.80 (s, 3H), 3.69
(m, 1H), 3.54 (dd, J ) 2.6, 9.4 Hz, 1H), 1.24 (s, 9H), 1.22 (s, 9H),
1.16 (s, 9H), 1.13 (s, 9H), 1.12 (s, 9H), 1.08 (s, 9H). 13C NMR
(125 MHz, CDCl3): δ 206.9, 177.9, 177.9, 177.5, 177.0, 176.7,
176.0, 159.2, 136.6, 129.3, 129.2, 128.4, 128.0, 100.3, 99.4. 78.0,
76.8, 73.9, 73.4, 71.8, 71.6, 71.5, 70.5, 70.1, 65.6, 62.0, 61.9, 55.2,
39.0, 38.95, 38.8, 38.7, 38.66, 30.9, 27.3, 27.29, 27.2, 27.1, 27.09,
27.06. HRMS calcd for C57H84O18Na ([M + Na]+) 1079.5550,
found 1079.5555.
1
hexane) to afford compound 9 (1.1 g, 97%) as a white foam. H
NMR (400 MHz, CDCl3): δ 5.68 (d, J ) 8.4 Hz, 1H), 5.42-5.38
(m, 2H), 5.25-5.21 (m, 3H), 5.13 (m, 1H), 5.00 (m, 1H), 4.93
(t, J ) 8.4 Hz, 1H), 4.46-4.40 (m, 2H), 4.31 (dd, J ) 12.0, 4.0
Hz, 1H), 4.12-3.99 (m, 6H), 3.91 (m, 1H), 3.81 (dd, J ) 6.4
Hz, 1H), 3.77 (m, 1H), 3.68 (m, 1H), 2.09 (s, 3H), 2.06 (s, 3H),
2.02 (s, 3H), 1.99 (s, 3H), 1.90 (s, 3H), 1.23-1.14 (m, 45H), 1.10
(s, 9H). 13C NMR (100 MHz, CDCl3): δ 177.8, 177.6, 176.8, 176.7,
176.0, 170.3, 170.1, 170.0, 169.69, 168.8, 100.2, 94.3, 91.7, 73.0,
72.1, 71.2, 70.4, 69.8, 67.5, 67.47, 67.1, 66.9, 65.3, 61.7, 61.0, 60.4,
39.0, 38.97, 38.9, 38.8, 38.7, 38.68, 27.3, 27.31, 27.2, 27.1, 27.0,
26.9, 21.0, 20.6, 20.58, 20.5. HRMS calcd for C58H90O27Na
([M + Na]+) 1241.5562, found 1241.5540.
Benzyl 2,4,6-Tri-O-pivaloyl-â-D-galactopyranosyl-(1f4)-2,3,6-
tri-O-pivaloyl-â-D-glucopyranoside (6). To a solution of com-
pound 5 (3.00 g, 2.83 mmol) in CH2Cl2 (27 mL) were added H2O
(3 mL) and DDQ (1.29 g, 5.55 mmol). The reaction mixture was
stirred at room temperature for 12 h until compound 3 was no longer
detectable in TLC. The mixture was diluted with CH2Cl2 (30 mL),
washed with water (3 × 40 mL), dried (Na2SO4), and evaporated.
Purified by silica gel flash chromatography (1:1 EtOAc-hexane)
2,3,4,6-Tetra-O-acetyl-r-D-galactopyranosyl-(1f3)-2,4,6-tri-
O-pivaloyl-â-D-galactopyranosyl-(1f4)-2,3,6-tri-O-pivaloyl-r-
D-glucopyranosyl-(1f1)-trichloroacetimide (10). Benzylamine
(0.2 mL, 1.8 mmol) was added to a solution of compound 9 (1.1 g,
0.90 mmol) in THF (20 mL). After being stirred overnight at room
temperature, the reaction mixture was concentrated in vacuo. The
residue was diluted with CH2Cl2 (20 mL) and then was washed
with 1 N HCl (10 mL), saturated aqueous NaHCO3 (10 mL), and
brine (10 mL), dried over Na2SO4, and concentrated. The crude
product was used for next step reaction without further purification.
The above crude residue was dissolved in dry CH2Cl2 (10 mL).
Then CCl3CN (0.9 mL, 9.0 mmol) and DBU (67 µL, 0.45 mmol)
were added successively. The reaction mixture was stirred for 4 h.
The solvent was removed in vacuo and the residue was purified
by silica gel flash chromatography (1:2 EtOAc-hexane) to afford
1
to give compound 6 (2.02 g, 76%) as a clear oil. H NMR (400
MHz, CDCl3): δ 7.32 (m, 5H), 5.34 (d, J ) 2.5 Hz, 1H), 5.28
(t, J ) 9.2 Hz, 1H), 4.95 (dd J ) 8.4, 9.2 Hz, 1H), 4.84 (m, 2H),
4.60 (m, 2H), 4.49 (d J ) 9.2 Hz, 1H), 4.33 (dd J ) 4.6, 11.9 Hz,
1H), 4.12 (m, 2H), 3.88 (m, 3H), 3.57 (m, 1H), 2.61 (br, 1H), 1.24
(m, 54H). 13C NMR (125 MHz, CDCl3): δ 178.7, 177.8, 177.6,
177.0, 176.5, 136.4, 128.3, 127.8, 99.7, 99.2, 73.8, 73.3, 72.6, 72.2,
71.7, 71.4, 71.2, 70.4, 69.2, 61.8, 61.6, 39.0, 38.8, 38.5, 30.7, 27.2,
26.9. HRMS calcd for C49H76O17Na ([M + Na]+) 959.4975, found
959.4957.
Benzyl 2,3,4,6-Tetra-O-benzyl-r-D-galactopyranosyl-(1f3)-
2,4,6-tri-O-pivaloyl-â-D-galactopyranosyl-(1f4)-2,3,6-tri-O-piv-
aloyl-â-D-glucopyranoside (8). A suspension of acceptor 6 (1.04
g, 1.1 mmol), perbenzylgalactosyl donor 7 (1.05 g, 1.66 mmol),
and 4 Å molecular sieves (2 g) in dry CH2Cl2 (20 mL) was stirred
at room temperature for 30 min. After the mixture was cooled to
-30 °C, N-iodosuccinimide (272 mg, 1.21 mmol) was added
followed by TMSOTf (20 µL, 0.2 mmol). The resulting mixture
was stirred at -30 °C for 2 h and then was allowed to warm slowly
to 10 °C. The mixture was diluted with CH2Cl2 (20 mL), and the
molecular sieves were filtered. The filtrate was washed with
saturated aqueous NaHCO3 (20 mL), 10% Na2S2O3 (20 mL), and
brine (20 mL), dried (Na2SO4), and concentrated. The residue was
purified by silica gel flash chromatography (1:8 EtOAc-hexane)
to furnish trisaccharide 8 (1.35 g, 84%) as a white foam. 1H NMR
(400 MHz, CDCl3): δ 7.36-7.19 (m, 25H), 5.51 (d, J ) 2.4 Hz,
1H), 5.16 (t, J ) 9.6 Hz, 1H), 5.08 (dd, J ) 10.0, 8.0 Hz, 1H),
4.90-4.77 (m, 5H), 4.67 (d, J ) 11,6 Hz, 1H), 4.61 (d, J ) 12.0
Hz, 1H), 4.56-4.50 (m, 3H), 4.47 (d, J ) 10.8 Hz, 1H), 4.44
(d, J ) 11.6 Hz, 1H), 4.35 (d, J ) 11.6 Hz, 1H), 4.26 (d, J ) 8.0
Hz, 1H), 4.24 (dd, J ) 12.0, 5.6 Hz, 1H), 3.98-3.92 (m, 3H),
3.89-3.85 (m, 3H), 3.81 (t, J ) 9.6 Hz, 1H), 3.65 (dd, J ) 10.4,
3.2 Hz, 1H), 3.53-3.49 (m, 3H), 3.41 (dd, J ) 6.8, 5.6 Hz, 1H),
1.23 (s, 9H), 1.21 (s, 9H), 1.18 (s, 9H), 1.16 (s, 9H), 1.14 (s, 9H),
1.11 (s, 9H), 1.08 (s, 9H). 13C NMR (125 MHz, CDCl3): δ 177.8,
177.0, 176.9, 176.7, 175.9, 138.9, 138.8, 138.7, 138.2, 136.6, 128.4,
128.3, 128.2, 128.1, 128.0, 127.95, 127.7, 127.6, 127.5, 127.4,
100.4, 99.4, 98.2, 78.9, 76.1, 75.2, 74.9, 73.9, 73.8, 73.5, 73.2, 73.1
72.1, 71.7 71.4, 70.7, 70.5, 70.3, 68.1, 67.5, 62.0, 61.97, 39.0, 38.9,
38.8, 38.7, 38.6, 30.9, 27.3, 27.3, 27.3, 27.2, 27.1. HRMS calcd
for C83H110O22Na ([M + Na]+) 1481.7381, found 1481.7327.
1
compound 10 (0.93 g, 78%) as a clear oil. H NMR (500 MHz,
CDCl3): δ 8.62 (s, 1H), 6.45, (d, J ) 3.5 Hz, 1H), 5.56 (t, J )
10.0 Hz, 1H), 5.39 (dd, J ) 8.0, 2.5 Hz, 1H), 5.25-5.22 (m, 2H),
5.15 (dd, J ) 10.0, 8.0 Hz, 1H), 5.01 (m, 1H), 4.94 (dd, J ) 10.5,
4.0 Hz, 1H), 4.45 (m, 2H), 4.35 (dd, J ) 10.0, 4.5 Hz, 1H), 4.14-
4.03 (m, 7H), 3.96 (t, J ) 9.5 Hz, 1H), 3.83 (t, J ) 6.5 Hz, 1H),
3.79 (dd, J ) 10.5, 3.0 Hz, 1H), 2.10 (s, 3H), 2.05 (s, 3H), 2.00 (s,
3H), 1.91 (s, 3H), 1.22 (s, 9H), 1.20 (s, 9H), 1.90 (s, 27H), 1.11 (s,
9H). 13C NMR (125 MHz, CDCl3): δ 177.7, 177.63, 177.6, 177.5,
176.8, 176.6, 176.4, 176.0, 171.1, 170.3, 170.1, 170.0, 169.7, 160.9,
160.6, 100.3, 94.2, 92.6, 90.8, 74.1, 72.9, 72.0, 71.5, 70.1, 69.8,
68.7, 67.5, 67.0, 66.8, 65.3, 61.5, 61.46, 61.4, 61.0, 60.4, 39.0,
38.97, 38.9, 38.8, 38.75, 38.7, 27.3, 27.2, 27.16, 27.14, 27.1, 27.0,
21.0, 20.6, 20.4, 14.2. HRMS ([M + Na]+) 1342.451 73, calcd.
1342.455 24.
2,3,4,6-Tetra-O-acetyl-r-D-galactopyranosyl-(1f3)-2,4,6-tri-
O-pivaloyl-â-D-galactopyranosyl-(1f4)-2,3,6-tri-O-pivaloyl-â-D-
glucopyranosyl(1f1)-(2S,3R,4E)-2-azido-3-O-(4-methoxybenzyl)-
4-octadecen-1,3-diol. (12). A suspension of trisaccharide donor 10
(450 mg, 0.34 mmol), sphingosine acceptor 11 (193 mg, 0.41
mmol), and 4 Å molecular sieves (2 g) in dry CH2Cl2 (10 mL) was
stirred at room temperature for 30 min. After cooling to -20 °C,
TMSOTf (14 µL, 0.068 mmol) was added. The resulting mixture
was stirred for 2 h and then diluted with CH2Cl2 (10 mL). Saturated
aqueous NaHCO3 (10 mL) was added to quench the reaction. The
molecular sieves were filtered through a Celite pad. The filtrate
was washed with brine (5 mL), dried over Na2SO4, and concen-
trated. The residue was purified by silica gel flash chromatography
(1:3 EtOAc-hexane) to provide 12 (485 mg, 87%) as a clear oil.

3494 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15
Xia et al.
1H NMR (500 MHz, CDCl3): δ 7.21 (d, J ) 8.5 Hz, 1H), 6.87
(d, J ) 8.5 Hz, 1H), 5.72 (dt, J ) 15.5, 6.5 Hz, 1H), 5.42 (dd, J
) 9.0, 2.0 Hz, 1H), 5.37 (dd, J ) 15.5, 8.5 Hz, 1H), 5.28-5.25
(m, 3H), 5.22 (t, J ) 9.8 Hz, 1H), 5.16 (dd, J ) 10.0, 8.0 Hz, 1H),
5.05 (m, 1H), 4.87 (dd, J ) 9.8, 7.5 Hz, 1H), 4.53 (d, J ) 7.5 Hz,
1H), 4.52 (m, 1H), 4.50 (d, J ) 11.5 Hz, 1H), 4.45 (d, J ) 8.0 Hz,
1H), 4.30 (dd, J ) 12.0, 5.5 Hz, 1H), 4.26 (d, J ) 11.5 Hz, 1H),
4.16 (m, 1H), 4.11-4.05 (m, 4H), 3.91 (t, J ) 9.5 Hz, 1H), 3.87-
3.79 (m, 4H), 3.82 (s, 3H), 3.63 (dd, J ) 10.5, 4.0 Hz, 1H), 3.59
(m, 1H), 3.53 (m, 1H), 2.17 (s, 3H), 2.10 (s, 3H), 2.07 (s, 3H),
1.94 (s, 3H), 1.38 (m, 2H), 1.32-1.26 (m, 22H), 1.25 (s, 9H), 1.24
(s, 9H), 1.23 (s, 9H), 1.22 (s, 9H), 1.20 (s, 9H), 1.16 (s, 9H), 0.89
(t, J ) 7.0 Hz, 3H). 13C NMR (125 MHz, CDCl3): δ 177.8, 177.7,
177.0, 176.8, 176.6, 176.0, 170.3, 170.1, 170.0, 169.7, 159.2, 137.9,
130.2, 129.2, 126.0, 113. 8, 100.7, 100.3, 94.4, 79.3, 76. 8, 74.5,
73.6, 73.5, 72.0, 71.6, 71.4, 70.0, 69.9, 68.6, 67.54, 67.5, 67.1, 66.9,
65.3, 64.2, 61.9, 61.6, 61.0, 55.2, 39.0, 38.95, 38.9, 38.7, 38.72,
38.67, 32.4, 31.9, 29.7, 29.65, 29.6, 29.5, 29.3, 29.2, 29.0, 28.0,
27.3, 27.2, 27.1, 27.09, 22.7, 21.0, 20.6, 20.5, 14.1. HRMS calcd
for C82H129N3O28Na ([M + Na]+) 1626.8655, found 1626.8696.
2,3,4,6-Tetra-O-acetyl-r-D-galactopyranosyl-(1f3)-2,4,6-tri-
O-pivaloyl-â-D-galactopyranosyl-(1f4)-2,3,6-tri-O-pivaloyl-â-D-
glucopyranosyl-(1f1)-(2S,3R,4E)-2-hexacosanoylamino-3-O-(4-
methoxybenzyl)-4-octadecen-1,3-diol (13). Triphenylphosphine
(154 mg, 0.59 mmol) was added to a solution of azide 12 (480
mg, 0.29 mmol) in benzene (20 mL) and water (0.2 mL). The
reaction mixture was stirred at 50 °C for 8 h. The solvent was
evaporated under reduced pressure and azetroped with benzene (2
× 20 mL). Then it was dissolved in dry THF (10 mL) and treated
with cerotic acid (167 mg, 0.42 mmol) and EDCI (81 mg, 0.42
mmol). After the mixture was stirred for 10 h at room temperature,
the solvent was evaporated and the residue was partitioned between
CH2Cl2 (20 mL) and water (10 mL). The organic layer was
separated and dried over Na2SO4. After the layer was concentrated,
the residue was purified by silica gel flash chromatography (1:3
EtOAc-hexane) to provide 13 (540 mg, 95%) as a foam. 1H NMR
(500 MHz, CDCl3): δ 7.17 (d, J ) 8.5 Hz, 1H), 6.82 (d, J ) 8.5
Hz, 1H), 5.61 (dt, J ) 15.0, 8.0 Hz, 1H), 5.50 (d, J ) 8.5 Hz, 1H),
5.39 (dd, J ) 6.0, 2.5 Hz, 2H), 5.30 (dd, J ) 15.5, 8.5 Hz, 1H),
5.24-5.17 (m, 3H), 5.13 (dd, J ) 10.0, 8.0 Hz, 1H), 5.02 (dd, J )
11.0, 3.5 Hz, 1H), 4.81 (dd, J ) 9.5, 8.0 Hz, 1H), 4.46 (d, J ) 9.0
Hz, 1H), 4.43 (d, J ) 4.0 Hz, 1H), 4.41 (d, J ) 8.0 Hz, 1H), 4.31
(dd, J ) 12.5, 5.0 Hz, 1H), 4.18 (d, J ) 11.0 Hz, 1H), 4.14-4.01
(m, 7H), 3.88 (t, J ) 9.5 Hz, 1H), 3.82 (t, J ) 6.5 Hz, 1H), 3.78
(s, 3H), 3.77-3.73 (m, 2H), 3.54 (m, 1H), 3.51 (dd, J ) 9.0, 3.0
Hz, 1H), 2.08 (s, 3H), 2.05 (s, 3H), 2.03 (s, 3H), 1.95 (s, 3H),
1.62-1.48 (m, 4H), 1.30-1.21 (m, 68H), 1.22 (s, 9H), 1.21
(s, 9H), 1.19 (s, 9H), 1.17 (s, 9H), 1.16 (s, 9H), 1.14 (s, 9H), 0.86
(t, J ) 6.5 Hz, 6H). 13C NMR (125 MHz, CDCl3): δ 177.8, 177.6,
176.9, 176.8, 176.76, 176.0, 172.4, 170.3, 170.1, 170.0, 169.7,
159.1, 136.8, 130.6, 129.1, 127.5, 113.7, 100.8, 100.1, 94.4, 79.4,
74.4, 73.5, 73.4, 72.0, 71.3, 70.1, 69.9, 68.1, 67.5, 67.5, 67.1, 66.9,
65.3, 61.8, 61.5, 61.0, 55.2, 51.6, 39.0, 39.0, 38.9, 38.8, 38.7, 38.66,
36. 9, 32.3, 31.9, 29.7, 29.7, 29.65, 29.5, 29.4, 29.39, 29.35, 29.3,
29.28, 27.8, 27.3, 27.2, 27.21, 27.13, 27.11, 27.10, 25.7, 22.68,
21.0, 20.6, 20.5, 14.1. HRMS calcd for C108H181NO29Na ([M +
Na]+) 1980.2645, found 1980.2728.
7.9 Hz, 1H), 5.67 (d, J ) 3.5 Hz, 1H), 5.07 (d, J ) 4.9 Hz, 1H),
5.03 (t, J ) 6.1 Hz, 1H), 4.89 (d, J ) 7.8 Hz, 1H), 4.77 (dd, J )
10.3, 4.6 Hz, 1H), 4.73 (dd, J ) 9.9, 3.7 Hz, 1H), 4.70 (m, 1H),
4.68 (d, J ) 2.7 Hz, 1H), 4.59 (d, J ) 2.7 Hz, 1H), 4.56-4.52
(m, 3H), 4.48-4.43 (m, 4H), 4.33 (dd, J ) 11.0, 5.1 Hz, 1H), 4.27-
4.21 (m, 4H), 4.18 (dd, J ) 10.5, 3.3 Hz, 1H), 4.06-4.03 (m, 2H),
3.88 (m, 1H), 2.48 (t, J ) 7.4 Hz, 2H), 1.93-1.83 (m, 5H), 1.58
(m, 1H), 1.42-1.21 (m, 68H), 0.90 (t, J ) 6.6 Hz, 3H), 0.89
(t, J ) 7.1 Hz, 3H). 13C NMR (125 MHz, pyridine-d5): δ 173.1,
105.3, 105.2, 97.5, 81.9, 79.9, 76.5, 76.3, 74.5, 72.6, 71.4, 71.1,
70.6, 70.3, 70.2, 65.8, 62.0, 61.8, 61.6, 54.7, 36.7, 34.7, 31.9, 31.89,
30.0, 29.9, 29.8, 29.77, 29.7, 29.68, 29.6, 29.5, 29.4, 29.37, 26.3,
26.2, 22.7, 14.0. HRMS calcd for C62H119NO18Na ([M + Na]+)
1188.8325, found 1188.8307.
(2S,3S,4S)-2-Azidooctadecan-1,3,4-triol (15). To a mixture
solution of CH2Cl2 (5 mL) and H2O (5 mL) containing NaN3 (2.05
g, 31.5 mmol) cooled at 0 °C was added dropwise Tf2O (1.1 mL,
6.3 mmol) in 20 min. After addition, the resulting mixture was
stirred for 3 h. The organic layer was separated, and the aqueous
portion was extracted with CH2Cl2 (2 × 2 mL). The combined
organic layer was washed with saturated aqueous Na2CO3.
To a suspension of phytosphingosine 14 (1.0 g, 3.1 mmol), K2-
CO3 (2.18 g, 15.8 mmol), and Cu2SO4 (20 mg) in a mixture of
MeOH (4 mL) and H2O (3 mL) was added the above organic layer,
which contained TfN3. More MeOH was added to make the mixture
a homogeneous solution. The reaction mixture was stirred overnight
at room temperature. The organic solvent was removed in vacuo.
The aqueous portion was extracted with ethyl acetate. After being
dried over anhydrous Na2SO4, it was purified by silica gel flash
chromatography (1:1 EtOAc-hexane) to give 0.96 g of product
1
15 in 89% yield. H NMR (400 MHz, CDCl3): δ 4.02 (dd, J )
11.6, 5.2 Hz, 1H), 3.92 (dd, J ) 11.6, 4.4 Hz, 1H), 3.82 (m, 1H),
3.78 (m, 1H), 3.69 (m, 1H), 1.64-1.54 (m, 3H), 1.46-1.28 (m,
23H), 0.91 (t, J ) 6.8 Hz, 3H). 13C NMR (100 MHz, CDCl3): δ
74.8, 72.6, 63.4, 62.0, 32.0, 31.9, 29.6, 29.5, 29.3, 25.7, 22.6, 14.0.
(2S,3S,4S)-2-Azido-3,4-di-O-benzyloctadecan-1,3,4-triol (16).
A solution of azidosphingosine 15 (0.40 g, 1.2 mmol), trityl chloride
(1.3 g, 4.7 mmol), and a catalytic amount of DMAP in dry pyridine
(5 mL) was stirred at 50 °C for 10 h. The reaction mixture was
diluted with CH2Cl2 and washed with cooled 1 N HCl, saturated
aqueous NaHCO3, and brine. The organic layer was dried over
anhydrous Na2SO4 and concentrated. The residue was then dissolved
in dry DMF (6 mL) and then treated with 60% NaH (106 mg, 2.6
mmol) for 10 min. BnCl (0.30 mL, 2.6 mmol) was added by syringe,
and the mixture was stirred overnight. After the solvent was
removed in vacuo, the residue was dissolved in water and extracted
with ethyl ether. The organic extraction was concentrated and then
dissolved in a mixture of CH2Cl2 and MeOH (2:1, 9 mL). pTsOH‚
H2O (95 mg, 0.5 mmol) was added, and the mixture was stirred
for 1 h at room temperature. After the mixture was concentrated,
the residue was purified by silica gel flash chromatography (1:6
EtOAc-hexane) to give 0.43 g of acceptor 16 in 68% yield for
three steps. 1H NMR (500 MHz, CDCl3): δ 7.37-7.28 (m, 10H),
4.73 (d, J ) 11.3 Hz, 1H), 4.69 (d, J ) 11.3 Hz, 1H), 4.65 (d, J )
11.4 Hz, 1H), 4.59 (d, J ) 11.4 Hz, 1H), 3.92 (dd, J ) 11.7, 5.1
Hz, 1H), 3.82 (dd, J ) 11.7, 5.1 Hz, 1H), 3.73 (m, 1H), 3.70
(m, 1H), 3.66 (m, 1H), 1.71 (m, 1H), 1.59 (m, 1H), 1.45 (m, 1H),
1.37-1.27 (m, 23H), 0.91 (t, J ) 6.8 Hz, 3H). 13C NMR (125
MHz, CDCl3): δ 138.0, 137.7, 128.6, 128.5, 128.1, 128.03, 128.0,
127.9, 80.5, 79.0, 73.7, 72.6, 63.1, 62.3, 32.0, 30.3, 29.7, 29.68,
29.6, 29.59, 29.4, 25.5, 22.7, 14.1.
r-D-Galactopyranosyl-(1f3)-â-D-galactopyranosyl-(1f4)-â-
D-glucopyranosyl-(1f1)-(2S,3R,4E)-2-hexacosanoylamino-4-oc-
tadecen-1,3-diol (1). A suspension of protected glycolipid 13
(42 mg, 0.021 mmol) and 10% Pd/C (10 mg) in ethanol (2 mL)
was stirred under H2 atmosphere (1 atm) for 2 h. The suspension
was filtered through a Celite pad, and the filtrate was concentrated.
The residue was dissolved in dry MeOH (2 mL), and freshly
prepared NaOMe (5 mg, 0.09 mmol) was added. The resulting
mixture was heated to reflux for 24 h. After the mixture was cooled
to room temperature, the precipitate was collected by centrifuge.
The precipitate was washed with MeOH (2 × 2 mL) and dissolved
in pyridine. The insoluble impurities were removed by centrifuge
and the clear solution was concentrated to give 1 (20 mg, 82%) as
a white powder. 1H NMR (500 MHz, pyridine-d5): δ 8.39 (d, J )
2,3,4,6-Tetra-O-acetyl-r-D-galactopyranosyl-(1f3)-2,4,6-tri-
O-pivaloyl-â-D-galactopyranosyl-(1f4)-2,3,6-tri-O-pivaloyl-â-D-
glucopyranosyl-(1f1)-(2S,3S,4S)-2-azido-3,4-di-O-benzylocta-
decan-1,3,4-triol (17). A suspension of trisaccharide donor 10 (82
mg, 0.06 mmol), phytosphingosine acceptor 16 (37 mg, 0.071
mmol), and 4 Å molecular sieves (0.5 g) in dry CH2Cl2 (2 mL)
was stirred at room temperature for 30 min. After the mixture was
cooled to -20 °C, TMSOTf (3 µL, 0.012 mmol) was added. The
resulting mixture was stirred for 2 h and then diluted with CH2Cl2
(10 mL). Saturated aqueous NaHCO3 (10 mL) was added to quench

Modification of the Ceramide Moiety
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15 3495
the reaction. The molecular sieves were filtered through a Celite
pad. The organic layer was separated, washed with brine (5 mL),
dried over Na2SO4, and concentrated. The residue was purified
by silica gel flash chromatography (1:3 EtOAc-hexane) to
provide 17 (83 mg, 81%) as a clear oil. 1H NMR (500 MHz,
CDCl3): δ 7.37-7.30 (m, 10H), 5.43 (dd, J ) 5.5, 2.0 Hz, 2H),
5.32-5.16 (m, 4H), 5.06 (dd, J ) 10.0, 3.0 Hz, 1H), 4.89 (dd, J )
9.5, 8.0 Hz, 1H), 4.67 (d, J ) 11.5 Hz, 1H), 4.62 (d, J ) 11.5 Hz,
1H), 4.58 (d, J ) 11.5 Hz, 1H), 4.52 (d, J ) 11.5 Hz, 1H), 4.51
(d, J ) 10.0 Hz, 1H), 4.45 (dd, J ) 7.5, 1.5 Hz, 2H), 4.30 (dd, J
) 12.0, 5.0 Hz, 1H), 4.17 (t, J ) 6.5 Hz, 1H), 4.14-4.07 (m, 4H),
4.03 (dd, J ) 15.5, 8.0 Hz, 1H), 3.92 (t, J ) 9.5 Hz, 1H), 3.86 (t,
J ) 6.8 Hz, 1H), 3.81 (dt, J ) 10.0, 3.0 Hz, 1H), 3.72 (m, 1H),
3.60 (m, 2H), 3.54 (m, 4H), 2.14 (s, 3H), 2.10 (s, 3H), 2.06 (s,
3H), 1.95 (s, 3H), 1.61 (m, 2H), 1.32-1.18 (m, 24H), 1.24 (s, 9H),
1.23 (s, 9H), 1.22 (s, 9H), 1.21 (s, 9H), 1.21 (s, 9H), 1.17 (s, 9H),
0.90 (t, J ) 7.0 Hz, 3H). 13C NMR (125 MHz, CDCl3): δ 177.8,
177.7, 177.1, 176.9, 176.6, 176.1, 170.3, 170.2, 170.1, 169.8, 163.5,
138.4, 138.0, 128.4, 128.0, 127.8, 127.78, 127.7, 100.8, 100.2, 94.5,
91.9, 79.3, 79.2, 74.5, 73.8, 73.5, 73.4, 72.0, 71.97, 71.6, 71.4, 70.0,
69.5, 67.54, 67.5, 67.1, 66.9, 65.4, 61.9, 61.5, 61.0, 39.1, 39.0, 38.9,
38.73, 38.72, 38.7, 31.9, 30.0, 29.73, 29.7, 29.66, 29.6, 29.57, 29.4,
27.4, 27.23, 27.2, 27.12, 27.11, 27.1, 25.4, 22.7, 21.0, 20.6, 20.5,
14.1. HRMS calcd for C88H135N3O28Na ([M + Na]+) 1704.9124,
found 1704.9106.
2,3,4,6-Tetra-O-acetyl-r-D-galactopyranosyl-(1f3)-2,4,6-tri-
O-pivaloyl-â-D-galactopyranosyl-(1f4)-2,3,6-tri-O-pivaloyl-â-D-
glucopyranosyl-(1f1)-(2S,3S,4S)-2-hexacosanoylamino-3,4-di-
O-benzyl-octadecan-1,3,4-triol (18). Triphenylphosphine (25 mg,
0.049 mmol) was added to a solution of azide 17 (83 mg, 0.049
mmol) in benzene (5 mL) and water (0.1 mL). The reaction mixture
was stirred at 50 °C for 10 h. The solvent was evaporated under
reduced pressure and azetroped with benzene (2 × 10 mL). Then
it was dissolved in dry THF (2 mL) and treated with cerotic acid
(31 mg, 0.078 mmol) and EDCI (15 mg, 0.078 mmol). After the
mixture was stirred for 10 h at room temperature, the solvent was
evaporated and the residue was partitioned between CH2Cl2 (10
mL) and water (10 mL). The organic layer was separated and dried
over Na2SO4. After the layer was concentrated, the residue was
purified by silica gel flash chromatography (1:3 EtOAc-hexane)
to provide 18 (84 mg, 84%) as a foam. 1H NMR (400 MHz,
CDCl3): δ 7.36-7.27 (m, 10H), 5.67 (d, J ) 8.2 Hz, 1H), 5.43
(br, 2H), 5.29-5.21 (m, 3H), 5.16 (dd, J ) 10.0, 8.0 Hz, 1H),
5.06 (dd, J ) 10.6, 2.9 Hz, 1H), 4.87 (dd, J ) 9.3, 7.9 Hz, 1H),
4.80 (d, J ) 11.3 Hz, 1H), 4.62 (d, J ) 11.7 Hz, 1H), 4.57 (d, J )
11.2 Hz, 1H), 4.51 (d, J ) 11.7 Hz, 1H), 4.45 (d, J ) 9.8, 7.9 Hz,
2H), 4.39-4.30 (m, 2H), 4.16-4.04 (m, 8H), 3.90 (t, J ) 9.5 Hz,
1H), 3.86-3.78 (m, 3H), 3.63 (m, 1H), 3.54 (m, 1H), 3.46
(m, 1H), 2.14 (s, 3H), 2.09 (s, 3H), 2.05 (s, 3H), 1.95 (s, 3H), 1.64
(m, 2H), 1.53 (m, 2H), 1.31-1.23 (m, 70H), 1.26 (s, 9H), 1.25
(s, 9H), 1.22 (s, 9H), 1.21 (s, 9H), 1.18 (s, 9H), 1.16 (s, 9H), 0.90
(t, J ) 6.4 Hz, 6H). 13C NMR (125 MHz, CDCl3): δ 177.8, 177.6,
176.9, 176.85, 176.8, 176.0, 172.4, 170.3, 170.2, 170.0, 169.7,
138.8, 138.6, 128.4, 128.3, 127.8, 127.7, 127.6, 127.5, 101.1, 100.1,
94.4, 80.0, 78.5, 74.4, 73.7, 73.5, 73.3, 72.0, 71.6, 71.3, 69.9, 68.9,
67.5, 67.49, 67.1, 66.9, 65.8, 65.3, 61.8, 61.5, 61.0, 49.8, 39.1, 39.0,
38.83, 38.8, 38.7, 38.68, 36.7, 31.9, 29.8, 29.7, 29.65, 29.6, 29.4,
29.37, 29.36, 29.3, 29.1, 27.3, 27.2, 27.18, 27.1, 27.09, 26.1,
25.6, 22.7, 21.0, 20.6, 20.5, 14.1. HRMS calcd for C114H185NO29-
Na ([M + Na]+) 2058.3115, found 2058.3051.
pyridine. The insoluble impurities were removed by centrifuge and
the clear solution was concentrated to give 2 (15 mg, 76%) as a
1
white powder. H NMR (500 MHz, pyridine-d5): δ 8.51 (d, J )
8.8 Hz, 1H), 7.72 (br, 1H), 7.38 (d, J ) 5.1 Hz, 1H), 7.07 (br,
1H), 6.71 (br, 1H), 6.64 (br, 1H), 6.50 (br, 2H), 6.43 (br, 2H),
6.12 (br, 1H), 5.86 (d, J ) 7.1 Hz, 1H), 5.81 (br, 1H), 5.65 (br,
1H), 5.12 (m, 1H), 5.03 (d, J ) 7.7 Hz, 1H), 5.00 (m, 1H), 4.87
(d, J ) 7.8 Hz, 1H), 4.76 (dd, J ) 10.5, 5.4 Hz, 1H), 4.73
(m, 1H), 4.66 (m, 1H), 4.58-4.49 (m, 4H), 4.47-4.40 (m, 4H),
4.37-4.34 (m, 2H), 4.31 (m, 1H), 4.26-4.18 (m, 4H), 4.01
(m, 2H), 3.81 (m, 1H), 2.42 (t, J ) 7.3 Hz, 2H), 2.21 (m, 1H),
1.93 (m, 2H), 1.81 (m, 2H), 1.67 (m, 1H), 1.43-1.19 (m, 66H),
0.86 (t, J ) 6.7 Hz, 3H), 0.85 (t, J ) 6.9 Hz, 3H). 13C NMR (125
MHz, pyridine-d5): δ 173.2, 105.2, 105.0, 97.5, 81.9, 90.0, 76.4,
76.3, 76.26, 75.6, 74.5, 72.6, 72.5, 71.4, 70.6, 70.5, 70.3, 70.2, 65.8,
62.0, 61.8, 61.6, 51.9, 36.7, 33.3, 31.9, 31.89, 30.1, 29.9, 29.8,
29.77, 29.7, 29.68, 29.6, 29.5, 29.4, 29.37, 26.4, 26.1, 22.7, 14.0.
HRMS calcd for C62H119NO19Na ([M + Na]+) 1204.8274, found
1204.8356.
Cell Stimulation and Cytokines Measurements. Methanol was
used to solubilize iGb3 and its analogues. The stimulation experi-
ments were performed at a final concentration of 1% methanol, a
concentration that is not toxic to cells. Glycoceramides at indicated
concentrations were incubated with hCD1d-transfected human B
lymphoblastoid (C1R). After 2 h, CD1d-restricted human VR24i
NKT cell clones (JS7) were added, and they were cocultured for
36 h. IL-4 and IFN-γ were quantified in cell culture supernatants
by Sandwich ELISA. First, capture antibody against the cytokine
was coated to the ELISA plate. After the antigen binding, a
biotinylated second antibody was used. Streptavidin-HRP conju-
gate was applied to finally quantitate the antigen amount.
Acknowledgment. This project was supported by The Ohio
State University (support to Peng George Wang) and by Swiss
National Foundation (Grant 3100A0-109918 to Gennaro De
Libero).
Supporting Information Available: Proton and carbon NMR
spectra of compounds 3-8, 10-12, 14-18; elemental analysis
results. This material is available free of charge via the Internet at
http://pubs.acs.org.
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